U.S. patent application number 16/746234 was filed with the patent office on 2020-05-14 for instrument and method for optical particle sensing.
The applicant listed for this patent is Invitrox, Inc.. Invention is credited to Don Gabriel.
Application Number | 20200150023 16/746234 |
Document ID | / |
Family ID | 47558526 |
Filed Date | 2020-05-14 |
United States Patent
Application |
20200150023 |
Kind Code |
A1 |
Gabriel; Don |
May 14, 2020 |
Instrument and Method for Optical Particle Sensing
Abstract
Devices for detecting particle sizes and distributions using
focused light scattering techniques, by passing a sample through a
focused beam of light, are disclosed. In one embodiment, the
devices include one or more lasers, whose light is focused into a
narrow beam and into a flow cell, and dispersions are passed
through the flow cell using hydrodynamic sample injection. In
another embodiment, a plurality of lasers is used, optionally with
hydrodynamic sample injection. Particles pass through and scatter
the light. The scattered light is then detected using scatter and
extinction detectors, and, optionally, fluorescence detectors, and
the number and size of the particles is determined. Particles in
the size range of 0.1 to 10 .mu..eta. can be measured. Using the
device, significantly smaller particles can be detected than if
techniques such as EQELS, flow cytometry, and other conventional
devices for measuring biological particles.
Inventors: |
Gabriel; Don; (Carrboro,
NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Invitrox, Inc. |
Durham |
NC |
US |
|
|
Family ID: |
47558526 |
Appl. No.: |
16/746234 |
Filed: |
January 17, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15244096 |
Aug 23, 2016 |
10539496 |
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16746234 |
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14233874 |
Mar 31, 2014 |
9423335 |
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PCT/US12/47766 |
Jul 20, 2012 |
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15244096 |
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61510447 |
Jul 21, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2015/0065 20130101;
G01N 2015/1006 20130101; G01N 2500/10 20130101; G01N 21/6428
20130101; G01N 2015/008 20130101; G01N 33/5026 20130101; G01N
2015/03 20130101; G01N 33/5044 20130101; G01N 2015/1087 20130101;
G01N 15/0211 20130101; G01N 33/5008 20130101; G01N 2015/0073
20130101; G01N 33/502 20130101; G01N 15/1459 20130101; G01N
2015/0084 20130101; G01N 33/483 20130101; G01N 15/1434
20130101 |
International
Class: |
G01N 15/14 20060101
G01N015/14; G01N 33/50 20060101 G01N033/50; G01N 33/483 20060101
G01N033/483; G01N 21/64 20060101 G01N021/64; G01N 15/02 20060101
G01N015/02 |
Claims
1-62. (canceled)
63. A method of identifying a biological particle of interest in a
sample medium, said method comprising: a) introducing a sample
medium, which may or may not include a biological particle of
interest, into a device for measuring the size and/or number of
particles in a sample medium, wherein the sample medium is, or is
derived from, one or more fluids selected from the group consisting
of blood, blood products, water, cerebrospinal fluid, ascites,
pleural fluid, and synovial fluid, wherein the device for measuring
the size and/or number of particles in a sample medium comprises:
i) one or more lasers which produce one or more beams of laser
light, each of which beams of laser light following a path, ii) a
first focusing lens positioned in the path of one of the beams of
laser light, which first focusing lens focuses the beams of laser
light such that the effective width in a direction transverse to
the path of the beams of laser light is between about 0.05 and 0.5
.mu.m, thereby forming a focused beam of laser light, iii) a flow
cell positioned in the path of the focused beam of laser light,
wherein the flow cell is adapted to receive and pass a sample
medium comprising a dispersion of particles, iv) a spatial filter
positioned in the path of the scattered beams of laser light, which
allows the scattered beams of laser light to pass through, and does
not allow the beams of laser light that are not scattered to pass
through, v) a collimating lens positioned in the path of the
scattered beams of laser light that passed through the spatial
filter, to collimate the scattered beams of laser light that passed
through the spatial filter, vi) a second focusing lens positioned
in the path of the collimated beams of laser light, for focusing
the collimated beams of laser light that passed through the
collimating lens, vii) a scatter detector positioned in the path of
the focused beams of laser light that passed through the second
focusing lens, viii) a mirror positioned between the flow cell and
the spatial filter, to reflect a portion of the scattered beams of
laser light, and ix) an extinction detector positioned in the path
of the reflected portion of the scattered beams of laser light, b)
passing the sample medium through one or more focused beams of
laser light in the device, so that the focused beam of laser light
is scattered when it interacts with one or more of the particles,
and the focused beam of laser light is not scattered if it does not
interact with the one or more particles, depending on whether the
one or more particles are in the path of the focused beam of laser
light, c) using focused light scattering techniques to prepare a
spectrum showing particle size distribution from within the sample
medium, wherein focused light scattering techniques comprise
passing a sample media through a particular path, where a focused
beam of light passes through the sample media, wherein the focused
beam is of a size such that a particle in the size range of 0.1 to
10 .mu.m is sufficient to block all of the beam, or a significant
enough part of the beam, so that the particle size can be measured,
and c) identifying the presence or absence of the biological
particle of interest by comparing the sizes of the particles in the
sample medium with the known size of the particle of interest.
64. The method of claim 63, wherein the size of the particles in
the sample medium is compared with a reference database comprising
a library of particle sizes obtained using focused light scattering
techniques, wherein the library comprises ten or more spectra.
65. The method of claim 63, wherein, after an initial determination
is made that a particle of interest is present in the sample
medium, a confirmatory assay is performed, wherein the confirmatory
assay involves taking an EQELS spectra of the sample medium, and
verifying the existence of a biological particle by identifying a
feature unique to the biological particle.
66. The method of claim 63, wherein the biological particle of
interest is a lymphocyte, erythrocyte, B-cell, T-cell, neutrophil,
monocyte, bacteria, fungi, viruses, protozoa, tumor cell, red blood
cell, white blood cell, granulocyte, platelet, cancer cell, or stem
cell.
67. The method of claim 63, wherein the biological microparticle
has a size ranging from about 0 .mu.m to about 20 .mu.m.
68. A method of determining the efficacy of a putative therapeutic
agent, comprising: a) introducing a sample medium, which sample
medium comprises a biological particle with a receptor to which a
putative therapeutic agent will bind, into a device for measuring
the size and/or number of particles in a sample medium, wherein the
device for measuring the size and/or number of particles in a
sample medium comprises: i) one or more lasers which produce one or
more beams of laser light, each of which beams of laser light
following a path, ii) a first focusing lens positioned in the path
of one of the beams of laser light, which first focusing lens
focuses the beams of laser light such that the effective width in a
direction transverse to the path of the beams of laser light is
between about 0.05 and 0.5 .mu.m, thereby forming a focused beam of
laser light, iii) a flow cell positioned in the path of the focused
beam of laser light, wherein the flow cell is adapted to receive
and pass a sample medium comprising a dispersion of particles, iv)
a spatial filter positioned in the path of the scattered beams of
laser light, which allows the scattered beams of laser light to
pass through, and does not allow the beams of laser light that are
not scattered to pass through, v) a collimating lens positioned in
the path of the scattered beams of laser light that passed through
the spatial filter, to collimate the scattered beams of laser light
that passed through the spatial filter, vi) a second focusing lens
positioned in the path of the collimated beams of laser light, for
focusing the collimated beams of laser light that passed through
the collimating lens, vii) a scatter detector positioned in the
path of the focused beams of laser light that passed through the
second focusing lens, viii) a mirror positioned between the flow
cell and the spatial filter, to reflect a portion of the scattered
beams of laser light, and ix) an extinction detector positioned in
the path of the reflected portion of the scattered beams of laser
light, b) passing the sample medium through one or more focused
beams of laser light in the device, so that the focused beam of
laser light is scattered when it interacts with one or more of the
particles, and the focused beam of laser light is not scattered if
it does not interact with the one or more particles, depending on
whether the one or more particles are in the path of the focused
beam of laser light, c) using focused light scattering techniques
to prepare a spectrum showing particle size and distribution from
within the sample medium, wherein focused light scattering
techniques comprise passing a sample media through a particular
path, where a focused beam of light passes through the sample
media, wherein the focused beam is of a size such that a particle
in the size range of 0.1 to 10 .mu.m is sufficient to block all of
the beam, or a significant enough part of the beam, so that the
particle size can be measured, b) incubating the sample medium with
a putative therapeutic agent, c) obtaining a second spectra showing
particle size and distribution on the incubated sample medium using
focused light scattering techniques, and d) determining whether the
particle size and distribution has been altered by the incubation
of the putative therapeutic agent, a change in the particle size
and/or distribution is indicative of a complex formation of the
putative therapeutic agent and the biological particle.
69. The method of claim 68, wherein the biological microparticle is
selected from tumor cells, red blood cells, white blood cells,
granulocytes, platelets, monocytes, neutrophils, lymphocytes,
cancer cells, bacteria, viruses, and fungi.
70. A method for determining whether a biological particle will
form a complex with a known therapeutic agent, comprising: a)
obtaining a spectra showing particle size and distribution using
focused light scattering techniques on a sample medium comprising a
biological particle with a receptor to which a known therapeutic
agent may or may not bind, using a device for measuring the size
and/or number of particles in a sample medium, wherein the device
for measuring the size and/or number of particles in a sample
medium comprises: i) one or more lasers which produce one or more
beams of laser light, each of which beams of laser light following
a path, ii) a first focusing lens positioned in the path of one of
the beams of laser light, which first focusing lens focuses the
beams of laser light such that the effective width in a direction
transverse to the path of the beams of laser light is between about
0.05 and 0.5 .mu.m, thereby forming a focused beam of laser light,
iii) a flow cell positioned in the path of the focused beam of
laser light, wherein the flow cell is adapted to receive and pass a
sample medium comprising a dispersion of particles, iv) a spatial
filter positioned in the path of the scattered beams of laser
light, which allows the scattered beams of laser light to pass
through, and does not allow the beams of laser light that are not
scattered to pass through, v) a collimating lens positioned in the
path of the scattered beams of laser light that passed through the
spatial filter, to collimate the scattered beams of laser light
that passed through the spatial filter, vi) a second focusing lens
positioned in the path of the collimated beams of laser light, for
focusing the collimated beams of laser light that passed through
the collimating lens, vii) a scatter detector positioned in the
path of the focused beams of laser light that passed through the
second focusing lens, viii) a mirror positioned between the flow
cell and the spatial filter, to reflect a portion of the scattered
beams of laser light, and ix) an extinction detector positioned in
the path of the reflected portion of the scattered beams of laser
light, wherein the sample medium is, or is derived from, a fluid
selected from the group consisting of blood, blood products, water,
cerebrospinal fluid, ascites, pleural fluid, and synovial fluid,
wherein focused light scattering techniques comprise passing a
sample media through a particular path, where a focused beam of
light passes through the sample media, and wherein the focused beam
is of a size such that a particle in the size range of 0.1 to 10
.mu.m is sufficient to block all of the beam, or a significant
enough part of the beam, so that the particle size can be measured,
b) incubating the sample medium with a known therapeutic agent, b)
obtaining a second spectra showing particle size and distribution
on the incubated sample medium using focused light scattering
techniques, and c) determining whether the particle size and
distribution has been altered by the incubation of the known
therapeutic agent, a change in the particle size and/or
distribution is indicative of a complex formation of the known
therapeutic agent and the biological particle.
71. The method of claim 70, wherein the biological microparticle is
selected from tumor cells, red blood cells, white blood cells,
granulocytes, platelets, monocytes, neutrophils, lymphocytes,
cancer cells, bacteria, viruses, and fungi.
72. A method for determining an effective dosage of a therapeutic
agent against a known cell, microbe or virus comprising: a)
generating a first spectrum showing particle size and distribution
using focused light scattering for a known cell, microbe or virus,
using a device for measuring the size and/or number of particles in
a sample medium, wherein the device for measuring the size and/or
number of particles in a sample medium comprises: i) one or more
lasers which produce one or more beams of laser light, each of
which beams of laser light following a path, ii) a first focusing
lens positioned in the path of one of the beams of laser light,
which first focusing lens focuses the beams of laser light such
that the effective width in a direction transverse to the path of
the beams of laser light is between about 0.05 and 0.5 .mu.m,
thereby forming a focused beam of laser light, iii) a flow cell
positioned in the path of the focused beam of laser light, wherein
the flow cell is adapted to receive and pass a sample medium
comprising a dispersion of particles, iv) a spatial filter
positioned in the path of the scattered beams of laser light, which
allows the scattered beams of laser light to pass through, and does
not allow the beams of laser light that are not scattered to pass
through, v) a collimating lens positioned in the path of the
scattered beams of laser light that passed through the spatial
filter, to collimate the scattered beams of laser light that passed
through the spatial filter, vi) a second focusing lens positioned
in the path of the collimated beams of laser light, for focusing
the collimated beams of laser light that passed through the
collimating lens, vii) a scatter detector positioned in the path of
the focused beams of laser light that passed through the second
focusing lens, viii) a mirror positioned between the flow cell and
the spatial filter, to reflect a portion of the scattered beams of
laser light, and ix) an extinction detector positioned in the path
of the reflected portion of the scattered beams of laser light;
wherein focused light scattering techniques comprise passing a
sample media through a particular path, where a focused beam of
light passes through the sample media, and wherein the focused beam
is of a size such that a particle in the size range of 0.1 to 10
.mu.m is sufficient to block all of the beam, or a significant
enough part of the beam, so that the particle size can be measured,
b) incubating a first concentration of a therapeutic agent with the
known cell, microbe or virus; c) generating a second spectrum
showing particle size and distribution using focused light
scattering of the combination of the therapeutic agent and the
known cell, microbe or virus; and d) comparing the first and second
spectra, wherein a change in the particle size and/or distribution
is indicative of binding of the therapeutic agent and the cell,
microbe or virus, and wherein binding is indicative of inhibition
of the known cell, microbe or virus; e) repeating steps a-e with
varying amounts of the therapeutic agent; and f) comparing the
spectra to determine the minimum amount of therapeutic agent
required to effectively bind the known cell, microbe or virus.
73. The method of claim 72, wherein the known cell, microbe or
virus is a cell selected from the group consisting of tumor cells,
red blood cells, white blood cells, granulocytes, platelets,
monocytes, neutrophils, lymphocytes, and cancer cells.
74. The method of claim 63, wherein the device further comprises a
hydrodynamic flow injector for introducing the sample medium into
the flow cell.
75. The method of claim 63, further comprising a detector
positioned in the path of the beams of light passing through the
second chromatic filter.
76. The method of claim 75, wherein the detector is a fluorescence
detector.
77. The method of claim 63, wherein the device further comprises:
k) a processor adapted to receive information from the various
detectors, and to responsively generate an output correlative of
the size and/or number of particles in the sample medium.
78. The method of claim 77, further comprising a memory map for
storing information on the size and/or number of particles in the
sample medium.
79. The method of claim 77, further comprising a video display
interface operatively coupled to the processor for outputting
information on the size and/or number of particles in the sample
medium.
80. The method of claim 63, wherein the first focusing lens focuses
the beams of light such that the effective width in a direction
transverse to the axis of the light beam is between about 0.05 and
0.25 .mu.m.
81. The method of claim 63, wherein the first focusing lens focuses
the beams of light such that the effective width in a direction
transverse to the axis of the light beam is between about 0.05 and
0.15 .mu.m.
Description
FIELD OF THE INVENTION
[0001] This application is generally in the area of single particle
optical light scattering devices, and techniques for using them,
particularly measure extremely small particles.
BACKGROUND OF THE INVENTION
[0002] It is often essential to characterize biological particles
by their size, surface condition, states of activation of any
surface receptors, distribution, and the like. This information is
useful in cell-based assays and other processes that rely upon
those characteristics. Additionally, it is useful in certain
diagnostic applications to detect known changes of the surface of a
biological particle. Accordingly, it can be desirable to detect the
surface and monitor changes to the surface in an efficient and
accurate manner.
[0003] "Electrophoretic Quasi-Elastic Light Scattering" (EQELS) is
one method for characterizing biological particles. This method
uses electrophoresis that is dependent on the particle's surface
charge density to identify and characterize suspended biological
particles. EQELS uses cells placed in an electric field, where the
surface charge of the particle will determine how that particle
moves in the electric field. Monitoring the electrophoretic
mobility of the cells provides information useful in distinguishing
among different particles in the field. One can screen and optimize
drug candidates which interact with the biological particles by
comparing the spectra of the particles alone, or bound to the drug
candidates.
[0004] Coulter counters can also be used to characterize biological
particles. These devices are primarily used to count and size cells
and other biological particles. The Coulter Counter works by
drawing fluid containing the biological particle through a small
opening located within a current between two electrodes. As the
fluid is drawn through the opening, the biological particles flow
through the current and measurably displace a portion of the
current. The measurable displacement is translated to a pulse that
is digitally processed by the Coulter Counter and translated to
allow one to characterize the size and number of biological
particles in the fluid.
[0005] Flow cytometry can also be used to characterize biological
particles. Flow cytometry uses a beam of light, such as a laser,
trained on a fluid to characterize, count and optionally sort
particles in the fluid. The fluid is focused into a stream, and
detectors at the intersection of the light and the fluid stream
determine scatter--both forward and side. Additionally, a
fluorescent detector may be present to detect fluorescent or
fluorescently-tagged particles. One can determine various physical
and chemical characteristics of each individual particle by
analyzing the detected pattern.
[0006] These methods are useful in detecting and characterizing
microparticles, including determining the number of particles,
density within a fluid medium, size, and surface characteristics of
the particle, confirming binding, or lack thereof, and the like.
The microparticles are generally in the size of between 0.1 .mu.m
and 100 .mu.m. However, developments in technology demand the
characterization of smaller biological particles, including, but
not limited to, nanoparticles.
[0007] The size of biological particles that can be analyzed using
currently available technology is limited. Accordingly, there is a
need for devices and processes for characterizing biological
particles that can detect biological particles of varying sizes,
including particles smaller than microparticles, and which can
characterize the detected particles with accuracy, quantify the
particles and/or monitor the particles. The present invention
provides such devices and processes.
SUMMARY OF THE INVENTION
[0008] The present invention relates to devices useful for, and
methods of detecting sizes and distributions of particles using
focused light scattering techniques. The devices use focused light
scattering techniques, and can be used, for example, to diagnose
disease, identify therapeutic agents, and obtain other useful
information about biological particles and/or therapeutic agents in
a sample medium. Representative particle sizes that can be measured
range from between about 0.1 .mu.m to about 100 .mu.m, more
typically in the range of between about 0.1 and about 20 .mu.m.
[0009] Briefly, focused light scattering techniques involve passing
a sample media through a particular path, where a focused beam of
light passes through the sample media. The focused beam is of a
size such that a particle in the size range of 0.1 to 10 .mu.m is
sufficient to block all of the beam, or a significant enough part
of the beam, so that the particle size can be measured.
[0010] When there are no particles passing through the pathway of
the beam, the beam passes through the media and onto a detector.
When a particle, or part of a particle, passes through the beam,
the beam is deflected. A diminished amount of light, or no light at
all, then reaches the detector, thus indicating that a particle (or
part of a particle) has interacted with the beam. The amount of
diminished light reaching the detector provides information about
the size of the particle. This is repeated as particles in the
sample medium pass through the beam, for example, until the sample
medium has entirely passed through the beam. Appropriate algorithms
then take the information, and the output is a spectrum showing the
particle size and particle distribution.
[0011] The device includes light from at least one, and, ideally,
at least two laser light sources. Where two or more laser light
sources are used, they each ideally provide light at different
wavelengths. Where two or more light sources are used, they can be
passed through a beam splitter, which combines the light beams. The
combined light beams pass through a focusing lens, which focuses
the light into a narrow beam. The beam is then passed through a
flow cell.
[0012] Dispersions including particles which are to be counted and
sized are passed through a flow cell, where they pass through the
focused beam of light. In one embodiment, the solutions are
introduced into the flow cell using a hydrodynamic sample injector.
When the focused light beam comes into contact with a particle, the
light is scattered.
[0013] The scattered light is then passed through a spatial filter,
such as a circular spatial filter. The filter allows light
reflected at certain angles, indicative of light hitting particles
of a certain size, to pass through. Ideally, the filter does not
permit light to pass through when it has not interacted with a
particle. However, a portion of this light can be reflected off of
a mirror, which can be a movable mirror, onto a detector (ideally
an extinction detector).
[0014] The light passing through the spatial filter is then passed
through a collimating lens, which focuses the beam from the
reflected angles into a straight line. Where one laser is used, the
light can then pass through a focusing lens, which then focuses the
light into a narrower beam, which is then passed on to a detector.
Where two lasers are used, the light can then pass through a second
beam splitter, which splits the beam into two beams.
[0015] Ideally, the splitter does not change the orientation of one
of the beams. That is, one beam proceeds directly through the
splitter, and the other is diverted, for example, at a 90 degree
angle. In this manner, rather than using a combination of a
collimating lens and focusing lens, one of the beams need only be
passed through a focusing lens. The other beam may be spread out
when it passes through the splitter, in which case it is
advantageous to use both a collimating lens and a focusing
lens.
[0016] The focused beams of light are passed through chromatic
filters, which filters out light of a wavelength produced by one of
the lasers, and passes light from the wavelength produced by the
other of the lasers onto a detector. This way, it is possible to
detect light from each of the lasers at separate detectors. Where
some of the particles may be labeled, and the label is detectable,
for example, by fluorescence, one can thus obtain information about
all particles, and about a subset of particles that fluoresces.
Those particles which fluoresce are typically those which form a
complex with a fluorescently-labeled material, such as a
fluorescently-labeled antibody. In one embodiment, a scattered
light detector is used to detect light reflected from all
particles, and a fluorescence detector is used to detect light
reflected from particles that either are fluorescent, or are
complexed with fluorescently-labeled materials, such as
fluorescently-labeled antibodies.
[0017] The device also includes a dynamic monitoring system and
method employed to monitor the size and/or number of particles,
and, optionally, additional information on a subset of the
particles, such as their number and/or size, which fluoresce when
complexed to a particular fluorescent molecule, in a single
particle optical sizing device as described herein. In one
embodiment, the dynamic monitoring system is a computer either
physically connected to the device, or connected remotely, for
example, using Bluetooth, infrared, or other such technology, and
capable of receiving information from the various detectors, as
well as software which handles the digital interface with the
device, wherein the computer and software are capable of counting
particles, sizing particles, and storing information on the number
and size of the particles in a given sample based on the
information received from the detectors and algorithms that use
this information to determine particle size and/or number of
particles.
[0018] Using focused light scattering techniques, significantly
smaller particles can be detected than if techniques such as EQELS,
flow cytometry, and other conventional methods of measuring
biological particles are used. Mathematical algorithms described
herein can enable one to not only detect small particles, but also
to determine a range of particle sizes, relative quantities of such
particles, and shapes of the particles.
[0019] Cells are one type of biological particle that can be
detected. The method can be used to determine the presence or
absence of a specific type of cell in a given solution. For
example, a sample of blood, urine, spinal fluid, and the like can
be evaluated for the presence or absence of bacteria, fungi,
viruses, and the like. The particle size, and, optionally, particle
shape, can also provide information about the specific type of
bacteria, fungi or virus.
[0020] In one embodiment, suitable information on the particles can
be obtained simply by obtaining a spectra using focused light
scattering of a sample medium, wherein the particle size and
distribution provides sufficient information about the presence or
absence of certain biological particles present in the sample
medium. For example, specific bacteria, fungi, or viruses can be
identified solely on the basis of their size, and liposomal
suspensions can be evaluated for agglomeration solely on the basis
of the size of the agglomerated particles.
[0021] In other embodiments, where there is an interest in
determining whether a particular agent forms a complex with a
particular type of biological particle, additional information may
be required. That is, one can determine the presence or absence of
a particular cell type, or an ejected particle from a type of cell,
by forming a complex between a) the cell or ejected particle and b)
an active agent conjugated to a microparticle or nanoparticle
("conjugate"). The complex has a larger particle size than the
cell, the ejected particle, or the conjugate, so the focused light
scattering technique can determine whether a complex was formed on
the basis of particle size.
[0022] The complex can be formed with a labeled material, such as a
fluorescently-labeled antibody. In one embodiment, the device
includes one or more additional lasers that shine a beam of light
at a frequency in which the fluorescently-labeled
antibody/antibodies absorb light. Thus, one source of light can be
used to count and size the number of particles, and another can be
used to determine the number of particles that formed a complex
with a desired fluorescently-labeled material.
[0023] In some aspects of this embodiment, the biological particle
is a cell that expresses a specific receptor, and the techniques
permit high throughput screening of putative therapeutic agents
that bind to the receptor.
[0024] In other aspects of this embodiment, the biological particle
comprises cells from a patient, for example, blood cells or cancer
cells, and these cells are incubated with putative therapeutic
agents. Agents that bind to the cells can potentially be useful as
therapeutic agents for the patient. Accordingly, this embodiment
provides personalized medicine approaches.
[0025] In some of these embodiments, two spectra are taken. The
first is taken on the sample media before complex formation, and
the second is taken after complex formation, so one can look for
the difference in particle size and distribution. However, in other
embodiments, where the complex has a known particle size, and all
that is required is to show that the complex formed, one can simply
incubate the biological particle and the substance which may or may
not form a complex with the biological particle, and use focused
light scattering techniques to determine whether the complex was
formed.
[0026] If the sample medium, with the biological particle and the
conjugate both present, is passed repeatedly through the focused
light scattering detector over a period of time, the kinetics of
complex formation can be observed.
[0027] If the sample medium is scanned with the biological particle
and the conjugate both present, but with different scans taken with
differing concentrations of the biological particle and/or
conjugate, one can determine additional information, relative to
binding affinity, minimum inhibitory concentration, and the
like.
[0028] If the sample medium includes cells of different sizes,
expressing different receptors, then information on the selectivity
of a putative therapeutic agent for one receptor over the other can
be obtained.
[0029] If the agent binding to the cells results in cell rupture,
then the efficacy of the active agent can be represented by a
decrease in particle (cell) density in the sample medium over
time.
[0030] Thus, complex formation provides useful information about
the biological particle, or the agent bound to the microparticle or
nanoparticle. For example, where the cell is a known cell, one can
screen putative therapeutic agents for their ability to bind to the
cell. Where the therapeutic agent is a known therapeutic agent, one
can determine whether a particular cell binds to the therapeutic
agent. This information can be useful in identifying personalized
medical approaches for a patient.
[0031] For example, it is critical to determine in a timely manner
whether a cancer patient will respond to a particular therapy. That
is, the tumors can grow and metastasize before the physician
determines that the patient does not respond to the therapy.
[0032] In one embodiment, the microparticles have a particle size
in the range of between about 0.1 and 10 .mu.m, and ideally have a
relatively consistent amount of active agent bound to them. One way
to produce particles with a relatively consistent amount of active
agent bound to them is to use dendrimers, where the dendrimers
include a known quantity of the active agent. Another way is to
produce polymer particles with a) a relatively narrow size
distribution, and b) a relatively consistent amount of protected
functional groups, so that after the polymers are produced, the
protecting groups can be removed, and the functional groups used to
conjugate the polymer particles to an active agent.
[0033] The active agent can be conjugated with the particle in such
a way that the portion of the active agent that is known to be
active (i.e., binds a receptor) is not significantly sterically
hindered by its conjugation with the particle. In some embodiments,
this will involve preparing an analogue of the active agent which
includes a further functional group which can be attached to the
particle.
[0034] In one embodiment, metallic particles, such as gold
particles, are used. Because these particles scatter a significant
amount of light, they can be conjugated with a specific active
agent, and used to identify even small molecules that bind to the
agent. That is, the amount of light that the particle scatters is
sufficiently large that the binding of the agent to the molecule of
interest can be measured, even though the molecule is not within
the size range of biological particles that can be measured. Means
for conjugating active agents to metallic particles are known to
those of skill in the art.
[0035] The present invention will be better understood with
reference to the following detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] FIG. 1 is a schematic illustration of a single particle
optical sizing device, referred to herein as a "surface antigen
detection enumerator" light scattering device capable of measuring
particles as small as around 0.01 .mu.m in diameter.
[0037] FIG. 2 is a schematic illustration of a device used for
focused light scattering, using a single light source and a single
detector.
[0038] FIG. 3 is a schematic illustration of a hydrodynamic flow
injector.
[0039] FIG. 4 is a schematic illustration of a device to apply
precise mechanical shear to a sample.
[0040] FIG. 5 is a flow chart showing the path of a fluid sample
from a pump controlled by robotics, through a shearing device, to a
scattering cell, where dynamic light scattering or electrophoretic
light scattering can be measured, to where the sample leaves the
device as effluent.
[0041] FIG. 6 is a chart showing the measurement of particles in a
sample composed of 6 differently-sized polystyrene beads, assessed
using the surface antigen detection enumerator light scattering
device in terms of particle count (number) by particle size
(.mu.m). The chart shows the remarkable resolution of very small
particle sizes. Current flow cytometers are not capable of
resolution to this degree.
[0042] FIG. 7 is a chart showing similar data from the data shown
in FIG. 6. The data in FIG. 7 is presented as points rather than as
histograms, and is reflected in particles per 10 ml sample, versus
particle size (.mu.m). Also, FIG. 7 separates the size distribution
into 3 different windows. In this embodiment, each window has a
separate detector that has been adjusted to detect particles in a
specific size range. The smallest particles are assessed from
scatted light focused onto a high-gain detector, the middle window
from scattered light focused onto a low gain detector, and the
window with the largest particles by a light extinction method.
DETAILED DESCRIPTION
[0043] The present invention relates to devices for measuring
particle size using focused light scattering techniques, and
methods for using focused light scattering techniques in biological
applications. Focused light scattering techniques provide one with
the ability to analyze a fluid and determine the size and number of
particles in a given sample and to, optionally, further
characterize the particles in the sample. Where the particle is a
biological particle, this information can be used to diagnose
disease, to conduct high throughput bioassays, and to obtain
information for personalized medical treatment.
[0044] The methods described herein provide numerous advantages
over the previous methods in the art, including the ability to
identify and characterize smaller particles, identify particles and
determine particle size, number or other characteristics without
using fluorescent antibodies or expensive flow cytometry, improving
the identification of the initial onset of the change in voltage
due, which would improve resolution of the generated spectra,
control of particle shearing, and improved information regarding
particle shape.
[0045] The methods also provide numerous characteristics of the
particles being evaluated, including, but not limited to:
identifying biological particles and distinguishing them from
various cells, quantifying particles, identifying surface epitopes,
identifying particle shape, and correlating this information with
platelet activation, thrombin production, disease states, and the
efficacy of putative therapeutic agents.
[0046] The present invention will be better understood with
reference to the following definitions.
Definitions
[0047] The term "cell" as used herein refers to any type of cell,
including human cells, animal cells (such as swine cells, rodent
cells, canine cells, bovine cells, ovine cells and/or equestrian
cells) cloned cells, plant cells, or the like. The cells may be
blood cells, cultured cells, biopsied cells, or cells that are
fixed with a preservative. The cells can be nucleated, such as
white blood cells or suspended endothelial cells, or non-nucleated,
such as platelets or red blood cells.
[0048] The term "focused light scattering" refers to a method for
sensing single particles, suspended in a solution, when the
solution is passed through a focused beam. When the beam passes
through the solution without being scattered by a particle, the
beam passes on to a photodetector and the intensity is measured.
When the beam is scattered, in whole or in part, by a particle, the
intensity of the beam hitting the photodetector is altered. The
particle size and concentration can be calculated, for example,
using light-extinction, light-scattering detection, or both.
[0049] A "focused light scattering device" is a multi-particle
optical sensor, which has high sensitivity and responds to
relatively concentrated suspensions, uses a relatively narrow light
beam to illuminate an optical sensing zone non-uniformly.
[0050] As used herein "particles" are small fragments or completely
intact biological cells, and related to a living organism when
referred to as "biological particles." Intact cells may range in
size from about 1 micron to 20 microns. Aggregates of intact cells
or fragments of cells may range in size from 2 microns to 100
microns. "Microparticles" are fragments of biological cells or
particles that generally range in size from about 0.1 .mu.m to
about 0.8 generally 0.1-20 Examples include, but are not limited to
blood cells, platelets (1-3 micron), cancer cells (5-15 micron),
red blood cells (.about.7 .mu.m), white blood cells (.about.5-10
.mu.m), bacteria (.about.0.5-1 .mu.m), tumors, granulocytes,
monocytes, neutrophils, lymphocytes, endothelial cells, stem cells,
viruses, and fungi.
[0051] "Light extinction" as used herein is a measurement of the
absorption and/or scattering of light in an electromagnetic field
by particles as they pass through the field. As a particle passes
through a field, there is a momentary reduction in the transmitted
light intensity due to the light refraction, absorption and/or
scattering. Measurement of light extinction by the particles
provides additional information regarding the characteristics of
the particles. A light extinction spectrum can be generated for
each particle.
[0052] "Light scattering" occurs when there is a momentary change
in the intensity of the incident light caused by the interaction of
the incident photons with the particle. In the case on focused
scattering device, the intensity of the scattered light reaching
the detector is proportional to the size of the particle. Thus,
when the particles being characterized are biological particles,
the method of light scattering will involve measuring voltage at
the detector this will be proportional to the particle size. An
exemplary focused light scattering system for detecting biological
particles is shown in FIG. 1.
[0053] "Nanoparticles" as used herein are particles or biological
particles that are generally smaller than 0.1 .mu.m in size.
Because of their small size, nanoparticles have a very high surface
area to volume ratio. Accordingly, nanoparticles often possess
unique physical characteristics. The present invention provides a
way to both quantify and monitor nanoparticles, in particular,
cellular nanoparticles, which are often believed to be responsible
for initiating further biochemical processes in living
organisms.
[0054] The types of devices that can be used to carry out these
diagnostic assays, and methods for performing these assays, are
described in more detail below.
I. Focused Light Scattering Devices and Algorithms for Measuring
Particle Size and Shape
[0055] The focused light scattering device includes the following
components:
[0056] a) one or more lasers which produce beams of laser
light,
[0057] b) a first focusing lens positioned in the path of the beams
of laser light, which focusing lens focuses the beams of light to a
size wherein a particle with a diameter of about 0.1 .mu.m is
sufficient to block all or substantially all of the light,
[0058] c) a flow cell positioned in the path of the focused light
source, wherein the flow cell is capable of receiving a sample
medium comprising a dispersion of particles, and passing the sample
medium through the focused light source, where light that interacts
with one or more of the particles is scattered, and light that does
not interact with one or more particles is not scattered,
[0059] d) a spatial filter, such as a circular spatial filter,
positioned in the path of the light, which allows scattered light
to pass through, and does not allow light that is not scattered to
pass through,
[0060] e) a collimating lens positioned in the path of light
passing through the circular spatial filter, through which
scattered light is collimated,
[0061] f) a second focusing lens positioned in the path of the
collimating lens, focusing the light passing through the
collimating lens,
[0062] g) a scatter detector positioned in the path of the focused
light passing through the focusing lens,
[0063] h) a mirror positioned between the flow cell and the
circular spatial filter, which mirror reflects a portion of the
scattered light, and
[0064] i) an extinction detector positioned in the path of the
reflected scattered light.
[0065] Where more than two laser light sources are used, additional
focusing lenses, collimating lenses, chromatic filters, and
detectors can be used so that each source of laser light can be
separated from the others. In one embodiment, the beam splitter
used in these embodiments is one that is capable of splitting the
beam into multiple beams, rather than just two beams. In another
embodiment, a series of beam splitters is used, each of which
splits a beam into two beams, so that multiple beams can be
obtained.
[0066] The device can also include a hydrodynamic flow injector for
introducing the sample medium into the flow cell. In one
embodiment, the device includes a hydrodynamic flow injector, but
only includes one source of laser light. In another embodiment, the
device includes two or more sources of laser light, but does not
include a hydrodynamic flow injector. In a third embodiment, the
device includes two or more sources of laser light and a
hydrodynamic flow injector.
[0067] In some embodiments, the device includes two or more lasers.
In these embodiments, the device further includes a beam splitter
that combines the light from the two lasers, and passes the
combined light beams through the first focusing lens. In these
embodiments, the device further includes a second beam splitter
positioned between the collimating lens and the second focusing
lens, splitting the beam into two beams, wherein one of the beams
is not diverted from its original path toward the second focusing
lens, and the other of the beams is diverted from its original path
toward the second focusing lens toward a second path.
[0068] In order to distinguish between light from the different
laser, so that light from only one laser passes through to a given
detector, chromatic filters are used to block out light from one or
the other of the lasers before the light impinges on a detector.
For example, a first chromatic filter can be positioned between the
second focusing lens and the scatter detector, which chromatic
filter permits light from one of the laser beams to pass through,
and which does not permit light from the other of the laser beams
to pass through, to the scatter detector. A second collimating lens
can be positioned along the second path, which lens collimates the
diverted light beam. A focusing lens can be positioned in the path
of light passing through the second collimating lens. A second
chromatic filter can be positioned in the path of light passing
through the focusing lens, which second chromatic filter permits
light from the laser beam that did not pass through the first
chromatic filter to pass through. A detector, for example, a
fluorescence detector, can be positioned in the path of the light
passing through the second chromatic filter.
[0069] The device also includes a dynamic monitoring system and
method employed to monitor the size and/or number of particles,
and, optionally, additional information on a subset of the
particles, such as their number and/or size, which fluoresce when
complexed to a particular fluorescent molecule, in a single
particle optical sizing device as described herein. In one
embodiment, the dynamic monitoring system is a computer either
physically connected to the device, or connected remotely, for
example, using Bluetooth, infrared, or other such technology, and
capable of receiving information from the various detectors, as
well as software which handles the digital interface with the
device, wherein the computer and software are capable of counting
particles, sizing particles, and storing information on the number
and size of the particles in a given sample based on the
information received from the detectors and algorithms that use
this information to determine particle size and/or number of
particles.
[0070] The monitoring system includes a data acquisition module
operatively coupled to the one or more detectors, and (iii) a
processing and display unit operatively coupled to the data
acquisition module for determining the size and/or number of
particles in a given sample and responsively outputting a graphical
representation of the size and/or number of the particles in the
sample. The data acquisition module uses the data obtained from the
detectors in the single particle optical sensing device described
herein, and an algorithm which correlates the data to the size
and/or number of particles in the sample medium.
[0071] The processing and display unit that is coupled to the data
acquisition module may utilize any suitable processing means, e.g.,
a general purpose programmable digital computer or central
processing unit (CPU) including memory and processor components.
The processor may be arranged to communicate with the memory by
means of an address/data bus, and can be constituted by a
commercially available or custom microprocessor. The memory can
include, without limitation, devices of varied type, such as cache,
ROM, PROM, EPROM, EEPROM, flash memory, SRAM, and DRAM.
[0072] The memory may include several categories of software and
data used in the data processing system: the operating system; the
application programs; the input/output (I/O) device drivers and the
data. The data may include a database of known profiles of particle
sizes, for example, a reference library of the size of platelets,
bacteria, viruses, fungi, cancer cells, stem cells, and complexes
of the cells with various molecules, including
fluorescently-labeled molecules, such as fluorescently-labeled
antibodies, and the like.
[0073] It will be appreciated that the operating system in the
processing and display unit can be of any suitable type for use
with a data processing system. Illustrative examples of operating
systems that can be usefully employed include, without limitation,
OS/2, AIX, OS/390 or System390 (International Business Machines
Corporation, Armonk, N.Y.), Windows CE, Windows NT, Windows95,
Windows98, Windows2000, or WindowsXP (Microsoft Corporation,
Redmond, Wash.), Unix or Linux or FreeBSD, Palm OS from Palm, Inc.,
Mac OS (Apple Computer, Inc.), Lab View or proprietary operating
systems.
[0074] The I/O device drivers typically include software routines
accessed through the operating system by the application programs
to communicate with devices such as I/O data port(s), data storage
and certain components of the memory.
[0075] The application programs are illustrative of the programs
that implement the various features of the system and can suitably
include one or more applications that support analysis of the data.
The data represent the static and dynamic data used by the
application programs, the operating system, the I/O device drivers,
and other software programs that may reside in the memory.
[0076] Any configuration of the processor capable of carrying out
the operations for the methodology of the invention can be
advantageously employed.
[0077] The I/O data port of the processing and display unit can be
used to transfer information between the processing and display
unit and another computer system or a network (e.g., the Internet)
or to other devices controllable by the processor.
[0078] The processing and display unit optionally, but ideally,
includes a display for graphically outputting information on the
size and/or number of particles in a sample, in the form of a
representation of the sample being assayed and the size and/or
number of particles in the sample. This representation may be a
graphic depiction, in which the size and/or number of particles are
schematically depicted in a graphical output, as a two-dimensional
column listing the size and/or number of particles, and the like.
Such type of depictions can provide an intuitive and readily
visually perceptible indication of the size and/or number of
particles in the sample.
[0079] Where the dynamic monitoring system is a computer, which is
either physically connected to the device, or capable of receiving
information remotely, for example, via Bluetooth or infrared, the
computer is capable of receiving information from the various
detectors. The computer also includes software which handles the
digital interface with the device. The computer and software are
capable of counting particles, sizing particles, and storing
information on the number and size of the particles in a given
sample based on the information received from the detectors. The
types of algorithms used to relate information from the various
detectors to particle size and/or particle number are described in
more detail below.
[0080] An exemplary apparatus useful for performing the methods
described herein is disclosed in U.S. Patent Application
Publication No. 20040011975, the contents of which are hereby
incorporated by reference in its entirety. The apparatus is
described therein is useful in performing particle analysis using
focused light scattering techniques. However, as described herein,
other similar apparatus can be employed, including detectors for
focused light scattering and/or light extinction.
[0081] The principal defining characteristic of the focused light
scattering method described in U.S. Patent Publication No.
20070010974, the contents of which are hereby incorporated by
reference, is not simply a significant reduction in the size of the
illuminated area, A.sub.O, resulting in a significant reduction in
V.sub.OSZ and improvement in sensitivity. Rather, it concerns the
nature of the illuminating beam and the resulting OSZ thereby
defined.
[0082] The term "focused light scattering" refers to a method for
sensing single particles, suspended in a solution, when the
solution is passed through a focused beam. When the beam passes
through the solution without being scattered by a particle, the
beam passes on to a photodetector and the intensity is measured.
When the beam is scattered, in whole or in part, by a particle, the
intensity of the beam hitting the photodetector is altered. The
particle size and concentration can be calculated, for example,
using light-extinction, light-scattering detection, or both.
[0083] In one embodiment, the beam is produced by a laser. The
laser beam interacts with the particles, and produces scattered
light when the laser beam interacts with a particle. In one aspect
of this embodiment, the apparatus includes two or more different
lasers, which can give off light at two or more different
wavelengths, and/or which can interact with the particles at
different angles. The use of light at different wavelengths can
enable one to identify specific epitopes. Particles can interact
with specific molecules, including fluorescently-labeled molecules,
and the fluorescence can be detected using a laser with light at a
predetermined wavelength that interacts with the fluorescent label.
The use of more than two lasers can enable the use of two or more
fluorescent labels, which labels fluoresce at different
wavelengths. This technique is described in more detail below.
[0084] A beam of light (usually laser light) of a single wavelength
is directed onto a hydrodynamically-focused stream of fluid that
includes the platelets. A number of detectors are aimed at the
point where the stream passes through the light beam. In one aspect
of this embodiment, one detector is in line with the light beam
(Forward Scatter or FSC) and one or more detectors are
perpendicular to it, including Side Scatter or SSC detectors and
one or more fluorescent detectors. Each suspended platelet passing
through the beam scatters the ray, and fluorescent chemicals either
present within the platelet or attached to the platelet are excited
into emitting light at a longer wavelength than the light source.
This combination of scattered and fluorescent light is picked up by
the detectors, and, by analyzing fluctuations in brightness at each
detector (one for each fluorescent emission peak), it is then
possible to derive various types of information about the physical
and chemical structure of each individual platelet.
[0085] Thus, in some embodiments of the apparatus described herein,
there are three or more detectors. For example, one can include one
detector for extinction [used to measure particles with a size
>0.7 microns], one for scattered light [used to measure
particles with a size in the range of 0.15 to 0.7 microns] and one
or more for fluorescence [used for phenotyping].
[0086] The sources of light can include lamps (i.e., mercury,
xenon); high-power water-cooled lasers (i.e., argon, krypton, dye
laser); low-power air-cooled lasers (i.e., argon (488 nm), red-HeNe
(633 nm), green-HeNe, HeCd (UV)); diode lasers (i.e., blue, green,
red, violet). The detectors can convert fluorescence signals from
light into electrical signals that can be processed by a
computer.
[0087] The process of collecting data from samples is termed
"acquisition." Acquisition is typically mediated by a computer
physically connected to the apparatus, and the software which
handles the digital interface with the apparatus. The software is
capable of adjusting parameters (i.e. voltage, compensation, etc.)
for the sample being tested, and also assists in displaying initial
sample information while acquiring sample data to insure that
parameters are set correctly. An interactive database can allow the
apparatus to be used in applications for both clinical and research
purposes. A wide variety of analysis software and
fluorescently-labeled antibodies has been developed, and are well
known to those of skill in the art.
[0088] The apparatus can include multiple lasers (between 2 and 5,
typically between two and four) and fluorescence detectors
(typically between 2 and 18, more typically between 2 and 10).
Increasing the number of lasers and detectors allows for multiple
antibody labeling, and can more precisely identify a target
population by their phenotypic markers.
[0089] Gating
[0090] The data generated by the apparatus can be plotted in a
single dimension, in two dimensions, or even in three dimensions.
The regions on these plots can be sequentially separated, based on
fluorescence intensity, by creating a series of subset extractions,
termed "gates." Specific gating protocols exist for diagnostic and
clinical purposes especially in relation to hematology.
[0091] The plots are often made on logarithmic scales. Because
different fluorescent dyes' emission spectra overlap, signals at
the detectors have to be compensated electronically as well as
computationally. Data accumulated using the apparatus can be
analyzed using software, e.g., Flowjo, FCS Express, VenturiOne or
CellQuest Pro. Data analysis can be performed on a separate
computer, if desired.
[0092] Computational Analysis
[0093] Automated population identification using computational
methods can be used as an alternative to traditional gating
strategies. Automated identification systems can potentially help
find rare and/or hidden populations. Representative automated
methods include FLOCK in Immunology Database and Analysis Portal
(ImmPort), FLAME in GenePattern and flowClust, in Bioconductor.
[0094] Fluorescent Labels
[0095] A wide range of fluorophores can be used as labels in flow
cytometry. Fluorophores, or simply "fluors", are typically attached
to an antibody that recognizes a target feature, epitope, on or in
the cell; they may also be attached to a chemical entity with
affinity for the cell membrane or another cellular structure. Each
fluorophore has a characteristic peak excitation and emission
wavelength, and the emission spectra of different labels often
overlap. Consequently, the combination of labels which can be used
depends on the wavelength of the lamp(s) or laser(s) used to excite
the fluorochromes and on the detectors available (Loken MR (1990).
Immunofluorescence Techniques in Flow Cytometry and Sorting (2nd
ed.). Wiley. pp. 341-53). The maximum number of distinguishable
fluorescent labels is thought to be 17 or 18, and this level of
plexy necessitates laborious optimization to limit artifacts, as
well as complex deconvolution algorithms to separate overlapping
spectra (Ornatsky, O.; Bandura, D.; Baranov, V.; Nitz, M.; Winnik,
M. A.; Tanner, S. (2010). "Highly multiparametric analysis by mass
cytometry". Journal of Immunological Methods 361 (1-2): 1-20)
[0096] Quantum dots are sometimes used in place of traditional
fluorophores because of their narrower emission peaks.
[0097] The fluorescent labels can be used, for example, to
determine the degree of protein expression and localization, the
existence of any protein modifications or intracellular antigens
(various cytokines, secondary mediators, etc.), membrane fluidity,
platelet viability, and platelet adherence.
[0098] Representative fluorescent labels are provided below:
TABLE-US-00001 Probe Ex (nm) Em (nm) Hydroxycoumarin 325 386
Aminocoumarin 350 445 Methoxycoumarin 360 410 Cascade Blue (375);
401 423 Pacific Blue 403 455 Pacific Orange 403 551 Lucifer yellow
425 528 NBD 466 539 R-Phycoerythrin (PE) 480; 565 578 PE-Cy5
conjugates 480; 565; 650 670 PE-Cy7 conjugates 480; 565; 743 767
Red 613 480; 565 613 PerCP 490 675 TruRed 490, 675 695 FluorX 494
520 Fluorescein 495 519 BODIPY-FL 503 512 TRITC 547 572 X-Rhodamine
570 576 Lissamine Rhodamine B 570 590 Texas Red 589 615
Allophycocyanin (APC) 650 660 APC-Cy7 conjugates 650; 755 767 Alexa
Fluor 350 343 442 Alexa Fluor 405 401 421 Alexa Fluor 430 434 540
Alexa Fluor 488 499 519 Alexa Fluor 500 503 525 Alexa Fluor 514 517
542 Alexa Fluor 532 530 555 Alexa Fluor 546 561 572 Alexa Fluor 555
553 568 Alexa Fluor 568 579 603 Alexa Fluor 594 591 618 Alexa Fluor
610 610 629 Alexa Fluor 633 632 648 Alexa Fluor 647 652 668 Alexa
Fluor 660 663 691 Alexa Fluor 680 680 702 Alexa Fluor 700 696 719
Alexa Fluor 750 752 776 Alexa Fluor 790 782 804 Cy2 489 506 Cy3
(512); 550 570; (615) Cy3B 558 572; (620) Cy3.5 581 594; (640) Cy5
(625); 650 670 Cy5.5 675 694 Cy7 743 767 DyLight 350 353 432
DyLight 405 400 420 DyLight 488 493 518 DyLight 549 562 576 DyLight
594 593 618 DyLight 633 638 658 DyLight 649 654 673 DyLight 680 692
712 DyLight 750 752 778 DyLight 800 777 794 Hoechst 33342 343 483
DAPI 345 455 Hoechst 33258 345 478 SYTOX Blue 431 480 Chromomycin
A3 445 575 Mithramycin 445 575 YOYO-1 491 509 Ethidium Bromide 493
620 Acridine Orange 503 530/640 SYTOX Green 504 523 TOTO-1,
TO-PRO-1 509 533 Thiazole Orange 510 530 Propidium Iodide (PI) 536
617 LDS751 543; 590 712; 607 7-AAD 546 647 SYTOX Orange 547 570
TOTO-3, TO-PRO-3 642 661 DRAQ5 647 681, 697 Indo-1 361/330 490/405
Fluo-3 506 526 DCFH 505 535 DHR 505 534 SNARF 548/579 587/635 Y66H
360 442 Y66F 360 508 EBFP 380 440 EBFP2 383 448 Azurite 383 447
GFPuv 385 508 T-Sapphire 399 511 TagBFP 402 457 Cerulean 433 475
mCFP 433 475 ECFP 434 477 CyPet 435 477 Y66W 436 485 dKeima-Red 440
616 mKeima-Red 440 620 TagCFP 458 480 AmCyan1 458 489 mTFP1 (Teal)
462 492 S65A 471 504 Midoriishi-Cyan 472 495 Wild Type GFP 396, 475
508 S65C 479 507 TurboGFP 482 502 TagGFP 482 505 TagGFP2 483 506
AcGFP1 484 510 S65L 484 510 Emerald 487 509 S65T 488 511 EGFP 488
507 Azami-Green 492 505 ZsGreen1 493 505 Dronpa-Green 503 518
TagYFP 508 524 EYFP 514 527 Topaz 514 527 Venus 515 528 mCitrine
516 529 YPet 517 530 TurboYFP 525 538 PhiYFP 525 537 PhiYFP-m 525
537 ZsYellow1 529 539 mBanana 540 553 Kusabira-Orange 548 559
mOrange 548 562 mOrange2 549 565 mKO 548 559 TurboRFP 553 574
tdTomato 554 581 DsRed-Express2 554 591 TagRFP 555 584 DsRed
monomer 557 592 DsRed2 ("RFP") 563 582 mStrawberry 574 596
TurboFP602 574 602 AsRed2 576 592 mRFP1 584 607 J-Red 584 610
mCherry 587 610 HcRed1 588 618 mKate2 588 633 Katushka (TurboFP635)
588 635 mKate (TagFP635) 588 635 TurboFP635 588 635 mPlum 590 649
mRaspberry 598 625 mNeptune 600 650 E2-Crimson 611 646
Monochlorobimane 380 461 Calcein 496 517
[0099] In some applications, particularly clinical applications, it
can be desirable to use microfluidics to introduce samples to the
apparatus. The microfluidic device can be disposable (i.e., used
once or perhaps a few times, followed by disposal and replacement)
and/or composed of a polymeric material. The microfluidic device
can be adapted to reduce the amount of sample used to determine
whether a patient can benefit from a particular anti-thrombotic
therapy. The microfluidic device preferably provides a tip adapted
for delivering the biological sample including the platelets into
the cell through which light passes, so that the platelets can then
travel through the light beam(s). In some embodiments, the tip is
adapted for sheath spraying. In other embodiments, the tip is
adapted for non-sheath spraying. In any of the embodiments herein
the apparatus may include a disposable inlet capillary.
[0100] The apparatus can also include an autodiluter, which can
start with the most dilute sample, rather than the most
concentrated sample, and can therefore use less sample.
Autodiluters are well known to those of skill in the art.
Representative autodiluters include the AutoDiluter-5.2, the CETAC
ADX-500 Autodiluter, the ProLiquid AutoDiluter, and the DYNATECH
Autodiluter III.
[0101] In high-throughput screening, it can be preferable to
include robotics, which can introduce the samples to the apparatus.
Ideally, the apparatus can then be cleaned in between samples, for
example, by flushing the various lines, and subsequent samples
introduced, enabling the screening to be automated. Information on
the screening results can be stored, for example, in a memory map,
and the information correlated with the patient's identity.
[0102] A "focused light scattering device" is a single-particle
optical sensor, which has high sensitivity and responds to
relatively concentrated suspensions, uses a relatively narrow light
beam to illuminate an optical sensing zone non-uniformly. It
differs from conventional SPOS devices in that it can handle more
concentrated solutions and smaller particle sizes.
[0103] In use, a solution including suspended platelets passes
through a zone. The zone is smaller than the flow channel, so that
the sensor responds to only a fraction of the total number of
platelets flowing through the channel, detecting a statistically
significant number of particles of any relevant diameter.
[0104] Because different particle trajectories flow through
different parts of the zone illuminated at different intensities,
it is necessary to deconvolute the result. Two methods of
deconvolution can be used: modified matrix inversion or successive
subtraction. Both methods use a few basis vectors measured
empirically or computed from a theoretical model, and the remaining
basis vectors are derived from these few. The sensor is compensated
for turbidity.
[0105] The sensor apparatus for single-particle optical sizing of
particles in a fluid suspension typically includes a means for
establishing flow of the suspension through a physically
well-defined measurement flow channel. There is also an
illumination means for effectively directing a relatively narrow
beam of light, having an axis, through the measurement flow channel
to form an optical sensing zone within the measurement flow
channel. The beam of light and the optical sensing zone are of such
size relative to the size of the measurement flow channel that the
sensor apparatus responds to only a fraction of the total number of
particles flowing through the measurement flow channel. In this
manner, the sensor apparatus responds effectively to a relatively
concentrated fluid suspension.
[0106] The beam has a maximum intensity portion and a continuum of
lesser intensities for positions spaced transverse to the axis from
the maximum intensity portion. In this manner, some of the
particles have trajectories through the maximum intensity portion,
others of the particles have trajectories through the lesser
intensity positions, and still others of the particles may have
trajectories outside the zone. Typically, the maximum intensity
portion of the beam is in a central portion of the beam.
[0107] The device also includes a detector means for
photo-detecting light from the zone to provide pulse height
signals. These signals each respond to a particle flowing through
the zone. The pulse height signals are functions of the sizes and
trajectories of detected particles. Particles of a given size
provide a maximum pulse height signal when flowing through the
maximum intensity portion, and lesser pulse height signals when
flowing through the lesser intensity positions of the zone. The
pulse height signals, collectively, form a pulse height
distribution PHD.
[0108] The device further includes a means for mathematically
deconvoluting the pulse height distribution to extract a particle
size distribution of the PSD particles in the fluid suspension. The
sensor apparatus can detect a statistically significant number of
particles of any given diameter or range of diameters that are
relevant to the fluid suspension.
[0109] In one embodiment, the measurement flow channel has a
thickness dimension axially of the beam of light, a width dimension
transverse to the beam, and a flow direction substantially
perpendicular to the thickness and width dimensions. The beam is
narrower than the measurement flow channel in the width direction.
The beam can be focused with a depth of field which is
substantially larger than the thickness dimension, and the beam
substantially has an effective width which does not vary
substantially over the thickness dimension.
[0110] In another embodiment, the beam has an effective width
between opposing positions transverse to the axis in the beam, at
which the lesser intensities have fallen to a given fraction of the
maximum intensity. The effective width is chosen so that the
largest particles of interest can be effectively sized. The given
fraction can be, for example, 1/e.sup.2 of the maximum intensity,
where e is the base of the natural system of logarithms, and the
effective width is substantially one half the size of the largest
particle to be sized.
[0111] In yet another embodiment, the apparatus uses hydrodynamic
sample injection, such as is described in Pelssers et al., Journal
of Colloid and Interface Science, Volume 137, Issue 2, July 1990,
Pages 350-361. Colloidal dispersions, such as platelets in serum or
other media, can be hydrodynamically focused into a narrow stream,
with widths ranging from about 3 to about 10, preferably about
.mu.m width. The use of a focused light scattering technique allows
one to measure relatively small particle sizes. However, where a
focused beam hits particles in a relatively wide sample stream (as
described above, where the beam is narrower than the measurement
flow channel in the width direction), the detection method relies
somewhat on statistics. That is, where the beam is substantially
narrower than the sample stream, an assumption is made that there
is an equal distribution of particles in the sample stream, so that
one can extrapolate the results of the interaction of the light in
the narrow beam with the particles in its path over the entire
width of the sample stream. By hydrodynamically focusing the sample
into a stream with a relatively narrow width, and using a focused
light source, it is possible to count all or most of the particles
in the sample stream, and rely to a lesser extent on
statistics.
[0112] The light beam can have, for example, a Gaussian intensity
profile, a circular cross-section, or an elliptical cross-section
being wider in a direction transverse to particle flow.
[0113] The detector means can be include a light extinction-type
detector, and can be a combination of detectors, for example, a
light-extinction detector type and a light-scattering type
detector. The light-scattering type detector means can include
means for passing a portion of scattered light from the zone
through a mask to select light scattered between a first and a
second angle to the beam and a means for directing a portion of the
light transmitted through the zone to a light-extinction type
detector.
[0114] The detector means can include a mirror for deflecting a
portion of the light from the optical-sensing zone to the
light-extinction detector. The illuminating means can include a
light source and optical fiber means for conveying light from the
light source to the optical sensing zone, and projecting the light
through the zone.
[0115] The detector means can also include an optical fiber means
for conveying the light passing through the optical sensing zone to
the light-extinction type detector. The detector means can also
include means for passing a portion of the light scattered from the
zone through a mask, to select light scattered between a first and
second angle to the beam, and an optical fiber means for conveying
the portion of the light to a light-scattering type detector. The
detector means can also include a light-scattering detector.
[0116] In one embodiment, the illumination means provides two light
beams directed through a pair of optical sensing zones positioned
within the measuring flow channel, and each beam has an effective
width determined by a desired maximum particle size.
[0117] The detector means can include a light-scattering detector
and a means for passing light scattered from the zone through a
mask means. The mask means can include a plurality of masks and
means for selecting one of the masks for passing the light
scattered from the zone, each mask defining different angles
between which the light is scattered. The masks can be located on a
rotatable wheel, and a mask can be selected by rotating the wheel
to a desired position.
[0118] The illuminating means can project a relatively wide
collimated beam through the optical sensing zone, and can include
an acceptance aperture to capture only those light rays that
closely surround the axis of the beam. This reduces the effective
width of the beam to a width in a direction transverse to the axis
of the light beam which is substantially one-half the size of the
largest particle to be sized. The illuminating means can also
include a means for coupling the light rays to the detector means.
This can be, for example, an optical fiber means.
[0119] In one aspect of the invention, a statistically significant
number of particles of each relevant size flow through all portions
and positions of the zone.
[0120] In another aspect of the invention, the fluid suspension is
relatively concentrated and the apparatus further comprises means
to compensate for turbidity of the suspension. In this aspect, the
detector means can operate on a light extinction principle, and
provide a signal having a baseline voltage level. The pulse height
signals appear as downwardly extending pulses from the baseline
voltage level, and the means for compensation for turbidity of the
suspension can include means to sense the baseline voltage level
and automatically increase the level to approximately the baseline
voltage level present in the absence of turbidity in the
suspension. The detector means can operate on a light extinction
principle, and provide a signal having a baseline voltage level,
wherein the means to compensate for turbidity can include a
computer means for correcting the pulse height signals in response
to the ratio of the baseline voltage level when the fluid
suspension is not turbid, to the baseline voltage level for the
turbid fluid suspension.
[0121] The detector means can also operate on a light extinction
principle and provide a signal having a baseline voltage level,
wherein the means to compensate for turbidity includes a means to
adjust the intensity of the beam of light by increasing the amount
of light produced by the illuminating means in response to the
ratio of the baseline voltage level when the fluid suspension is
not turbid, to the baseline voltage level for the turbid fluid
suspension.
[0122] The particle trajectories can be substantially uniformly
distributed across the width of the measurement flow channel.
[0123] The means for deconvoluting the pulse height distribution
can include basis vectors, each corresponding to a particular
particle size, and a source vector representing a measured pulse
height distribution for a fluid suspension as detected by the
detector means. There can also be a means using a deconvolution
algorithm to derive the particle size distribution from the pulse
height distribution. At least some of the basis vectors can have
values based upon measurements of particles of known size. Some of
the basis vectors can also have values based upon measurements of
particles of known size and others of the basis vectors can be
computed from the sum of the basis vectors by interpolation and/or
extrapolation.
[0124] The basis vectors can be computed, and the basis vectors can
be column basis vectors of a matrix, where the means using a
deconvolution algorithm performs matrix inversion and vector
multiplication, or the means using a deconvolution algorithm can
perform successive subtraction.
[0125] The means using a deconvolution algorithm can provide a
deconvoluted pulse height distribution dPHD, and the apparatus
further comprises means providing a calibration curve of the
relationship of pulse height and diameter, and means using the
calibration curve to transform each deconvoluted pulse height value
in the dPHD into a unique particle diameter associated with this
pulse height value. This can yield a "raw" particle size
distribution PSD. There can also be a means for converting the raw
PSD into a final PSD by renormalizing the raw PSD by multiplying by
the value 1/PHI.sub.d, where PHI.sub.d is the fraction of particles
actually detected by the device for particles of each size.
[0126] The particle trajectories can be distributed non-uniformly
across the width of the measurement flow channel, and the basis
vectors can be based upon the response of particles of known size
flowing through the measurement flow channel with the same
non-uniform distribution of particle trajectories as the fluid
suspension.
[0127] The sensor apparatus may respond only to a fraction of the
total number of particles flowing through the measurement flow
channel.
[0128] One can prepare a matrix for deconvoluting pulse height
distributions derived from particles of unknown size flowing along
different trajectories through a non-uniform light field of a
single-particle optical sizing device. This can enable one to size
particles in a fluid suspension. To do this, one can determine the
value of at least one empirical basis vector for the matrix by
measuring the response of particles of known size flowing through
the single-particle optical sizing device. Then, one can compute
other basis vectors for the matrix corresponding to particles of
other sizes, by interpolating and/or extrapolating the other basis
vectors from the empirical basis vector.
[0129] One can also determine the value of additional empirical
basis vectors for the matrix by measuring the response of particles
of known sizes flowing through the single-particle optical sizing
device, and computing the other basis vectors for the matrix
corresponding to particles of other sizes from the at least one
empirical basis vector and the additional empirical basis
vectors.
[0130] Another way to prepare a matrix for deconvoluting pulse
height distributions derived from particles of unknown size flowing
along different trajectories through a non-uniform light field of a
single-particle optical sizing device for sizing particles in a
fluid suspension involves determining the value of at least one
computed basis vector corresponding to particles of at least one
size for the matrix. One can compute other basis vectors for the
matrix corresponding to particles of other sizes from computed
basis vectors.
[0131] Also disclosed is a method of deconvoluting a pulse height
distribution derived from particles of unknown size flowing along
different trajectories through a non-uniform light field of a
single-particle optical sizing device for sizing particles in a
fluid suspension. The method involves setting up a matrix having a
plurality of columns, each containing a basis vector comprising a
pulse height distribution of particles of a known size
corresponding to the response of a photo-detector of the device to
the particles of known size. Each successive column contains a
basis vector for particles of successively larger sizes. The matrix
also has a like plurality of rows, each corresponding to a
successive pulse height channel, each channel including a range of
pulse heights, with successive rows corresponding to successively
larger pulse heights, and with each column having a maximum count
pulse height value at a location for a row which relates to a pulse
height corresponding to the particle of known size associated with
the column. The maximum count pulse height values for successive
columns are arranged on a diagonal of the matrix. The matrix is
modified by setting all terms corresponding to pulse height values
greater than the maximum count pulse height value in a column to
zero. A deconvolution algorithm is used to perform matrix inversion
and vector multiplication of the pulse height distribution and the
matrix as modified.
[0132] Before the modifying step, one can renormalize the values of
the basis vectors in the columns by setting the maximum count pulse
height value to equal 1.0 and all other count pulse height values
in the column to a value maintaining the same proportionate value
to 1.0 that the other count pulse height values had to the maximum
count pulse height value of the column.
[0133] The response of the photo-detection to the particles of
known size is developed empirically for some of the basis vectors
by sending particles of the substantially known size through the
device and providing a response by the device to the particles of
known size. The response to the photo-detector for the remaining
basis vectors can be computed by interpolating and/or extrapolating
the response for the remaining basis vectors from the some of basis
vectors.
[0134] The response of the photo-detector to the particles of known
size can be computed for some of the basis vectors and the response
to the photo-detector for the remaining basis vectors can be
computed by interpolating and/or extrapolating the response from
the some basis vectors.
[0135] A pulse height distribution ("PHD") can be derived from
particles of unknown size flowing along different trajectories
through a non-uniform light field of a single-particle optical
sizing device for sizing particles in a fluid suspension can be
deconvoluted by setting up a matrix having a plurality of columns.
Each column includes a basis vector comprising a pulse height
distribution of particles of a substantially known size
corresponding to the response of a photo-detector of the device to
the particles of known size, and each successive column contains a
basis vector for particles of successively larger sizes. The matrix
can also include a like plurality of rows, each corresponding to a
successive pulse height channel, each channel including a range of
pulse heights, successive rows corresponding to successively larger
pulse heights, each column having a maximum count pulse height
value at a location for a row which relates to a pulse height
corresponding to the particle of known size associates with the
column. The maximum count pulse height values for successive
columns can be arranged on a diagonal of the matrix. A successive
subtraction algorithm can be implemented, by starting with the
basis vector with its maximum count value in the largest row
number; scaling a column basis vector by a factor corresponding to
the value of the row in the PHD that matches the column number;
subtracting the scaled basis vector from the PHD to form an element
of a deconvoluted PHD (dPHD), leaving an intermediate PHD vector
with a smaller number of total particles; and repeating this
process using the remaining basis vectors until the entire PHD has
been consumed and all the elements of the deconvoluted dPHD have
been formed.
[0136] A single-particle optical sizing sensor for sizing particles
in a relatively concentrated fluid suspension sample for turbidity
of the suspension sample can be compensated using a sensor
operating on a light extinction principle whereby a photo-detector
produces signal V.sub.LE(t) having a baseline voltage level and a
response to blockage of light by a particle as a downwardly
extending pulse from the baseline voltage level. The compensation
method involves passing a non-turbid suspension through the sensor;
measuring a baseline voltage level V.sub.0 produced in response to
the non-turbid suspension; passing the relatively concentrated
suspension sample through the sensor; measuring a baseline voltage
V.sub.0.sup.T produced in response to the relatively concentrated
suspension sample, calculating the ratio V.sub.oV.sub.0.sup.T; and
adjusting the sensor in response to G to compensate for the
turbidity when the relatively concentrated suspension sample passes
through the sensor.
[0137] The baseline voltage V.sub.0.sup.T can effectively be
subtracted from the signal V.sub.LE(t), the remaining signal can be
inverted to produce a pulse height signal 2 V.sub.LE.sup.T(t), and
an adjustable gain amplifying means can be used to amplify the
pulse height signal 3 V.sub.LE.sup.T(t). The adjustable gain
amplifying means can be controlled by the ratio G to provide a
compensated pulse height signal .DELTA.V.sub.LE(t).
[0138] The signal V.sub.LE(t) produced by the sensor in response to
the relatively concentrated suspension sample can be amplified by
adjustable gain amplifier means, the gain of which is controlled by
the ratio G to provide a compensated signal V.sub.LE(t) having a
compensated baseline voltage V.sub.0, subtracting the baseline
voltage V.sub.0 from the compensated signal V.sub.LE(t), and
inverting the remaining signal to produce compensated pulse height
signal .DELTA.V.sub.LE(t).
[0139] In one embodiment, the single-particle optical sizing sensor
comprises a light source producing a light beam of adjustable
intensity, wherein the intensity is increased in response to the
ratio G to compensate for the turbidity.
[0140] Particles in a fluid suspension can also be optically sized
by establishing a flow of the suspension through a physically
well-defined measurement flow channel of a single-particle optical
sizing sensor apparatus wherein a beam of light, having an axis, is
directed through the measurement flow channel to form an optical
sensing zone within the measurement flow channel. The beam of light
and the optical sensing zone are ideally of such size relative to
the size of the measurement flow channel that the sensor apparatus
responds to only a fraction of the total number of particles
flowing through the measurement flow channel. The sensor apparatus
can respond effectively to a relatively concentrated fluid
suspension. The beam can have a maximum intensity portion in the
beam and a continuum of lesser intensities for positions in the
beam spaced transversely from the axis, whereby some of the
particles have a trajectory through the maximum intensity portion,
others of the particles have trajectories through the lesser
intensity positions, and still others of the particles may have
trajectories outside the zone. Light from the zone can be detected
to provide pulse height signals, each responsive to a particle
flowing through the zone. The pulse height signals are functions of
the sizes and trajectories of detected particles, and the pulse
height signals collectively form a pulse height distribution PHD.
The PDH can be mathematically deconvoluted and processed to extract
from the PHD a particle size distribution PSD of the particles in
the fluid suspension.
[0141] The step of mathematically deconvoluting the PHD can involve
determining the value of at least one empirical basis vector by
measuring the response to particles of known size flowing through
the single-particle optical sizing device. Other basis vectors
corresponding to particles of other sizes can be computed by
interpolating and/or extrapolating the other basis vectors from the
empirical basis vector.
[0142] The value of additional empirical basis vectors for
particles of known sizes flowing through the single-particle
optical sizing device can be determined; and the other basis
vectors for the matrix corresponding to particles of other sizes
can be calculated by interpolating and/or extrapolating the other
basis vectors from at least one empirical basis vector and the
additional empirical basis vectors. The method can further involve
determining the value of at least one computed basis vector
corresponding to particles of at least one size. Other basis
vectors corresponding to particles of other sizes can also be
computed by interpolating and/or extrapolating the other basis
vectors from computed basis vectors.
[0143] The step of deconvoluting and processing the PHD can involve
setting up a matrix having a plurality of columns, each containing
a basis vector comprising a pulse height distribution of particles
of a known size corresponding to the response of a photo-detector
of the device to the particles of known size, each successive
column containing a basis vector for particles of successively
larger sizes. The matrix can also have a like plurality of rows,
each corresponding to a successive pulse height channel, each
channel including a range of pulse heights, successive rows
corresponding to successively larger pulse heights, each column
having a maximum count pulse height value at a location for a row
which relates to pulse heights corresponding to the particle of
known size associated with the column. The maximum count pulse
height values for successive columns can be arranged on a diagonal
of the matrix. The matrix can be modified by setting all terms
corresponding to pulse height values greater than the maximum count
pulse height value in a column to zero. A deconvolution algorithm
can be used to perform matrix inversion and vector multiplication
of the pulse height distribution and the inverted matrix as
modified. The response of the photo-detector to the particles of
known size can be developed empirically for some of the basis
vectors by directing a flow of particles of the known size through
the device and providing a response by the device to the particles
of known size. The response to the photo-detector for the remaining
basis vectors can be calculated by interpolating and/or
extrapolating the response for the remaining basis vectors from the
some of basis vectors.
[0144] The step of mathematically deconvoluting the PHD can also
involve using a deconvolution algorithm to provide a deconvoluted
pulse height distribution dPHD. The method can further involve
providing a calibration curve of the relationship of pulse height
and diameter, and using the calibration curve to translate each
deconvoluted pulse height value in the dPHD into a unique particle
diameter associated with this pulse height value yielding a "raw"
particle size distribution in PSD. The raw PSD can be converted
into a final PSD by renormalizing the raw PSD by multiplying by the
value 1/PHI.sub.d, where PHI.sub.d is the fraction of particles
actually detected by the device for particles of each size.
[0145] In use, a focused laser light beam passes through a chamber
through which fluid flows, and the laser light scatters as the
particles pass through the focused laser beam. An extinction
detector determines when particles have passed through the beam. In
the absence of a particle interfering with the beam of light, the
light would pass, uninterrupted, to the extinction detector. When a
particle blocks the passage of light, the resulting loss of light
hitting the extinction detector signals that a particle has passed
through the beam. The light hitting the particles is reflected, and
passed through a scatter collimating lens, which re-focuses the
light, which then passes through a scatter focus lens, which sends
a single beam through to a scatter detector.
[0146] A representative focused light scattering device is shown in
FIG. 1. A first laser (1) emits light at a first wavelength, and a
second laser (2) emits light at a second wavelength. Both beams of
light pass through a first beam splitter (3) and through a first
focusing lens (4) before they enter into a flow cell (15). The flow
cell includes a site (5) for hydrodynamic injection of the sample.
As the platelets in the flow cell pass through the beams of light,
the light is scattered as it hits the platelets. The scattered
light passes through a circular spatial filter (6) and then through
a first collimating lens (7). The light beam passes through a
second beam splitter (16), which splits the light into two beams. A
first beam passes through a second focusing lens (8) and through a
first chromatic filter (9) that passes scattered light from the
first laser (1) through a first detector (10). The second beam
passes through a second collimating lens (11), a third focusing
lens (12) and a second chromatic filter (13) that passes scattered
light from the second laser (2) to a second detector (14).
[0147] The two photodetectors (10 and 14) each are able to detect
light at a certain frequency, so that light transmitted at
different frequencies (as a result of the two lasers hitting
particles, and which may interact with fluorescent tags on the
particles) can be separately determined.
[0148] A third detector (an extinction detector) (18) receives a
portion of the light passing through the flow cell. A portion of
the light passing through the flow cell is reflected off of a
movable mirror (17) and onto the third detector.
[0149] As is shown in FIG. 2, there are two important features
inherent in the optical design. First, the incident beam alone (in
conjunction with the front and back windows 36 and 37 of the
measurement flow channel 35) defines the OSZ. The side walls 38 and
39 that confine the fluid-particle suspension along the x-axis are
no longer of any consequence with respect to definition of the OSZ.
Second, the physical volume associated with the OSZ can no longer
be described by a single value; rather, it now depends on the size
of the particles being measured.
[0150] The approach shown schematically in FIG. 2 involves
illuminating measurement flow channel 35 with a light beam 41 from
a laser light source 40 which is focused by a lens 42 to form a
beam 44 of relatively narrow cross section--i.e., smaller than a
typical illuminated width, a, of the flow cell in a conventional
LE-type sensor. The resulting OSZ is therefore defined
approximately by a "pencil" beam of light 46, together with the
front and back windows of the flow cell, separated by dimension
"b." The schematic diagram in FIG. 2 provides a simplified picture
of the OSZ defined by focused light beam 46. First, the region of
illumination that comprises the OSZ is not sharply defined, as
implied by the approximately cylindrical zone indicated in FIG. 1.
Rather, the outer boundary of the OSZ is "fuzzy," extending well
beyond the zone indicated, as discussed below. Second, the beam
passing through the flow channel 10, assuming that it has been
focused, typically is not uniform in width. Rather, in the case of
a focused beam, its width varies over the depth of the measurement
flow cell 35. The extent to which the beam waist varies over the
depth of the channel depends on the depth of field of the focused
beam, defined as the distance (y-axis) between the points at which
the beam waist grows to 2 times its minimum value. Ideally, the
depth of field is significantly greater than the channel thickness,
b, resulting in a relatively uniform beam width throughout the flow
channel.
[0151] Consequently, focused light scattering devices may include a
fundamentally different sensor. In the conventional design, the
physical width of the flow channel 10 and the effective width
(x-axis) of the OSZ are one and the same, equal to dimension "a."
By contrast, the physical width of the flow channel in a sensor
used for focused light scattering devices (also defined as "a") is
typically much larger than the nominal width, 2w, of the incident
light beam and therefore has no significant influence on the OSZ.
Hence, the spacers (or shims) 38 and 39 that separate the front and
back windows 36 and 37, determining the depth, b, of the flow cell
(and OSZ), no longer need to be opaque or smooth on an optical
scale to avoid scattering by the edges. This is a significant
advantage, making fabrication of the flow cell easier and less
expensive.
[0152] It is usually convenient and effective to employ a
"circularized" light beam, in which the incident intensity ideally
depends only on the radial distance, r, from the beam axis
(coincident with the y-axis, with x=z=0, as seen in FIG. 1).
Typically, one employs a "Gaussian" light beam--i.e. one having a
Gaussian intensity profile, described in the focal plane (minimum
beam waist), at y=b/2, by I(r)=I.sub.0exp(-2r.sup.2/w.sup.2) (7)
where r.sup.2=X.sup.2+Z.sup.2 for the assumed circular beam.
[0153] Quantity 2w is the diameter of an imaginary cylinder
containing most of the incident light flux. The intensity on its
surface equals 1/e.sup.2, where e is the base for natural
logarithms, or 0.135 times its value, I.sup.0, at the center of the
beam (r=0). Essentially 100% (apart from losses due to reflections
at optical interfaces and extinction by particles in the beam) of
the light flux contained in the incident beam traverses the
fluid-particle mixture in the flow channel and impinges on the
distant detector D.sub.LE. This causes detector D.sub.LE to provide
a light extinction signal V.sub.LE in the form of a downwardly
extending pulse.
[0154] This behavior is in sharp contrast to the illumination
scheme employed in a conventional LE-type sensor. There, the
starting light beam is expanded greatly along the lateral (x) axis
of the flow cell, so that its width (1/e.sup.2 intensity) is much
larger than the width, a, of the front window (and OSZ). As a
result, there is relatively little variation in the incident
intensity along the x-axis (i.e. for y=z=0) where the beam enters
the flow cell, because the light is captured at the top of the
x-expanded Gaussian beam. Therefore, a particle passing through the
OSZ will experience substantially the same maximum beam intensity
(i.e. at z=0), regardless of its trajectory. The specific values of
x and y defining the trajectory ideally have no influence on the
resulting sensor response, i.e. the pulse height.
[0155] There is a sharp contrast between the conventional optical
design and the scheme employed in the sensor used for focused light
scattering devices. There is a large variation in the incident
intensity as a function of position (x-axis) across the width of
the flow channel. In the case in which the incident light beam has
a symmetric (circular) Gaussian profile, the intensity variation is
given by Equation 7, with r=x. The maximum intensity, I.sub.0, is
achieved at the center of the beam (x==0), where for simplicity x=0
represents the midpoint of the channel (with the side walls at
x=.+-.a/2). As noted, the intensity occurring at x=.+-.w, z=0 is
reduced substantially, to 0.135 I.sub.0. The intensity drops
steeply with increasing distance from the beam, falling, for
example, to 0.018 I.sub.0 at x=.+-.2w, z=0 and 0.00033 I.sub.0 at
x=.+-.4 w, z=0.
[0156] The consequences for the light-extinction signal thus
generated by the passage of particles through the new OSZ are
far-reaching. First, as for a conventional LE-type sensor, the
pulse height, .DELTA.V.sub.L E, generated by passage of a particle
through the OSZ in general increases with increasing particle size,
all other factors being equal. In general, the larger the particle,
the larger the fraction of light "removed" from the incident beam,
thus unable to reach the detector D.sub.LE. However, with the new
sensor the fraction of light removed from the beam now depends on
the precise trajectory of the particle--specifically, the minimum
distance, |x|, of the particle to the center of the beam, x=0. (To
first approximation, the response of the sensor will not vary
significantly with changes in the y-axis value of the trajectory,
assuming that the beam width is approximately constant over the
depth of the flow channel, given an appropriately large depth of
field, as discussed above.)
[0157] For a particle of given size and composition (hereinafter
assumed to be spherical and homogeneous, for simplicity), the
maximum "signal," or pulse height, is achieved when the particle
passes through the center of the beam, x=0. A particle of given
effective cross-sectional area, AA, blocks the largest amount of
incident light at the center of the beam, where the intensity is
greatest. Particles of identical size that pass through the flow
channel along different trajectories, with different minimum
distances, |x|, from the beam axis, are exposed to varying, but
smaller, maximum levels of illumination. The greater the distance
from the beam axis, the lower the integrated intensity incident on
a particle and, hence, the less light flux removed from the beam,
and the smaller the resulting pulse height. The response therefore
consists of a continuous "spectrum" of pulse heights, ranging from
a maximum value, for trajectories that pass through the center of
the beam, to essentially zero (i.e. indistinguishable from noise
fluctuations), for trajectories located very far from the incident
beam (|x|>>w). The maximum pulse height depends on the beam
waist, 2w, and the size of the particles, as well as in some cases
the refractive indices of the particles and surrounding fluid.
(This depends on the extent to which light scattering is
significant relative to refraction and reflection in contributing
to the overall light extinction signal.) A crucial assumption is
that the particle trajectories are distributed randomly (i.e. occur
with equal frequency) within the flow channel. This assumption is
usually valid, given the typical dimensions of the flow channel and
the relatively low flow rates utilized. It is also assumed that the
number of particles passing through the sensor is sufficiently
large that the statistical fluctuations in the number of particles
having trajectories with any given x-axis value (i.e. over any
(narrow) range of x values) can be ignored.
[0158] The relationship between particle size and pulse height for
the sensor in a focused light scattering device is therefore
radically different from that obtained for a sensor of conventional
design. In the latter case, irrespective of their trajectories,
particles of a given size (and composition) give rise to pulses of
nearly uniform height. This behavior is important for sensor design
for the conventional SPOS method. The typically small variations in
pulse height that occur, for example, when measuring polystyrene
latex "standard" particles of essentially uniform size are caused
by variations in the incident beam intensity within the OSZ along
the x- and y-axes, for a given z-axis value. These variations
ultimately determine the resolution of the sensor. The resulting
width of the PSD is therefore mostly a consequence of residual
non-uniformity of illumination across the OSZ, rather than an
actual range of particle diameters.
[0159] By contrast, there is an obvious deterioration in the
particle size resolution for sensor design for focused light
scattering devices. When a single particle passes through the
sensor, it gives rise to a light-extinction pulse with a height,
.DELTA.V.sub.LE that can vary between a given maximum value and
essentially zero. Conversely, given a single detected pulse, it is
impossible to determine the size of the particle that has produced
it, solely from knowledge of the pulse height. For example, a
particle that is relatively small, but which passes directly
through the beam axis, yields the maximum pulse height possible for
a particle of that size (and composition). Alternatively, a
particle that is much larger but which passes relatively far from
the beam axis yields a pulse height that could actually be the
same, depending on its size and trajectory. Even though the large
particle is able to intercept a much larger area of incident
illumination than the small one, the average intensity incident on
it is smaller than the intensity incident on the small particle.
Hence, the resulting pulse height could turn out to be the same as
that produced by the small particle. Obviously, there are an
infinite number of pairs, {d, |x|}, of particle diameters and
minimum beam-trajectory distances that can give rise to the same
pulse height. The particle diameter, d, and the resulting pulse
height, .DELTA.V.sub.LE, are effectively "decoupled" from each
other. This is the problem of "trajectory ambiguity", which for
more than twenty years has motivated the search for new
light-scattering based schemes for particle size determination
using Gaussian beams.
[0160] The effects of trajectory ambiguity described above might
present a difficulty in measuring the size of a single particle, or
a relatively small number of particles. However, the apparently
poor size resolution associated with the sensor used for focused
light scattering devices can be restored to a very acceptable level
by means of appropriate mathematical deconvolution of the
pulse-height data. The resulting dramatic improvement in the
effective sensor resolution is possible by virtue of the fact that
the sensor in a focused light scattering device is intended to be
exposed to a large, statistically significant number of particles
of every relevant diameter, or range of diameters, contained in the
sample of interest. This is the circumstance that renders the new
sensing method very useful for particle size analysis, in sharp
contrast to the situation that holds for "contamination"
applications. There, the sensor is exposed to relatively small
numbers of particles of any given size, for which statistical
significance is often not achieved.
[0161] The "raw" response of the sensor used in a focused-beam
device, like its conventional SPOS predecessor, consists of the
pulse height distribution (PHD)--a histogram of particle "counts"
vs pulse height, .DELTA.V.sub.LE. The pulse-height scale is
typically divided into a relatively large number (e.g. 32, 64 or
128) of "channels," or "bins," each of which encompasses an
appropriately narrow range of pulse height voltages, thus defining
the voltage resolution of the PH). It is usually convenient to
establish channels that are evenly spaced on a logarithmic voltage
scale. Measurement of a new pulse causes the number of particle
counts stored in the appropriate pulse height channel in the
histogram to be incremented by one. Data are ideally collected from
the particle suspension of interest for a sufficiently long time
that the resulting PHD becomes statistically reliable, and thus
smooth and reproducible. This means that the number, N.sub.I, of
particle counts collected in the I-th pulse-height channel is
statistically significant, dominating the fluctuations due to
statistical "noise," for all I, e.g. for 1.ltoreq.I.ltoreq.128, in
the case of 128 channels. Assuming Poisson statistics, this means
that N.sub.I>>N.sub.I, for all I.
[0162] Relatively high levels of particle concentration are
possible because the sensor responds to only a small fraction of
the total number of particles passing through it. For example,
concentrations in the range of hundreds of thousands of
particles/ml, in sample sizes of tens of mls, can be measured. That
is, millions of particles can be present, a portion of which is
passed through the beam of light and counted. The fraction of
particles that are actually counted, relative to the number of
particles present in the sample (Np), is known as phi.sub.d, or
"sensor efficiency," and is calculated by taking the ratio of the
particles actually detected over the number of particles in the
sample. The number of particles detected over the number of
particles in the sample typically ranges from about 0.5 to about
5%.
[0163] The fact that the sensor efficiency is so relatively small
is not surprising. In the case of a tightly focused beam, the
width, a, of the flow channel is invariably much larger than the
nominal width, 2w, of the focused beam. Therefore, most of the
particles passing through the sensor are exposed to negligible
levels of light intensity, because their trajectories are located
so relatively far from the beam axis--i.e. |x|>>w.
Consequently, only a small fraction of the total number of
particles is able to "block" enough light to give rise to
detectable pulses, relative to the prevailing noise level. The
great majority of particles pass undetected through the sensor.
[0164] While this limitation may appear to be serious, in practice
it is of little concern, for two reasons. First, the fraction,
phi.sub.d, of particles that produce detectable, measurable pulses
will be fixed for a given sensor width, a, even though the value
changes with particle diameter, d. Second, the new sensing method
is intended for use in determining the particle size distribution
(PSD) for samples that, by definition, are highly concentrated to
begin with. Even following dilution, if required, the concentration
of particles of any given size (i.e. within any (narrow) size
range) is, by definition, relatively high. Assuming a suitable flow
rate and data collection time, the resulting PHD will possess an
acceptable signal/noise ratio, with a low level of statistical
fluctuations. Hence, even though only a small fraction of the
available particles will contribute to the raw data, the resulting
PHD will be representative of the much larger number of particles
in the sample that are ignored. Therefore, a reliable and accurate
PSD, representative of the entire sample, can be obtained from the
"inefficient" sensor used in the focused light scattering devices
described herein.
[0165] Several additional features of the PHD that can be obtained
are noteworthy. First, as a consequence of the fact that the
particle trajectories span a large range of |x| values, passage of
uniform particles through the sensor indeed results in a PHD
containing a wide range of pulse heights. In this case, these range
from the threshold of individual pulse detection (dictated by the
prevailing r.m.s. noise level), roughly 5 millivolts (mV), to a
maximum of approximately 326 mV for the nominal "end" of the
distribution. (This excludes a small number of "outlier" pulses,
due to agglomerates and over-size primaries that extend to 500 mV).
Given the uniformity of the particles, this 65-fold range of pulse
heights can only be ascribed to differences in particle trajectory.
(To a first approximation, one can neglect variations in the beam
width over the depth of the flow channel, as discussed
earlier.)
[0166] Second, as expected, the PHD is highly asymmetric, skewed
greatly in the direction of smaller pulse heights. Clearly, there
are many particle trajectories that sample a large range of |x|
values (and, hence, beam intensities), but only relatively few that
probe the central portion of the Gaussian profile, where the
intensity is substantially uniform. The PHD exhibits a broad,
smooth upswing in the number of particles with increasing pulse
height, accelerating to a relatively sharp peak, followed by a
dramatic decline to the baseline, representing zero pulse events.
This sharp "cut-off" at the upper end of the distribution defines
the maximum pulse height, referred to hereafter as
.sup.M.DELTA.V.sub.LE. The counts collected at this maximum value
represent particles that passed through, or very close to, the
center of the beam--i.e. trajectories with x approximately equal to
0--where the fraction of total incident light flux "blocked" by the
particles is the largest value possible. The counts collected in
smaller pulse height channels represent particles that passed
further from the beam axis; the greater parameter |x|, the smaller
the resulting pulse heights.
[0167] There is a relationship between the particle trajectory and
the resulting pulse height. Trajectory "A" gives rise to extinction
pulses having the maximum pulse height, .sup.M.DELTA.V.sub.LE,
immediately preceding the upper cut-off of the PHD. Trajectories
"B," "C," "D" and "E" located progressively further from the beam
axis, give rise to pulses with correspondingly lower pulse heights
and progressively lower numbers of particle counts. Eventually, the
number of particle counts per channel approaches zero, as the pulse
height reaches the detection limit (approximately equal to 5
mV).
[0168] The reproducibility of the PHD depends only on the degree to
which the number of counts contained in the various channels is
large compared to statistical fluctuations. Therefore, the
"reliability" (i.e. the smoothness and reproducibility) of the PHD
should depend on the total number of particles counted during a
measurement. For a given particle size there will obviously exist a
minimum number of pulses that should be counted and analyzed, below
which the PHD should be expected to exhibit significant,
irreproducible "structure" from one measurement to the next, due to
statistical noise. Again, the PHDs produced by the new sensor have
meaning only to the extent that relatively large, statistically
meaningful numbers of particles of the same size are detected
during the data collection period. Only if this is true can one
expect to obtain optimal, reproducible PHD results, and
correspondingly accurate, representative particle size distribution
(PSD) results derived from the latter using the methods discussed
below.
[0169] To confirm that the data measured is significant, one can
overlay two or more PHDs taken from measuring the same sample in
multiple runs.
[0170] Exposing the sensor to larger particles will yield a PHD
that is shifted to larger pulse heights. Specifically, the maximum
pulse height, .sup.M.DELTA.V.sub.LE, corresponding to particle
trajectories passing through, or very close to, the beam axis,
increases.
[0171] An LS-type sensor can be used in place of, or in addition
to, an LE sensor. The LS-type sensor uses a light collection
means--typically one or more lenses--in order to gather scattered
light rays originating from individual particles passing through
the OSZ, created by the incident light beam.
[0172] The lens system is designed to collect scattered light over
a particular, optimal range of angles, typically encompassing
relatively small angles of scattering. In the scheme shown in FIG.
2, a mask 50 has been placed in front of the first collection lens.
Mask 50 comprises an outer opaque ring 52 and an inner opaque area
54, which form a transparent ring 56. Mask 50 allows only light
rays with scattering angles, theta, located within an imaginary
annular cone defined by angles theta.sub.1 and theta.sub.2 (i.e.
theta.sub.1.ltoreq.theta.sub.2) to impinge on the first collection
lens 62. Typically, this lens is centered on the axis of the
incident beam, at an appropriate distance (i.e. its focal length)
from the center of the flow channel, causing a portion of the
diverging scattered light rays from the OSZ to be captured by the
lens and become approximately collimated. A second lens 64 can then
be used to focus the resulting parallel scattered rays onto a
suitable (small-area) detector DLs. The resulting signal is
"conditioned" by one or more electronic circuits, typically
including the functions of current-to-voltage conversion and
amplification.
[0173] There is a crucial difference between the signal, V.sub.LS,
created by this optical scheme and the signal, V.sub.LE, produced
by the LE-type sensor. Unlike the latter, the LS-type sensor, by
design, prevents the incident light beam emerging from the back
window of the flow cell from reaching the detector, D.sub.LS.
Instead, the incident beam is either "trapped" by means of a
suitable small opaque beam "stop" (e.g., the inner opaque area 54)
or deflected by a small mirror away from the lens that is used to
collect the scattered light rays originating from the OSZ.
Consequently, the relatively large "baseline" level, V.sub.0,
necessarily present in the overall signal, V.sub.LE, produced by
the LE-type sensor is now absent from the LS signal, V.sub.LS.
Ideally, the new "baseline" signal level is zero--i.e. there should
be no scattered light generated from sources within the OSZ in the
absence of a particle. In practice, of course, there will be some
amount of background light caused by light scattered from the
surfaces of the front and/or back windows of the flow channel, due
to imperfections on, or contaminants attached to, the latter
surfaces. In addition, there may be fluctuating background light
due to scattering from small contaminant particles suspended in the
diluent fluid. Also, for some samples there may be fluctuations in
background light produced by a "sea" of ultra-fine particles which
comprise a major fraction of the overall particle population, but
which are too small to be detected individually.
[0174] When a particle of sufficient size passes through the OSZ,
defined by the incident Gaussian light beam and front and back
windows of flow channel 35, a momentary pulse occurs in the output
signal produced by the detector, D.sub.LS, and associated
signal-conditioning circuit. In general, one might naively expect
that the larger the particle, the greater the amount of light
scattered by it, assuming the same trajectory, and therefore the
greater the height of the signal pulse.
[0175] In practice, the actual pulse height depends not only on the
size of the particle, but also its composition--specifically, its
index of refraction (and that of the surrounding fluid) and
absorbance, if any, at the incident wavelength. The pulse height
also depends on the wavelength of the beam and the orientation of
the particle as it passes through the OSZ, if it is not spherical
and homogeneous. Finally, for particles comparable in size to, or
larger than, the wavelength, the scattering intensity varies
significantly with the scattering angle. Consequently, in this case
the pulse height depends on the range of angles over which the
scattered light is collected and measured.
[0176] The relationship between the scattered light "radiation
pattern" (i.e. intensity vs angle) and all of these variables is
described by classical Mie scattering theory, which takes into
account the mutual interference of the scattered light waves within
the particle. In general, the larger the particle, the more complex
(i.e. non-isotropic) the angular dependence of the scattered
intensity resulting from intra-particle interference. In order to
optimize the response and performance of the LS-type sensor, one
must confine the collection of scattered light to a range of
angles, theta, for which the net integrated response,
.DELTA.V.sub.LE, increases monotonically with the diameter, d, of
particles of a given composition (i.e. refractive index) over the
largest possible, or expected, size range. This requirement can
usually be satisfied by choosing a range of relatively small
angles, theta.sub.1<theta<theta.sub.2, close to the forward
direction. In this way, one avoids "reversals" in the integrated
scattering intensity with increasing particle size due to
variations of the intensity with changes in angle, especially
significant at larger angles as a consequence of Mie intra-particle
interference.
[0177] There are two properties of the signal, V.sub.LS, produced
by the new LS-type sensor that are qualitatively different from the
properties of the signal, V.sub.LE, produced by the corresponding
LE-type sensor. First, the signal pulse caused by passage of a
particle through the OSZ and the "overall" signal, V.sub.LS, are
essentially the same in the case of the LS-type sensor. The
relatively high background signal level that accompanies the pulse
of interest in the LE-type sensor is absent: (The same situation
clearly holds for a conventional LS-type sensor).
[0178] Therefore, in the case of relatively small particles that
give rise to pulses of low magnitude, the signal/noise ratio
achieved in practice using the LS method should be significantly
better than that realized using the LE method. This advantage
becomes more important the smaller the particle and the weaker the
resulting pulse, as the latter approaches the prevailing noise
fluctuations. Another way of appreciating the inherent advantage of
the LS method over its LE counterpart is to realize that the former
is based on "detection at null." That is, quantitative detection of
a pulse ideally is carried out in the presence of zero background
signal. From a signal/noise perspective, this is in sharp contrast
to the situation that obtains for the LE method, which requires
high "common-mode rejection." The "common-mode" signal, V.sub.0, is
always present in the raw signal, V.sub.LE, and must be subtracted,
or otherwise suppressed, in order to extract the (often small)
signal pulse of interest.
[0179] There is a second important and distinguishing property of
the LS signal, V.sub.LS. The signal/noise ratio associated with the
measurement of .DELTA.V.sub.LS can in principle be improved by
increasing the power of the incident light beam, so as to increase
the light intensity incident on a particle at all points within the
OSZ. Therefore, in principle one can reduce the lower size
detection limit for the new LS sensor by increasing the power of
the light source, as for a conventional LS sensor. Eventually, a
lowest size limit will be reached, based on noise fluctuations
associated with the suspending fluid and/or the light source and
detection system. Of course, as discussed above, the lower particle
size limit can also be improved for the new LS-type sensor by
reducing the width, 2w, of the incident beam, assuming no change in
the power of the latter. This action will obviously increase the
maximum intensity incident on the particles that pass through the
beam axis (x=0), and therefore the height of the largest resulting
pulse for a particle of given size, as well. However, this method
of improving the sensitivity eventually reaches a point of
diminishing return, due to limitations imposed by diffraction
theory (establishing a minimum beam width) and excessive variation
of the focused beam width over the depth, b, of the flow cell due
to excessively-long depth of field.
[0180] By contrast, an increase in the power of the light source
has relatively little effect on the lowest particle size that can
be measured using the LE method. For example, a doubling of the
power of the light source will result in a doubling of the baseline
signal level (FIG. 2), to 2V.sub.0. The height of the pulse,
.DELTA.V.sub.LE, produced by a particle of the same size and
trajectory will also be doubled, assuming no change in the beam
width. However, the root-mean-square magnitude of the noise
fluctuations associated with the relatively high baseline signal
level will typically also be approximately doubled, because these
fluctuations are usually associated with the light source and
therefore scale with the output power. Hence, one expects little or
no improvement in the signal/noise level for the LE-type sensor.
Consequently, there should be little or no reduction in the lower
size detection limit achievable by the LE method as a consequence
of increasing the power of the light source. An improvement will be
realized only if the signal/noise ratio associated with the light
source improves with increased power.
[0181] When uniform size particles flow through the new LS-type
sensor, depending on their trajectories they are individually
exposed to different values of maximum incident intensity, given by
Equation 7, with r=x, z=0. (For simplicity, it can be assumed that
the particles are much smaller than the beam width, so that every
point in a given particle is exposed to the same intensity at any
given time.) Therefore, as with the new LE-type sensor, the height,
.DELTA.V.sub.LS, of the resulting pulse generated by a particle of
given size depends on the distance, |x|, of closest approach (z=0)
to the axis of the incident beam. The smaller the distance |x|, the
larger the value of .DELTA.V.sub.LS. Hence, like its LE
counterpart, the LS-type sensor generates a distribution of widely
varying pulse heights, .DELTA.V.sub.LS, when a suspension of
uniform particles passes through it at an appropriate flow rate.
The shape of the resulting PHD bears a strong qualitative
resemblance to the highly asymmetric shape of the PHDs obtained
using the new LE method, exemplified in FIGS. 4, 6 and 7. That is,
the number of pulse counts (y-axis) is relatively small at the
smallest measurable pulse height just above the noise fluctuations)
and rises with increasing pulse height, .DELTA.V.sub.LS. The pulse
count value culminates in a peak value at a maximum pulse height,
referred to as .sup.M.DELTA.V.sub.LS, corresponding to particle
trajectories for which |x|.apprxeq.0. Above .DELTA.V.sub.LS the
number of pulse counts ideally falls to zero, assuming that the
particle concentration is below the coincidence concentration
(discussed earlier) for particles of that size, so that at most one
particle effectively occupies the OSZ at any given time. Of course,
a PHD obtained using the new LS method usually pertains to
particles that are smaller--often significantly so--than those used
to generate a typical PHD using the new LE method.
[0182] As noted above, the shape of the PHD--number of pulse counts
vs .DELTA.V.sub.LS--generated for uniform particles using the new
LS method is qualitatively similar to the shape of the PHD obtained
for uniform (typically larger) particles using the new LE method.
Both kinds of PHDs share the distinguishing characteristic of a
sharp "cut-off" following their respective peak number of pulse
counts, coinciding with their maximum pulse height values,
.sup.M.DELTA.V.sub.LS and .sup.M.DELTA.V.sub.LE. However, it should
be appreciated that there are quantitative differences in the
shapes of the two kinds of d=l, notwithstanding their qualitative
similarities, even for the same particle size--e.g. d=1 .mu.m. The
"front end" design of the new LS-type sensor--i.e. the focused
light beam and relatively thin flow cell--is essentially the same
as that utilized for the new LE-type sensor. Therefore, what
distinguishes one type of sensor from the other concerns the means
and manner of light detection and the type and magnitude of the
response pulses generated by each method, even in the case of
particles of the same size. For the new LS method, the response is
due only to light scattering, and its magnitude, .DELTA.V.sub.LS,
is proportional to the intensity of the light incident on the
particle, all other relevant variables being the same.
[0183] By contrast, for the new LE method the magnitude of the
response, .DELTA.V.sub.LE, is a more complex function of the
intensity incident on the particle. First, the response is due to a
combination of physical effects--refraction (and reflection) plus
light scattering. However, the scattering phenomenon asserts itself
in an "inverse" sense. That is, a small fraction of the incident
light flux is removed from the beam before it reaches the detector.
Second, over the typical size range for which the new LE method is
applicable, there is a substantial variation in the incident
intensity across the particle. Therefore, it should not be
surprising that the fractional change of pulse height due to a
given change in |x|, dependent on both particle size and
trajectory, is generally different for the two methods. Similarly,
the fractional change in pulse height with particle diameter,
dependent on both particle size and trajectory, is also generally
different for the two methods.
[0184] The task of converting the "raw" data--the PHD--obtained
from a sample of suspended particles into the object ultimately
desired--the particle size distribution, or PSD, is described in
detail below.
[0185] It is useful to compare this task conceptually with the
operation required in the case of a conventional LE- or LS-type
sensor. There, the height of the pulse due to passage of a particle
through the OSZ is nearly independent of its trajectory, because
the intensity of the incident beam is designed to be approximately
constant across the flow channel (i.e. along the x-axis) for a
given z-axis value (e.g. z=0). Consequently, particles of a given
size ideally give rise to pulses of substantially the same height,
and the resulting PHD is therefore, in effect, equivalent to the
final desired PSD. There is a one-to-one correspondence between a
given, measured pulse height, .DELTA.V.sub.LE (or .DELTA.V.sub.LS),
and the particle diameter, d. If particles of a larger or smaller
size pass through the sensor, the resulting pulse heights are
larger or smaller, respectively. A "calibration curve," consisting
of pulse height vs particle diameter, is all that is needed to
obtain, by simple interpolation, the PSD from the PHD. Obtaining
the raw PHD data using the conventional SPOS method is equivalent
to determining the final, desired PSD.
[0186] By contrast, as discussed earlier, the response of the LE-
(or LS-) type sensor is much more "convoluted." Even in the
simplest case of particles of a single size, the resulting PHD
consists of a broad spectrum of pulse heights, from the smallest
values just above the prevailing noise fluctuations, to the maximum
value, .sup.M.DELTA.V.sub.LE (or .sup.M.DELTA.V.sub.LS), associated
with that size. Therefore, in the typical case of particles of
widely varying size, the resulting PHD consists of an even wider
assortment of pulse heights. No longer is there a simple
correspondence between pulse height and particle size. It is
therefore no longer a simple, straightforward procedure to
transform the set of particle counts vs pulse-height values
contained in the PHD into the desired size distribution--particle
counts vs particle diameter.
[0187] It typically involves three procedures to convert the PHD to
the desired PSD. First, the raw PHD must be inverted, or
deconvoluted, using a specialized mathematical algorithm. Its
purpose is to convert the "wide-spectrum" PHD produced by the new
LE- (or LS-) type sensor into a "sharp", idealized PHD, equivalent,
in effect, to what would have been obtained using a conventional
LE- (or LS-) type sensor. Such an idealized, deconvoluted
PHD--hereinafter referred to as the dPHD--has the property that all
pulses of a given height, .DELTA.V.sub.LE (or .DELTA.V.sub.LS),
belong exclusively to particles of a given size (assuming, always,
particles of a given composition). The dPHD is equivalent to what
would have been obtained if all of the particles contributing to
the original PHD had passed through the center (axis) of the
incident beam.
[0188] A second straightforward procedure is then carried out. A
preliminary, or "raw", PSD is obtained from the dPHD by simple
interpolation of the calibration curve that applies to the specific
new LE- (or LS-) type sensor utilized--e.g. the curve shown in FIG.
8A. This procedure permits a one-to-one translation of each
deconvoluted pulse height value in the dPHD into a unique particle
diameter associated with this value, thus yielding the raw PSD. A
third procedure is then needed to convert the raw PSD thus obtained
into a final PSD that is quantitatively accurate. The number of
particle counts in each diameter channel of the raw PSD is the
number of this size that actually contributed to the measured PHD.
As discussed above, this is typically only a small fraction of the
total number of particles of the same size (i.e. within the size
range defined by the diameter channel) residing in the volume of
sample suspension that passed through the sensor during data
collection. This fraction, phi.sub.d, of particles actually
detected by the new LE- (or LS-) type sensor varies significantly
with the particle diameter, d.
[0189] The third procedure involves multiplying the number of
particles contained in each diameter channel of the raw PSD by the
value of 1/phi.sub.1 that applies for that channel. This operation
yields the final, desired PSD, describing the number of particles
of each size estimated to reside in the quantity of sample
suspension that passed through the sensor during data acquisition.
Values of l/phi.sub.d for each value of diameter, d, can be
obtained from the sensor efficiency curve, phi.sub.d vs d, by
interpolation.
[0190] There are two independent algorithms presented herein for
deconvoluting a measured PHD, to obtain the dPHD, hereinafter
referred to as "matrix inversion" and "successive subtraction."
Implementation of either procedure is based on the property that
the response of the new LE- (or LS-) type sensor--like its
conventional SPOS counterpart--is additive. Because the particles
passing through the sensor give rise to signal pulses one at a
time, the resulting PHD can be considered to be composed of a
linear combination, or weighted sum, of individual PHDs
corresponding to uniform particles of various sizes, referred to as
"basis vectors." (This term is well known in linear algebra.) Each
of these basis vectors represents the response of the system to a
statistically significant number of particles of a single, given
size.
[0191] In one embodiment, the focused light scattering device
described herein incorporates both the new LE- and LS-type SPOS
sensors in a single sensor, having two independent output signals,
V.sub.LE and V.sub.LS. The resulting dual "LE+LS" design offers
increased capability and flexibility, providing single-particle
counting and sizing over a relatively large range of particle
sizes. The LS-type sensor subsystem can be used to extend the size
range below the lower detection limit provided by the new LE-type
sensor subsystem. The extent to which the lower particle size limit
can be extended depends on a variety of parameters. These include:
the width, 2w, of the narrow (typically focused) beam within the
measurement flow cell; the power of the light source; the range of
angles over which scattered light is collected for implementation
of the new LS-type sensing function; and the physical properties,
including the refractive index, of both the particles and the
suspending fluid.
[0192] The dual LE+LS sensor includes a light source, preferably
consisting of a laser diode module, typically having an output
wavelength in the range of 600 to 1100 nanometers (nm). The beam
produced by the light source means preferably is collimated
(parallel) and "circularized"--i.e. the intensity is a function
only of the distance, r, from the central axis. Furthermore, the
beam preferably has a Gaussian intensity profile, along any axis
normal to the axis of propagation of the beam. The new LE+LS sensor
also includes a focusing means, typically a single- or
multi-element lens, capable of focusing the starting collimated
light beam to the desired beam width, 2w, at the center of the
measurement flow channel in the OSZ, consistent with the desired
particle size range. It is assumed that the focusing means has an
appropriate focal length, thus yielding acceptable values for both
the width and depth of field of the focused beam. The latter is
preferably significantly longer than the thickness, b, of the flow
channel, in order to optimize the resolution of the resulting
PSD.
[0193] A measurement flow cell is typically fabricated from a
suitable transparent material, such as glass, quartz or sapphire,
or alternative semi-transparent material, such as PTFE (e.g.
Teflon.TM., manufactured by DuPont) or other suitable plastic that
is sufficiently transparent at the operating wavelength and
compatible with the fluid-particle mixture. A suitable fluidics
system, including a flow pump means and optional means for
automatic dilution of the starting sample suspension (if needed),
are typically required to facilitate the steady flow of the
particle-fluid suspension through the flow cell. The flow rate, F,
is usually chosen to be the same as, or close to, the value used to
generate the calibration curve for the LE- or LS-type sensor.
[0194] The thickness, b, of the flow channel should be small enough
to achieve a high coincidence concentration limit and as uniform a
beam width as possible (ideally with b<<depth of field),
resulting in improved resolution for the final PSD. However, it
must be large enough to prevent frequent clogging by over-size
particles (e.g. agglomerated primaries and contaminants in the
fluid/diluent). The width, a, of the flow channel is also chosen to
strike a compromise between two competing effects. A relatively
large value reduces the impedance to the flowing fluid-particle
mixture and lowers the velocity (and increases the pulse width) for
a given flow rate, F. However, the larger parameter a, the smaller
the sensor efficiency, phi.sub.d, for any given particle diameter,
d. This results in a smaller fraction of particles in the sample
actually contributing to the measured PHD and final PSD, which, if
too small, may be undesirable.
[0195] The new LE+LS sensor contains two separate light collection
and detection subsystems, used independently to extract the desired
LE- and LS-type signals. The LE-type signal can be captured using a
small light reflecting means M (e.g. mirror), positioned so as to
intercept the narrow beam of incident light after it passes through
the flow cell and fluid-particle mixture. The resulting transmitted
beam, thus deflected away from the optical axis of the combined
sensor, is caused to impinge on a nearby light detection means
D.sub.LE. The latter typically consists of a small-area,
solid-state (silicon) detector, operating in a linear region and
having a spectral response that is matched to the wavelength of the
light source, thus providing an output signal with an acceptable
signal/noise (S/N) ratio. The output of the detector means is
typically a current (the "photocurrent"), which can be conditioned
by a current-to-voltage converter ("transimpedance" amplifier),
yielding an output signal in the generally desired form of a
time-varying voltage, V.sub.LE(t).
[0196] Alternatively, a small detector element can be placed
directly in the path of the light beam after it emerges from the
flow cell, thus eliminating the need for the intermediate light
reflecting means discussed above. Regardless of whether a mirror or
detector element is used to "capture" the transmitted light beam,
there are two requirements. First, the means used must function as
an effective beam "stop." That is, it must be able to prevent any
significant fraction of the arriving light flux from being
reflected back toward the flow cell, thus becoming a source of
"stray" light. Through unintended internal reflections from the
various optical surfaces, a portion of the stray light can find its
way to the scattering detection means D.sub.LS, thus corrupting the
resulting LS signal, by contributing a portion of the incident
intensity to the latter. Second, the means used to capture the LE
signal must be small enough not to intercept, and therefore block,
scattered light rays at any angles that are intended to be captured
and redirected to the light detection means D.sub.LS, as discussed
below.
[0197] Separately, scattered light originating from particles
passing through the OSZ is collected over a range of scattering
angles, theta, with theta.sub.1<theta<theta.sub.2, where
angles theta.sub.1 and theta.sub.2 are defined by a suitable
aperture means, such as an annular mask fabricated from a
photographic negative with an outer opaque portion, a transparent
intermediate portion, and an inner opaque portion. The scattered
rays selected by the mask are allowed to impinge on a collecting
lens of appropriate focal length and location, which converts the
diverging scattered rays into an approximately parallel beam. A
second lens is then typically used to refocus the rays onto a
relatively small light detection means D.sub.LS. As in the case of
the LE subsystem, the output signal of D.sub.LS is typically a
current, which can be optionally conditioned, typically by means of
a transimpedance amplifier, so that the final output is in the form
of a time-varying voltage, V.sub.LS(t).
[0198] The signals V.sub.LE(t) and V.sub.LS(t) can be organized
into respective pulse height distributions PHD by pulse height
analyzers. The PHDs are then respectively deconvoluted in computer
deconvolution means, which ultimately compute a pair of respective
particle size distributions ("PSD").
[0199] This embodiment can be implemented as an LE-type or LS-type
sensor only, simply by removing (or not installing in the first
place) the optical elements, detection means and signal
conditioning circuitry associated with the unwanted subsystem. In
this case, it may be useful to adjust the width, 2w, of the focused
beam within the measurement flow channel, in order to optimize the
resulting performance of the LE- or LS-type sensor. This parameter
will impact the usable particle size range, coincidence
concentration limit and minimum detectable particle size
differently for the two sensing modes, as discussed earlier.
[0200] Hydrodynamic Sample Injection
[0201] In one embodiment, the apparatus uses hydrodynamic sample
injection, such as is described in Pelssers et al., Journal of
Colloid and Interface Science, Volume 137, Issue 2, July 1990,
Pages 350-361. Colloidal dispersions, such as platelets in serum or
other media, can be hydrodynamically focused laser. into a narrow
stream, with widths ranging from about 3 to about 10, preferably
about .mu.m width. The use of a focused light scattering technique
allows one to measure relatively small particle sizes. However,
where a focused beam hits particles in a relatively wide sample
stream (as described above, where the beam is narrower than the
measurement flow channel in the width direction), the detection
method relies somewhat on statistics. That is, where the beam is
substantially narrower than the sample stream, an assumption is
made that there is an equal distribution of particles in the sample
stream, so that one can extrapolate the results of the interaction
of the light in the narrow beam with the particles in its path over
the entire width of the sample stream. By hydrodynamically focusing
the sample into a stream with a relatively narrow width, and using
a focused light source, it is possible to count all or most of the
particles in the sample stream, and rely to a lesser extent on
statistics.
[0202] A representative injector for carrying out hydrodynamic
injection is shown in more detail in FIG. 3. As shown in FIG. 3, As
shown in FIG. 3, a sample passes through an inlet port (100),
through a fluid sheath (110), and outward through the bottom of the
injector into a flow cell (120). The sample travels through the
sample injection tube, with fluid from a fluid sheath surrounding
the stream, and hydrodynamic focusing within the flow cell forcing
particles into a single-particle-file stream where laser light
intercepts the stream at a sample interrogation point. The design
of the flow cell, when a hydrodynamic injector is used, permits
particles to flow through the center of the flow cell. Increasing
the sample pressure increases the core diameter and the flow
rate.
[0203] The device described herein is similar to the device
disclosed in U.S. Patent Application Publication No. 20040011975,
the contents of which are hereby incorporated by reference in its
entirety. The main differences between the device disclosed in U.S.
Patent Application Publication No. 20040011975 and the instant
device are that the instant device further includes a hydrodynamic
injector, or additional lasers and detectors, such as detectors
that can detect fluorescent labels.
[0204] The apparatus is described therein is useful in performing
particle analysis using focused light scattering techniques.
However, as described herein, other similar apparatus can be
employed, including detectors for focused light scattering and/or
light extinction.
[0205] The principal defining characteristic of the focused light
scattering method described herein is not simply a significant
reduction in the size of the illuminated area, A.sub.O, resulting
in a significant reduction in V.sub.OSZ and improvement in
sensitivity. Rather, it concerns the nature of the illuminating
beam and the resulting OSZ thereby defined.
[0206] The term "focused light scattering" refers to a method for
sensing single particles, suspended in a solution, when the
solution is passed through a focused beam. When the beam passes
through the solution without being scattered by a particle, the
beam passes on to a photodetector and the intensity is measured.
When the beam is scattered, in whole or in part, by a particle, the
intensity of the beam hitting the photodetector is altered. The
particle size and concentration can be calculated, for example,
using light-extinction, light-scattering detection, or both.
[0207] In one embodiment, the beam is produced by a laser. The
laser beam interacts with the particles, and produces scattered
light when the laser beam interacts with a particle. In one aspect
of this embodiment, the apparatus includes two or more different
lasers, which can give off light at two or more different
wavelengths, and/or which can interact with the particles at
different angles. The use of light at different wavelengths can
enable one to identify specific epitopes. Particles can interact
with specific molecules, including fluorescently-labeled molecules,
and the fluorescence can be detected using a laser with light at a
predetermined wavelength that interacts with the fluorescent label.
The use of more than two lasers can enable the use of two or more
fluorescent labels, which labels fluoresce at different
wavelengths. This technique is described in more detail below.
[0208] A beam of light (usually laser light) of a single wavelength
is directed onto a stream of a fluid, in one embodiment, a
hydrodynamically-focused stream of a fluid, which includes
particles whose size and number is to be determined.
[0209] A number of detectors are aimed at the point where the
stream passes through the light beam. In one aspect of this
embodiment, one detector is in line with the light beam (Forward
Scatter or FSC) and one or more detectors are perpendicular to it,
including Side Scatter or SSC detectors and one or more fluorescent
detectors. Each suspended particle passing through the beam
scatters the ray, and fluorescent chemicals either present within
the particle or attached to the particle are excited into emitting
light at a longer wavelength than the light source. This
combination of scattered and fluorescent light is picked up by the
detectors, and, by analyzing fluctuations in brightness at each
detector (one for each fluorescent emission peak), it is then
possible to derive various types of information about the physical
and chemical structure of each individual particle.
[0210] Thus, in some embodiments of the apparatus described herein,
there are three or more detectors. For example, one can include one
detector for extinction [useful for measuring particles with a size
>0.7 microns], one for scattered light [useful for measuring
particles with a size between 0.15 and 0.7 microns] and one or more
for determining fluorescence [used for phenotyping].
xaxv
[0211] Light Sources
[0212] The sources of light can include lamps (i.e., mercury,
xenon); high-power water-cooled lasers (i.e., argon, krypton, dye
laser); low-power air-cooled lasers (i.e., argon (488 nm), red-HeNe
(633 nm), green-HeNe, HeCd (UV)); diode lasers (i.e., blue, green,
red, violet). The detectors can convert fluorescence signals from
light into electrical signals that can be processed by a
computer.
[0213] Data Acquisition
[0214] The process of collecting data from samples is termed
"acquisition." Acquisition is typically mediated by a computer
physically connected to the apparatus, and the software which
handles the digital interface with the apparatus. The software is
capable of adjusting parameters (i.e. voltage, compensation, etc.)
for the sample being tested, and also assists in displaying initial
sample information while acquiring sample data to insure that
parameters are set correctly. An interactive database can allow the
apparatus to be used in applications for both clinical and research
purposes. A wide variety of analysis software and
fluorescently-labeled antibodies has been developed, and are well
known to those of skill in the art.
[0215] The apparatus can include multiple lasers (between 2 and 5,
typically between two and four) and fluorescence detectors
(typically between 2 and 18, more typically between 2 and 10).
Increasing the number of lasers and detectors allows for multiple
antibody labeling, and can more precisely identify a target
population by their phenotypic markers.
[0216] Gating
[0217] The data generated by the apparatus can be plotted in a
single dimension, in two dimensions, or even in three dimensions.
The regions on these plots can be sequentially separated, based on
fluorescence intensity, by creating a series of subset extractions,
termed "gates." Specific gating protocols exist for diagnostic and
clinical purposes especially in relation to hematology.
[0218] The plots are often made on logarithmic scales. Because
different fluorescent dyes' emission spectra overlap, signals at
the detectors have to be compensated electronically as well as
computationally. Data accumulated using the apparatus can be
analyzed using software, e.g., Flowjo, FCS Express, VenturiOne or
CellQuest Pro. Data analysis can be performed on a separate
computer, if desired.
[0219] Computational Analysis
[0220] Automated population identification using computational
methods can be used as an alternative to traditional gating
strategies. Automated identification systems can potentially help
find rare and/or hidden populations. Representative automated
methods include FLOCK in Immunology Database and Analysis Portal
(ImmPort), FLAME in GenePattern and flowClust, in Bioconductor.
[0221] Microfluidics
[0222] In some applications, particularly clinical applications, it
can be desirable to use microfluidics to introduce samples to the
apparatus. The microfluidic device can be disposable (i.e., used
once or perhaps a few times, followed by disposal and replacement)
and/or composed of a polymeric material. The microfluidic device
can be adapted to reduce the amount of sample used to determine
whether a patient can benefit from a particular therapy. The
microfluidic device preferably provides a tip adapted for
delivering the biological sample including the platelets into the
cell through which light passes, so that the platelets can then
travel through the light beam(s). In some embodiments, the tip is
adapted for sheath spraying. In other embodiments, the tip is
adapted for non-sheath spraying. In any of the embodiments herein
the apparatus may include a disposable inlet capillary.
[0223] Auto-Diluters
[0224] The apparatus can also include an autodiluter, which can
start with the most dilute sample, rather than the most
concentrated sample, and can therefore use less sample.
Autodiluters are well known to those of skill in the art.
Representative autodiluters include the AutoDiluter-5.2, the CETAC
ADX-500 Autodiluter, the ProLiquid AutoDiluter, and the DYNATECH
Autodiluter III.
[0225] Mechanical Shearing Devices (for EQELS and/or ISADE
Devices)
[0226] Certain biological and non-biological species require
mechanical shear for activation. Species may be a cell, a protein,
ribo- or deoxyribonucleic acid, polysaccharides, aggregated cells
or molecules or the like. Representative examples of cells include,
but are not limited to, endothelial cells and platelets, and
representative examples of molecules include von Willebrand factor
and DNA. Both biological and non-biological species may aggregate
to exhibit an effect on the mechanical properties of a fluid.
Examples would be thixotropy (shear thinning) and rheopexy (shear
thickening). Biological cells, molecules like fibrin, vWF, tubulin,
myosin, and the like, and non-biological materials, like paints or
inks, are examples.
[0227] The provision of mechanical shear can be accomplished by
integrating a mechanical shearing device into the apparatus
described herein. Although there are many means for introducing
mechanical shear, any one of which can be used, in one embodiment,
a suitable device is shown in FIG. 4. In this device, a sample is
placed in a suitable container. The sample in the container may
then be extricated from the container manually, or by a robotic and
then introduced into a pump. In one aspect of this embodiment, the
pump is a precision pump that can produce a precise and continuous
flow rate of the sample within the shearing device (200). The
shearing device (200) is composed of a hollow fiber that may be
linear or coiled, though in the embodiment shown in FIG. 4, it is
coiled. In one aspect of this embodiment, the hollow fiber is
composed of materials that minimize interaction with the sample or
under certain other cases or circumstances may interact with the
sample in a specific manner. For example, the inner surface of the
hollow fiber may be coated with collagen that interacts with vWF or
with platelets. The length of the hollow fiber and the inter
diameter of the hollow fiber can be precisely known. Further, the
pressure drop across the fiber can be precisely known, from the
flow rates, volume flux, and/or from pressure sensors located at
each end of the hollow fiber. From these data, the mechanical shear
rate experienced by the sample can be calculated. The effluent
sample can then be passed on to the scattering chamber of EQELS or
the hydrodynamic injector of the ISADE device described herein by
means of a linking hollow fiber. In this manner, the entire system
is closed, and the sample can pass in a continuous manner from the
original sample container though the shearing device and into the
respective scattering chambers or hydrodynamic injector, then into
an effluent chamber via an exit port (210).
[0228] In use, the sample follows a path from a storage device, via
a pump (310) controlled by robotics (300) through a shearing device
(320), into a cell where scattered light can be detected (330), and
then to an effluent container (340). Although a scattering chamber
of an EQELS device is shown in FIG. 5, when the shearing device is
used in connection with the hydrodynamic injector of an ISADE
device as described herein, the hydrodynamic injector can
substitute for the scattering chamber shown in FIG. 5.
[0229] In an alternative embodiment, shear can be provided using
ultrasound; vibration; radiowaves; cone-plate, parallel plate or
coutte shearing surfaces; a coaxial plunger device and the
like).
[0230] Robotics
[0231] In high-throughput screening, it can be preferable to
include robotics, which can introduce the samples to the apparatus.
Ideally, the apparatus can then be cleaned in between samples, for
example, by flushing the various lines, and subsequent samples
introduced, enabling the screening to be automated. Information on
the screening results can be stored, for example, in a memory map,
and the information correlated with the patient's identity.
[0232] A "focused light scattering device" is a single-particle
optical sensor, which has high sensitivity and responds to
relatively concentrated suspensions, uses a relatively narrow light
beam to illuminate an optical sensing zone non-uniformly. It
differs from conventional single particle optical sizing (SPOS)
devices in that it can handle more concentrated solutions and
smaller particle sizes.
[0233] In use, a solution including suspended particles passes
through a zone. The zone is smaller than the flow channel, so that
the sensor responds to only a fraction of the total number of
particles flowing through the channel, detecting a statistically
significant number of particles of any relevant diameter.
[0234] Because different particle trajectories flow through
different parts of the zone illuminated at different intensities,
it is necessary to deconvolute the result. Two methods of
deconvolution can be used: modified matrix inversion or successive
subtraction. Both methods use a few basis vectors measured
empirically or computed from a theoretical model, and the remaining
basis vectors are derived from these few. The sensor is compensated
for turbidity.
[0235] The sensor apparatus for single-particle optical sizing of
particles in a fluid suspension typically includes a means for
establishing flow of the suspension through a physically
well-defined measurement flow channel. There is also an
illumination means for effectively directing a relatively narrow
beam of light, having an axis, through the measurement flow channel
to form an optical sensing zone within the measurement flow
channel. The beam of light and the optical sensing zone are of such
size relative to the size of the measurement flow channel that the
sensor apparatus responds to only a fraction of the total number of
particles flowing through the measurement flow channel. In this
manner, the sensor apparatus responds effectively to a relatively
concentrated fluid suspension.
[0236] The beam has a maximum intensity portion and a continuum of
lesser intensities for positions spaced transverse to the axis from
the maximum intensity portion. In this manner, some of the
particles have trajectories through the maximum intensity portion,
others of the particles have trajectories through the lesser
intensity positions, and still others of the particles may have
trajectories outside the zone. Typically, the maximum intensity
portion of the beam is in a central portion of the beam.
[0237] The device also includes a detector means for
photo-detecting light from the zone to provide pulse height
signals. These signals each respond to a particle flowing through
the zone. The pulse height signals are functions of the sizes and
trajectories of detected particles. Particles of a given size
provide a maximum pulse height signal when flowing through the
maximum intensity portion, and lesser pulse height signals when
flowing through the lesser intensity positions of the zone. The
pulse height signals, collectively, form a pulse height
distribution PHD.
[0238] The device further includes a means for mathematically
deconvoluting the pulse height distribution to extract a particle
size distribution of the PSD particles in the fluid suspension. The
sensor apparatus can detect a statistically significant number of
particles of any given diameter or range of diameters that are
relevant to the fluid suspension.
[0239] In one embodiment, the measurement flow channel has a
thickness dimension axially of the beam of light, a width dimension
transverse to the beam, and a flow direction substantially
perpendicular to the thickness and width dimensions. The beam is
narrower than the measurement flow channel in the width direction.
The beam can be focused with a depth of field which is
substantially larger than the thickness dimension, and the beam
substantially has an effective width which does not vary
substantially over the thickness dimension.
[0240] In another embodiment, the beam has an effective width
between opposing positions transverse to the axis in the beam, at
which the lesser intensities have fallen to a given fraction of the
maximum intensity. The effective width is chosen so that the
largest particles of interest can be effectively sized. The given
fraction can be, for example, 1/e.sup.2 of the maximum intensity,
where e is the base of the natural system of logarithms, and the
effective width is substantially one half the size of the largest
particle to be sized.
[0241] The light beam can have, for example, a Gaussian intensity
profile, a circular cross-section, or an elliptical cross-section
being wider in a direction transverse to particle flow.
[0242] The detector means can be include a light extinction-type
detector, and can be a combination of detectors, for example, a
light-extinction detector type and a light-scattering type
detector. The light-scattering type detector means can include
means for passing a portion of scattered light from the zone
through a mask to select light scattered between a first and a
second angle to the beam and a means for directing a portion of the
light transmitted through the zone to a light-extinction type
detector.
[0243] The detector means can include a mirror for deflecting a
portion of the light from the optical-sensing zone to the
light-extinction detector. The illuminating means can include a
light source and optical fiber means for conveying light from the
light source to the optical sensing zone, and projecting the light
through the zone.
[0244] The detector means can also include an optical fiber means
for conveying the light passing through the optical sensing zone to
the light-extinction type detector. The detector means can also
include means for passing a portion of the light scattered from the
zone through a mask, to select light scattered between a first and
second angle to the beam, and an optical fiber means for conveying
the portion of the light to a light-scattering type detector. The
detector means can also include a light-scattering detector.
[0245] In one embodiment, the illumination means provides two light
beams directed through a pair of optical sensing zones positioned
within the measuring flow channel, and each beam has an effective
width determined by a desired maximum particle size.
[0246] The detector means can include a light-scattering detector
and a means for passing light scattered from the zone through a
mask means. The mask means can include a plurality of masks and
means for selecting one of the masks for passing the light
scattered from the zone, each mask defining different angles
between which the light is scattered. The masks can be located on a
rotatable wheel, and a mask can be selected by rotating the wheel
to a desired position.
[0247] The illuminating means can project a relatively wide
collimated beam through the optical sensing zone, and can include
an acceptance aperture to capture only those light rays that
closely surround the axis of the beam. This reduces the effective
width of the beam to a width in a direction transverse to the axis
of the light beam which is substantially one-half the size of the
largest particle to be sized. The illuminating means can also
include a means for coupling the light rays to the detector means.
This can be, for example, an optical fiber means.
[0248] In one aspect of the invention, a statistically significant
number of particles of each relevant size flow through all portions
and positions of the zone.
[0249] In another aspect of the invention, the fluid suspension is
relatively concentrated and the apparatus further comprises means
to compensate for turbidity of the suspension. In this aspect, the
detector means can operate on a light extinction principle, and
provide a signal having a baseline voltage level. The pulse height
signals appear as downwardly extending pulses from the baseline
voltage level, and the means for compensation for turbidity of the
suspension can include means to sense the baseline voltage level
and automatically increase the level to approximately the baseline
voltage level present in the absence of turbidity in the
suspension. The detector means can operate on a light extinction
principle, and provide a signal having a baseline voltage level,
wherein the means to compensate for turbidity can include a
computer means for correcting the pulse height signals in response
to the ratio of the baseline voltage level when the fluid
suspension is not turbid, to the baseline voltage level for the
turbid fluid suspension.
[0250] The detector means can also operate on a light extinction
principle and provide a signal having a baseline voltage level,
wherein the means to compensate for turbidity includes a means to
adjust the intensity of the beam of light by increasing the amount
of light produced by the illuminating means in response to the
ratio of the baseline voltage level when the fluid suspension is
not turbid, to the baseline voltage level for the turbid fluid
suspension.
[0251] The particle trajectories can be substantially uniformly
distributed across the width of the measurement flow channel.
[0252] Deconvoluting the Pulse Height Distribution
[0253] The means for deconvoluting the pulse height distribution
can include basis vectors, each corresponding to a particular
particle size, and a source vector representing a measured pulse
height distribution for a fluid suspension as detected by the
detector means. There can also be a means using a deconvolution
algorithm to derive the particle size distribution from the pulse
height distribution. At least some of the basis vectors can have
values based upon measurements of particles of known size. Some of
the basis vectors can also have values based upon measurements of
particles of known size and others of the basis vectors can be
computed from the sum of the basis vectors by interpolation and/or
extrapolation.
[0254] The basis vectors can be computed, and the basis vectors can
be column basis vectors of a matrix, where the means using a
deconvolution algorithm performs matrix inversion and vector
multiplication, or the means using a deconvolution algorithm can
perform successive subtraction.
[0255] The means using a deconvolution algorithm can provide a
deconvoluted pulse height distribution dPHD, and the apparatus
further comprises means providing a calibration curve of the
relationship of pulse height and diameter, and means using the
calibration curve to transform each deconvoluted pulse height value
in the dPHD into a unique particle diameter associated with this
pulse height value. This can yield a "raw" particle size
distribution PSD. There can also be a means for converting the raw
PSD into a final PSD by renormalizing the raw PSD by multiplying by
the value 1/PHI.sub.d, where PHI.sub.d is the fraction of particles
actually detected by the device for particles of each size.
[0256] The particle trajectories can be distributed non-uniformly
across the width of the measurement flow channel, and the basis
vectors can be based upon the response of particles of known size
flowing through the measurement flow channel with the same
non-uniform distribution of particle trajectories as the fluid
suspension.
[0257] The sensor apparatus may respond only to a fraction of the
total number of particles flowing through the measurement flow
channel.
[0258] One can prepare a matrix for deconvoluting pulse height
distributions derived from particles of unknown size flowing along
different trajectories through a non-uniform light field of a
single-particle optical sizing device. This can enable one to size
particles in a fluid suspension. To do this, one can determine the
value of at least one empirical basis vector for the matrix by
measuring the response of particles of known size flowing through
the single-particle optical sizing device. Then, one can compute
other basis vectors for the matrix corresponding to particles of
other sizes, by interpolating and/or extrapolating the other basis
vectors from the empirical basis vector.
[0259] One can also determine the value of additional empirical
basis vectors for the matrix by measuring the response of particles
of known sizes flowing through the single-particle optical sizing
device, and computing the other basis vectors for the matrix
corresponding to particles of other sizes from the at least one
empirical basis vector and the additional empirical basis
vectors.
[0260] Another way to prepare a matrix for deconvoluting pulse
height distributions derived from particles of unknown size flowing
along different trajectories through a non-uniform light field of a
single-particle optical sizing device for sizing particles in a
fluid suspension involves determining the value of at least one
computed basis vector corresponding to particles of at least one
size for the matrix. One can compute other basis vectors for the
matrix corresponding to particles of other sizes from computed
basis vectors.
[0261] Also disclosed is a method of deconvoluting a pulse height
distribution derived from particles of unknown size flowing along
different trajectories through a non-uniform light field of a
single-particle optical sizing device for sizing particles in a
fluid suspension. The method involves setting up a matrix having a
plurality of columns, each containing a basis vector comprising a
pulse height distribution of particles of a known size
corresponding to the response of a photo-detector of the device to
the particles of known size. Each successive column contains a
basis vector for particles of successively larger sizes. The matrix
also has a like plurality of rows, each corresponding to a
successive pulse height channel, each channel including a range of
pulse heights, with successive rows corresponding to successively
larger pulse heights, and with each column having a maximum count
pulse height value at a location for a row which relates to a pulse
height corresponding to the particle of known size associated with
the column. The maximum count pulse height values for successive
columns are arranged on a diagonal of the matrix. The matrix is
modified by setting all terms corresponding to pulse height values
greater than the maximum count pulse height value in a column to
zero. A deconvolution algorithm is used to perform matrix inversion
and vector multiplication of the pulse height distribution and the
matrix as modified.
[0262] Before the modifying step, one can renormalize the values of
the basis vectors in the columns by setting the maximum count pulse
height value to equal 1.0 and all other count pulse height values
in the column to a value maintaining the same proportionate value
to 1.0 that the other count pulse height values had to the maximum
count pulse height value of the column.
[0263] The response of the photo-detection to the particles of
known size is developed empirically for some of the basis vectors
by sending particles of the substantially known size through the
device and providing a response by the device to the particles of
known size. The response to the photo-detector for the remaining
basis vectors can be computed by interpolating and/or extrapolating
the response for the remaining basis vectors from the some of basis
vectors.
[0264] The response of the photo-detector to the particles of known
size can be computed for some of the basis vectors and the response
to the photo-detector for the remaining basis vectors can be
computed by interpolating and/or extrapolating the response from
the some basis vectors.
[0265] A pulse height distribution ("PHD") can be derived from
particles of unknown size flowing along different trajectories
through a non-uniform light field of a single-particle optical
sizing device for sizing particles in a fluid suspension can be
deconvoluted by setting up a matrix having a plurality of columns.
Each column includes a basis vector comprising a pulse height
distribution of particles of a substantially known size
corresponding to the response of a photo-detector of the device to
the particles of known size, and each successive column contains a
basis vector for particles of successively larger sizes. The matrix
can also include a like plurality of rows, each corresponding to a
successive pulse height channel, each channel including a range of
pulse heights, successive rows corresponding to successively larger
pulse heights, each column having a maximum count pulse height
value at a location for a row which relates to a pulse height
corresponding to the particle of known size associates with the
column. The maximum count pulse height values for successive
columns can be arranged on a diagonal of the matrix. A successive
subtraction algorithm can be implemented, by starting with the
basis vector with its maximum count value in the largest row
number; scaling a column basis vector by a factor corresponding to
the value of the row in the PHD that matches the column number;
subtracting the scaled basis vector from the PHD to form an element
of a deconvoluted PHD (dPHD), leaving an intermediate PHD vector
with a smaller number of total particles; and repeating this
process using the remaining basis vectors until the entire PHD has
been consumed and all the elements of the deconvoluted dPHD have
been formed.
[0266] A single-particle optical sizing sensor for sizing particles
in a relatively concentrated fluid suspension sample for turbidity
of the suspension sample can be compensated using a sensor
operating on a light extinction principle whereby a photo-detector
produces signal V.sub.LE(t) having a baseline voltage level and a
response to blockage of light by a particle as a downwardly
extending pulse from the baseline voltage level. The compensation
method involves passing a non-turbid suspension through the sensor;
measuring a baseline voltage level V.sub.0 produced in response to
the non-turbid suspension; passing the relatively concentrated
suspension sample through the sensor; measuring a baseline voltage
V.sub.0.sup.T produced in response to the relatively concentrated
suspension sample, calculating the ratio V.sub.oV.sub.0.sup.T; and
adjusting the sensor in response to G to compensate for the
turbidity when the relatively concentrated suspension sample passes
through the sensor.
[0267] The baseline voltage V.sub.0.sup.T can effectively be
subtracted from the signal V.sub.LE(t), the remaining signal can be
inverted to produce a pulse height signal 2 V.sub.LE.sup.T(t), and
an adjustable gain amplifying means can be used to amplify the
pulse height signal 3 V.sub.LE.sup.T(t). The adjustable gain
amplifying means can be controlled by the ratio G to provide a
compensated pulse height signal .DELTA.V.sub.LE (t).
[0268] The signal V.sub.LE(t) produced by the sensor in response to
the relatively concentrated suspension sample can be amplified by
adjustable gain amplifier means, the gain of which is controlled by
the ratio G to provide a compensated signal V.sub.LE(t) having a
compensated baseline voltage V.sub.0, subtracting the baseline
voltage V.sub.0 from the compensated signal V.sub.LE(t), and
inverting the remaining signal to produce compensated pulse height
signal .DELTA.V.sub.LE(t).
[0269] In one embodiment, the single-particle optical sizing sensor
comprises a light source producing a light beam of adjustable
intensity, wherein the intensity is increased in response to the
ratio G to compensate for the turbidity.
[0270] Particles in a fluid suspension can also be optically sized
by establishing a flow of the suspension through a physically
well-defined measurement flow channel of a single-particle optical
sizing sensor apparatus wherein a beam of light, having an axis, is
directed through the measurement flow channel to form an optical
sensing zone within the measurement flow channel. The beam of light
and the optical sensing zone are ideally of such size relative to
the size of the measurement flow channel that the sensor apparatus
responds to only a fraction of the total number of particles
flowing through the measurement flow channel. The sensor apparatus
can respond effectively to a relatively concentrated fluid
suspension. The beam can have a maximum intensity portion in the
beam and a continuum of lesser intensities for positions in the
beam spaced transversely from the axis, whereby some of the
particles have a trajectory through the maximum intensity portion,
others of the particles have trajectories through the lesser
intensity positions, and still others of the particles may have
trajectories outside the zone. Light from the zone can be detected
to provide pulse height signals, each responsive to a particle
flowing through the zone. The pulse height signals are functions of
the sizes and trajectories of detected particles, and the pulse
height signals collectively form a pulse height distribution PHD.
The PDH can be mathematically deconvoluted and processed to extract
from the PHD a particle size distribution PSD of the particles in
the fluid suspension.
[0271] The step of mathematically deconvoluting the PHD can involve
determining the value of at least one empirical basis vector by
measuring the response to particles of known size flowing through
the single-particle optical sizing device. Other basis vectors
corresponding to particles of other sizes can be computed by
interpolating and/or extrapolating the other basis vectors from the
empirical basis vector.
[0272] The value of additional empirical basis vectors for
particles of known sizes flowing through the single-particle
optical sizing device can be determined; and the other basis
vectors for the matrix corresponding to particles of other sizes
can be calculated by interpolating and/or extrapolating the other
basis vectors from at least one empirical basis vector and the
additional empirical basis vectors. The method can further involve
determining the value of at least one computed basis vector
corresponding to particles of at least one size. Other basis
vectors corresponding to particles of other sizes can also be
computed by interpolating and/or extrapolating the other basis
vectors from computed basis vectors.
[0273] The step of deconvoluting and processing the PHD can involve
setting up a matrix having a plurality of columns, each containing
a basis vector comprising a pulse height distribution of particles
of a known size corresponding to the response of a photo-detector
of the device to the particles of known size, each successive
column containing a basis vector for particles of successively
larger sizes. The matrix can also have a like plurality of rows,
each corresponding to a successive pulse height channel, each
channel including a range of pulse heights, successive rows
corresponding to successively larger pulse heights, each column
having a maximum count pulse height value at a location for a row
which relates to pulse heights corresponding to the particle of
known size associated with the column. The maximum count pulse
height values for successive columns can be arranged on a diagonal
of the matrix. The matrix can be modified by setting all terms
corresponding to pulse height values greater than the maximum count
pulse height value in a column to zero. A deconvolution algorithm
can be used to perform matrix inversion and vector multiplication
of the pulse height distribution and the inverted matrix as
modified. The response of the photo-detector to the particles of
known size can be developed empirically for some of the basis
vectors by directing a flow of particles of the known size through
the device and providing a response by the device to the particles
of known size. The response to the photo-detector for the remaining
basis vectors can be calculated by interpolating and/or
extrapolating the response for the remaining basis vectors from the
some of basis vectors.
[0274] The step of mathematically deconvoluting the PHD can also
involve using a deconvolution algorithm to provide a deconvoluted
pulse height distribution dPHD. The method can further involve
providing a calibration curve of the relationship of pulse height
and diameter, and using the calibration curve to translate each
deconvoluted pulse height value in the dPHD into a unique particle
diameter associated with this pulse height value yielding a "raw"
particle size distribution in PSD. The raw PSD can be converted
into a final PSD by renormalizing the raw PSD by multiplying by the
value 1/PHI.sub.d, where PHI.sub.d is the fraction of particles
actually detected by the device for particles of each size.
II. Particles that can be Detected
[0275] Using the techniques described herein, various biological
particles can be detected. Cells are one type of biological
particle that can be detected. The method can be used to determine
the presence or absence of a specific type of cell in a given
solution. For example, a sample of blood, urine, spinal fluid, and
the like can be evaluated for the presence or absence of bacteria,
fungi, viruses, and the like. The particle size, and, optionally,
particle shape, can also provide information about the specific
type of bacteria, fungi or virus.
III. Microparticles and Nanoparticles Suitable for Use in Focused
Light Scattering
[0276] In some embodiments, where the complex between an active
agent and a biological particle does not result in a change in
particle size (i.e., no particle agglomeration or cell rupture), it
may be necessary to conjugate the active agent with a microparticle
(such as a nanoparticle, polystyrene bead, gold particle, and the
like). Thus, when the agent forms a complex with the biological
particle, the complex increases the size of the biological particle
by the size of the microparticle, and this increased particle size
is measurable using the techniques described herein.
[0277] In one embodiment, the particles have a particle size in the
range of between about 0.1 and 10 .mu.m, and ideally have a
relatively consistent amount of active agent bound to them. That
is, if all that is important is to determine that the biological
particles bound to an active agent of interest, then one can simply
incubate the biological particle with the conjugate, and look for
the decrease in peak corresponding to the biological particle. This
will confirm that a complex of the biological particle and the
active agent was formed.
[0278] If there is an interest in quantifying how much of the
biological particle was present, then it may be important to use
particles with a nearly uniform particle size, defined as having
90% or more of the particles within 5% of the mean particle size,
more preferably around 99% uniformity or better. In addition to a
uniform particle size, in some embodiments, it may be desirable to
have uniform substitution on the particles themselves. That is,
rather than having particles of a relatively uniform particle size
form complexes of different particle sizes with the biological
particle of interest, it may be desirable to form complexes with a
relatively uniform particle size, to ease their quantification.
[0279] One way to produce particles with a relatively constant
particle size, and with a relatively consistent amount of active
agent conjugated to the particles, is to use dendrimers. The
dendrimers can include a known quantity of the active agent, by
virtue of the active functional groups at the terminus on the
dendrimers.
[0280] Another way is to produce polymer particles with: a) a
relatively narrow size distribution, and b) a relatively consistent
amount of protected functional groups, so that after the polymers
are produced, the protecting groups can be removed, and the
functional groups used to conjugate the polymer particles to an
active agent.
[0281] The active agent can be conjugated with the particle in such
a way that the portion of the active agent that is known to be
active (i.e., binds a receptor) is not significantly sterically
hindered by its conjugation with the particle. In some embodiments,
this will involve preparing an analogue of the active agent which
includes a further functional group which can be attached to the
particle.
[0282] In one embodiment, metallic particles, such as gold
particles, are used. Because these particles scatter a significant
amount of light, they can be conjugated with a specific active
agent, and used to identify even small molecules that bind to the
agent. That is, the amount of light that the particle scatters is
sufficiently large that the binding of the agent to the molecule of
interest can be measured, even though the molecule is not within
the size range of biological particles that can be measured. Means
for conjugating active agents to metallic particles are known to
those of skill in the art.
[0283] Metallic particles, such as gold particles, can also be
used. These can be conjugated with active agents using known
methodology to form particles capable of forming a complex with a
biological particle, including particles too small to detect using
focused light scattering techniques. Because the particles are
highly dense, they produce enough light scattering to be detected,
despite their small size. Binding of even a small molecule to the
particles can be detected because of the intense scattering from
the metallic bead so that enough light is scattered for the complex
formation to be measured.
IV. Methods for Detecting the Presence or Absence of Specific
Particles in a Solution
[0284] A sample medium can be evaluated for the presence or absence
of specific particles. For example, a sample of blood, urine,
spinal fluid, amniotic fluid, pleural fluid, peritoneal fluid and
the like, can be evaluated for the presence or absence of specific
microbes (cells (lymphocytes, B-cells, T-cells, neutrophils,
monocytes, and the like), bacteria, fungi, viruses, and the like),
and/or for the presence of relatively high concentrations of white
blood cells or other biological particles indicative of a disease
state. The methods also permit one to determine presence or absence
of shed particles.
[0285] The particle (intact cell, microparticle, bacteria, virus,
fungus, and the like) can be further identified and characterized
by addition of probe particles that are coated with a specific
agent that recognizes and binds to the specific surface epitope on
the unknown particle surface. If the recognition and binding of the
probe particle occurs, a new particle size will be created and
appear, thus identifying the unknown particle. For example if the
unknown particle was a stem cell and the probe particle was labled
with an anti-CD34 antibody, binding of the probe particle with the
stem cell would occur and a new sized particle would appear that
would identify the presence of CD34 positive stem cells.
[0286] Once the particles are identified by size, one can confirm
the identity of the particles, for example, by using a reference
library correlating the particle size to a given biological
species. Thus, in one embodiment, the method involves comparing
information obtained on a biological particle using focused light
scattering techniques, with a library of data on biological
particles obtained using similar focused light scattering
techniques. The library can include information on two or more
biological particles, preferably, ten or more particles, more
preferably, one hundred or more particles, and, most preferably,
more than a thousand particles.
[0287] After a preliminary identification has been made on the type
of biological particle, other biological techniques can optionally
be used to confirm the identity of the particle. For example, an
EQELS spectra of the particle can be taken, and compared to a
library of EQELS spectra of known particles, to confirm the
identity of the particle.
[0288] Antibodies or other molecules specific for the specific
biological particle can be added to the solution, and if the
particles bind to the antibodies, the method will detect the
absence of the particles. Ideally, the molecules will be conjugated
with a microparticle or nanoparticle, such as a latex particle. As
the conjugate interacts with a biological particle, peaks
representing the particle and the conjugate will decrease, and
peaks corresponding to the complex of the particle and the
conjugate will increase.
[0289] In one embodiment, the biological particle is a microbe. The
technique can be used to identify the type of microbe (i.e.,
bacteria, fungi, or virus), and, ideally, the specific class of
microbe (i.e., Pneumonia, Clostridia, and the like). In this
embodiment, once a preliminary identification of the microbe is
made, a confirmation assay can be conducted by first taking an
EQELS spectra of the microbe in solution, subjecting the microbe to
an antibody specific for the microbe, and taking a second EQELS
spectra. If the EQELS spectra are different, this provides
confirmation that the microbe was properly identified and was bound
by the antibody. Further, once a microbe has been identified, a
putative antimicrobial compound can be put in solution/suspension
along with the microbe, to determine whether it is able to kill the
microbe.
[0290] In one embodiment, the methods can be used to identify a
potential therapeutic agent capable of interacting with a known
cell, such as a cancer cell, bacteria, fungi, or virus. In this
embodiment, one first uses focused light scattering techniques to
generate a spectrum showing the particle size and distribution for
the known cancer cell, bacteria, fungi, or virus in a sample
medium. Then, a putative therapeutic agent conjugated to a
microparticle or nanoparticle is added to the sample medium and
allowed to incubate with the known cell, microbe or virus. A second
spectrum is generated using focused light scattering techniques. If
peaks corresponding to a complex of the microparticle-conjugated
therapeutic agent and the known cell or microbe are observed, then
the therapeutic agent has bound to the cell. This is indicative of
the potential utility of the putative therapeutic agent against the
known cell, microbe or virus.
[0291] In one aspect of this embodiment, spectra of a microbe in a
sample medium are compared with a reference database of spectra of
known microbes, thus providing a rapid means for identifying a
particular microbe. The spectra can also provide an initial
determination of particle size and/or particle density in the
medium.
[0292] Bacterial Detection
[0293] In one example of identifying microbes, one can determine
whether the microbes are mono-dispersed or poly-dispersed by their
number and size. Since E. coli tend to mono-disperse and
Streptococcus tend to poly-disperse, this embodiment can be used to
observe particle size in a sample, where observation of clumping
identifies presence bacteria known to clump (i.e.
Streptococcus).
[0294] Detection of Particle Shedding:
[0295] In another embodiment, the method is used to detect particle
shedding. Representative biological particles which shed smaller
particles include tumors, red blood cells, white blood cells,
granulocytes, platelets, monocytes, neutrophils, lymphocytes,
endothelial cells, cancer cells, stem cells, bacteria, viruses, and
fungi. Particle shedding may result from cell interactions, a
change of cellular state such as activation or deactivation, as a
result of expression, cell death, etc. The methods described herein
can be used to identify such particle shedding.
[0296] In one embodiment, a drug or ligand is added to a vessel
with a cell. Where the drug or ligand reacts with the cell, the
cell may shed membrane particles or other particles. As described
in detail herein, the method can be used to detect the size, number
and/or type of particles shed by the reaction. Therefore, where the
cell is known, this technique may be used to detect efficacy of
unknown drug agent; and where the drug is known, the technique may
be used to identify the presence of a specific cell type.
[0297] The ejected particles can be observed using the methods
described herein. The ejected particles can similarly be
characterized by comparing the size and/or shape with a library of
data collected using focused light scattering techniques on known
ejected particles, and/or by binding some or all of the ejected
particles to an antibody or other such molecule.
V. Detection of Microparticles Ejected from Cells
[0298] In another embodiment, the ISADe device can be used to
detect particle shedding. Representative biological particles which
shed smaller particles include tumors, red blood cells, white blood
cells, granulocytes, platelets, monocytes, neutrophils,
lymphocytes, endothelial cells, cancer cells, stem cells, bacteria,
viruses, and fungi. Particle shedding may result from cell
interactions, a change of cellular state such as activation or
deactivation, as a result of expression, cell death, etc. The
methods described herein can be used to identify such particle
shedding.
[0299] In one embodiment, a drug or ligand is added to a vessel
with a cell. Where the drug or ligand reacts with the cell, the
cell may shed membrane particles or other particles. As described
in detail herein, the method can be used to detect the size, number
and/or type of particles shed by the reaction. Therefore, where the
cell is known, this technique may be used to detect efficacy of
unknown drug agent; and where the drug is known, the technique may
be used to identify the presence of a specific cell type.
[0300] The ejected particles can be observed using the devices and
methods described herein. The ejected particles can similarly be
characterized by comparing the size and/or shape with a library of
data collected using focused light scattering techniques on known
ejected particles, and/or by binding some or all of the ejected
particles to an antibody or other such molecule.
[0301] The ISADe device described above can be used to identify
particles in the given size range (i.e., the size of the ejected
(shed) microparticles and the size of the platelets). As shown in
FIG. 6, a sample composed of 6 differently-sized polystyrene beads
was introduced to the device, and assessed using the device in
terms of particle count (number) by particle size (.mu.m). The
chart shows the remarkable resolution of very small particle sizes.
Current flow cytometers are not capable of resolution to this
degree.
[0302] FIG. 7 is a chart showing similar data from the data shown
in FIG. 6. The data in FIG. 7 is presented as points rather than as
histograms, and is reflected in particles per 10 ml sample, versus
particle size (.mu.m). Also, FIG. 7 separates the size distribution
is separated into 3 different windows. In this embodiment, each
window has a separate detector that has been adjusted to detect
particles in a specific size range. The smallest particles are
assessed from scatted light focused onto a high-gain detector, the
middle window from scattered light focused onto a low gain
detector, and the window with the largest particles by a light
extinction method.
[0303] Detecting Individual Molecules
[0304] In one embodiment, the methods permit one to detect the
presence of specific molecules, where the molecules are of a size
below the threshold limit of detection for focused light scattering
techniques. In this embodiment, highly reflective metallic
particles, such as gold particles, are covalently linked to a
ligand that binds to the molecules of interest. Because the
metallic particles are dense and highly efficient light scattering
particles, the amount of light scattering when the ligand binds to
a molecule of interest can be measured using the focused light
scattering technique, thus confirming the presence or absence of a
molecule of interest in the solution. In another embodiment,
microparticles conjugated to an active agent known to bind in a
specific manner to a particular secies of shed particles are used,
rather than metallic particles. The complex of the shed particle
and the microparticle is then measured. Representative agents
include antibodies and small molecules known to bind to the
particular shed particles.
[0305] Detection of Particle Aggregation
[0306] In another embodiment, the method is used to determine
particle aggregation and types of aggregation. Such aggregation may
include, but is not limited to aggregation of a particle with other
particles, or aggregation of a particle with a drug.
[0307] Particularly with respect to liposomal therapy,
agglomeration of particles (such as liposomes) can result in
significant mortality or morbidity when the agglomerated particles
are administered. Accordingly, the method can be used to evaluate a
sample of liposomes before administration to ensure that the
particles have not agglomerated before a patient is treated.
VI. Methods for Detecting Binding of a Particle to a Known
Molecule
[0308] In some embodiments, one knows the identity of a known
molecule, for example, a drug molecule that is known to interact
with the receptor on a biological particle that may or may not be
present in the sample medium. By incubating the sample medium with
a conjugate of the drug molecule and a microparticle, one can look
for complex formation indicative of the binding of the drug
molecule with biological particles in the sample medium. The
complex formation can be observed over time, or simply after a
suitable incubation period.
[0309] The methods can be performed by using, as a starting
material, either a drug or a biological microparticle in a vessel,
and then adding a known material (either a known cell or microbe,
for example) to test for interaction with drug; or a known drug to
test for interaction with microparticle). Then, focused light
scattering techniques can be performed to look for change in
particle size from size of starting material, where an increase or
decrease in particle size is indicative of interaction and
binding.
[0310] In order to preserve the ability of the known molecule to
bind to the particle of interest, in those embodiments where a
molecule is conjugated to a microparticle, it is conjugated in a
way that does not adversely impact its ability to bind to the
particle of interest. This may involve developing a modified
molecule, wherein the molecule is modified to include a functional
group capable of being conjugated to the microparticle, such that
the molecule still maintains its ability to form a complex with the
particle of interest. Such modifications are routine in the
art.
[0311] By using a biological particle known to form a complex with
the active agent, one can evaluate such modified compounds for
their ability to maintain their binding affinity for the particle
of interest by incubating the modified compound, or the conjugate
of the compound with the microparticle with the particle. Those
compounds which maintain the ability to bind the particle of
interest can be identified using focused light scattering
techniques, because the particle size of the complex is larger than
the particle size of the non-complexed particle and non-complexed
conjugate.
[0312] Column-Based Approaches to Removing Biological Particles
[0313] Rather than forming a complex of the biological particle and
a conjugate of an active agent and a microparticle, one can
optionally prepare a column including microparticles conjugated to
agents that bind to the biological particle of interest, and pass
the sample medium through a column including the microparticles.
The sample, minus any complexed biological particles, will elute
from the column. By performing the focused light scattering method
on the eluted material, one can identify changes in particle
number/population density, as compared to the starting material. A
decrease in particle number/population density is indicative of
interaction and binding in the column, and, therefore, an
indication that the biological particle of interest was present in
the sample.
[0314] Magnetic Bead-Based Approaches to Remove Biological
Particles
[0315] Rather than forming a complex of the biological particle and
a conjugate of an active agent and a microparticle, and using
focused light scattering methods to identify the presence of the
complex between the biological particle and the conjugate, one can
optionally use a magnetic microparticle conjugated to the active
agent. Thus, one can first obtain a focused light scattering
spectra using the methods described herein, then use magnetic
particles to complex with the biological particle of interest. The
complex can be removed from the sample media using a magnet. Then,
one can obtain a second focused light scattering spectra, and
identify whether the number of biological particles of interest has
been reduced.
VII. Methods for Determining Binding of a Known Particle to an
Unknown Compound
[0316] In other embodiments, it is desired to learn whether a
putative therapeutic agent can bind to known biological particles.
In these embodiments, putative therapeutic agents are conjugated to
a microparticle or metallic nanoparticle, such as a gold
nanoparticle, and incubated with the known biological particles.
Thus, one can determine whether a compound forms a complex with the
biological particles.
[0317] In some aspects of this embodiment, it is desirable to know
the minimum inhibitory concentration, or binding affinity, of an
active agent. The agent can be complexed with the biological
particle at differing concentrations, and this information can be
obtained. Some degree of extrapolation may be required, since the
agent is conjugated to a microparticle, and therefore may behave
differently than the native drug.
[0318] In other aspects of this embodiment, it is desired to know
the selectivity of an active agent for one receptor over another.
In this aspect, the agent can be complexed with a plurality of
biological particles expressing differing receptors, and binding
information can be obtained for each of the receptors. Thus,
selectivity can be determined.
[0319] Determining Therapeutic Activity/Efficacy/Selectivity
[0320] In another embodiment, the method is used to identify
therapeutic agents. While small molecules, proteins and peptides
are not likely to be large enough to see, even with this technique,
they can either be coupled to a microparticle, such as a latex
particle or magnetic bead, which can be incubated with the
molecules, or placed on a column.
[0321] In one aspect of this embodiment, a biological particle with
a target for the therapeutic agent (i.e., a receptor) is placed in
suspension, and microparticles conjugated to one or more putative
therapeutic agents are added to the suspension.
[0322] If the therapeutic agent binds to the biological particle,
the binding can be observed because the size of the complex of the
biological particle and the conjugate is greater than that of the
conjugate or the biological particle.
[0323] This technique can be used, for example, to determine the
efficacy of specific antibacterial or other drug candidates for a
particular infection, or to identify agents useful for treating
specific types of cancer. In one aspect of this embodiment, the
techniques are useful for personalized medicine, where a particular
patient's bacterial infection, platelet or cancer cells, or
erythrocytes, are analyzed for their ability to bind to and
interact with specific therapeutic agents. In another aspect of
this embodiment, one can generate a plurality of spectra and
compare the results, to determine minimum inhibitory concentrations
and, therefore, useful dosage ranges for a given drug (where the
drug is an inhibitor).
VIII. Methods for Identifying Patients Likely to Benefit from
Treatment
[0324] Certain patients respond to therapy due to an interaction of
a cellular receptor with a drug molecule. However, certain other
patients have genetic mutations which do not permit the patients
cells to bind to the drug molecule, thus rendering the drug
ineffective. To determine whether a patient will respond to a given
therapy, a solution of the patient's cells can be combined with a
drug molecule of interest, where the molecule is bound to a probe
particle, which can be a microparticle or nanoparticle. If the drug
molecule of interest binds to the patient's cells, then the
concentration of the conjugate, and/or the patient's cells will be
lower, and a new peak corresponding to the complex of the cells and
the conjugate will be observed. The absence of a new peak
corresponding to the complex of the cells and the conjugate is
indicative that the patient will not respond to the particular drug
therapy. Thus, the method can confirm that the patient will or will
not achieve a benefit using the particular drug therapy. This
enables a personalized medicine approach using rapid and
inexpensive methods.
[0325] In one aspect of this embodiment, the cells are cancer cells
from an individual patient, and sample media containing the cancer
cells are incubated with a series of therapeutic agents bound to a
microparticle or nanoparticle. Those therapeutic agents that form a
complex with the cancer cells are potential candidates for
personalized treatment of the patient, since they bind to and
interact with the cancer cells. This technique can provide a
relatively quick and inexpensive method for identifying patients
who have certain mutations, such as HER2 positive patients,
patients whose cancer cells have vitamin D receptors, patients with
estrogen-responsive cancer cells, and the like.
[0326] In another aspect of this embodiment, the cells are blood
cells from a patient suffering from atherosclerosis, and who is
being evaluated to see if his blood cells will respond to treatment
with clopridogrel bisulfate (Plavix.RTM.). The interaction with the
blood cells and clopidogrel bisulfate is a surface interaction, but
a small percentage of patients have a mutation in their blood cells
that inhibits the surface interaction with this compound. For most
of those patients, there is an alternative therapeutic agent, but
it is important to identify those patients before the symptoms
worsen, possibly leading to a heart attack. In this aspect, blood
cells of a patient are incubated with microparticles conjugated to
clopidogrel bisulfate, and a spectrum is obtained using focused
light scattering techniques. For example interaction of the probe
particle can be between the ADP receptor and Plavix target, P2Y12
or to the activation of the of VASP pathway. The presence of a
complex between the clopidogrel bisulfate and the blood cells can
be observed. Further, if the patient is responsive to Plavix, no
activation will occur when ADP or other specific agonists are added
to the patient's platelets. Thus, no activation will occur and
specific activation epitopes, like CD62, glycoprotein alpha2,
beta3, etc., will be detected by specific probe particles. Because
the size of blood cells and the microparticle/clopidogrel bisulfate
conjugate are known, only one spectrum needs to be obtained, and
the only peak of interest is the peak corresponding to the complex
of the conjugate with the blood cells.
IX. Methods for Performing High Throughput Bioassays
[0327] Any and all of these assays can be optimized for high
throughput screening using suitable robotics. Liquid handlers can
transfer samples to a multi-tube or multi-well plate, and a "memory
map" can be used to correlate the samples to their location on the
plate. Information on each sample can then be stored, and used to
provide information about drug candidates, patient diagnoses, and
proposed patient treatment options.
[0328] Robotics systems are known in the art, and can be used to
move samples taken from individual patients to known positions in a
multi-tube or multi-well plate. Once information on the sample is
obtained using the focused light scattering techniques described
herein, the information can be correlated to the individual patient
via the stored information correlating the location of the tube and
the patient identification. Liquid handlers can take portions of
the sample and evaluate a plurality (i.e., at least two) of
different screening assays, for example, by incubating portions of
the sample with different microparticles, bound to different active
agents.
[0329] Automated processes using known robotics can be used to pull
and place samples (like high throughput screening) with use of a
"memory map". A user can then pick desired screens to be run and
the robotic apparatus will implement desired processes.
[0330] In another aspect of the embodiments described herein, the
methods can be automated using robotics to pull and place samples
(analogous to conventional high throughput screening methods),
optionally in conjunction with a "memory map". A user can then pick
desired screens to be performed, and the robotic apparatus can
implement the desired processes. In this embodiment, a laboratory
can be set up to automatically screen numerous samples.
[0331] In a preferred embodiment, the personalized medicine
processes described herein are automated, to provide relatively
inexpensive, and relatively fast, high throughput screening methods
to identify preferred therapies for patients suffering from
disease.
X. Reference Libraries
[0332] A reference database of information gained by performing
focused light scattering on known compounds can be used. One can
compare the sample to the reference database in order to identify
or characterize the particles in the tested sample. A reference
database includes at least two, preferably more than ten, more
preferably greater than a hundred, and most preferably, greater
than a thousand bits of information on particle size that can be
used to correlate the particle size measured using the techniques
described herein with particle sizes for known biological particles
that are stored in the reference database.
[0333] The present invention will be better understood with
reference to the following non-limiting examples.
Example 1: Representative Focused Light Scattering Device
[0334] A representative focused light scattering device is shown in
FIG. 1. A first laser (1) emits light at a first wavelength, and a
second laser (2) emits light at a second wavelength. Both beams of
light pass through a first beam splitter (3) and through a first
focusing lens (4) before they enter into a flow cell (15). The flow
cell includes a site (5) for hydrodynamic injection of the sample.
As the platelets in the flow cell pass through the beams of light,
the light is scattered as it hits the platelets. The scattered
light passes through a circular spatial filter (6) and then through
a first collimating lens (7). The light beam passes through a
second beam splitter (16), which splits the light into two beams. A
first beam passes through a second focusing lens (8) and through a
first chromatic filter (9) that passes scattered light from the
first laser (1) through a first detector (10). The second beam
passes through a second collimating lens (11), a third focusing
lens (12) and a second chromatic filter (13) that passes scattered
light from the second laser (2) to a second detector (14).
[0335] The two photodetectors (10 and 14) each are able to detect
light at a certain frequency, so that light transmitted at
different frequencies (as a result of the two lasers hitting
particles, and which may interact with fluorescent tags on the
particles) can be separately determined.
[0336] A third detector (an extinction detector) (18) receives a
portion of the light passing through the flow cell. A portion of
the light passing through the flow cell is reflected off of a
movable mirror (17) and onto the third detector.
Example 2: Detection of Microparticle's (MP) Present in a
Biological Sample
[0337] In this example, a specimen is subject to particle sizing
and counting. After an appropriate dilution of the sample, the
diluted specimen is introduced into the device for analysis. As
counting proceeds, counts will accumulate in the size region less
that 1 micron. The appearance of MP in this size region will
indicate the presence of MPs.
Example 3: Microparticle Characterization
[0338] Once MPs are detected, as described in Example 2, it may be
important to determine the source of the MP (from platelets
neutrophils, tumor cell, etc.). This Example provides two different
options for characterizing the MP.
[0339] The first option is to use MP sizing and counts. In this
case, the specimen is incubated with a second particle with a
specific ligand conjugated to its surface. The choice of the ligand
will depend on the specific MP be characterized. For example, if
the MP of interest has coagulation tissue factor (TF) on its
surface, the conjugated particle can be conjugated with an antibody
against TF-particle or with coagulation factor FVII. Either ligand
will specifically bind to MPs with TF on their surfaces.
[0340] When the conjugated particles are incubated with the anti-TF
conjugated particles, a new size corresponding to the
TF-MP+anti-TF-particles will appear when the MP and the probe
particle are counted. In a like manner, other MPs can be
characterized by developing probe particles with a conjugated
ligand specific for the MP of interest.
[0341] The second option is to use an EQELS device to analyze the
MPs. The basic procedure is the same, except that differences in
the particles electrophoretic mobility can be analyzed.
[0342] Specifically, a baseline electrophoretic mobility of the MP
can be obtained. The MP will then be mixed with the probe particle
that is conjugated with the specific ligand for the MP of interest,
or just with the ligand without conjugation to a probe particle.
When the probe particle binds to the MP of interest, a difference
in the electrophoretic mobility is observed. These data will
provide a specific identification of the MP. If the ligand alone
was used to bind to the MP, the EQELS data, in addition to IP of
the particle, will also provide binding constants for the ligand to
the MP.
Example 4: Determination of Cellular Activation
[0343] In this Example, the cellular activation, such as platelet
activation, is determined. A baseline resting platelet run on the
sizing-counting device is determined. The platelet or cell is then
activated using appropriate agonists. Once activation occurs, new
surface epitopes appear on the surface. In the case of the platelet
DC62, CD41, various integrins, and the like, begin to appear.
[0344] A probe particle with a ligand for the specific epitope of
interest is then added to the activated platelets (or other cells).
The ligand can be an antibody, or small molecule, that specifically
binds to the activated epitope. When binding occurs, a particle is
created (the activated cell+the probe particle) which has a larger
size than either the activated cell or the probe particle. The
appearance of these new particles represents the appearance of the
activated platelet+the bound probe particle.
Example 5: Microbe Identification
[0345] In this Example, microbes are identified using the
techniques described herein. The microbe may be bacterial, viral,
fungal, or protozoa. A sample containing a microbe is analyzed
using the counting/sizing device described herein. The sample can
be from a water supply, from a patient (human or animal), or other
source. The size distribution will be determined, and compared to a
database, to determine whether one or more of the particles in the
sample fall into any of the sizes typical for known microbes. If
so, a probe particle conjugated with a ligand that specifically
binds to certain microbes can be added. The identity of the microbe
is confirmed when a new size distribution is obtained when the
sample is incubated with the probe particle.
[0346] Additional verification can be obtained if the specimen is
examined by EQELS, where a swim rate is determined for flagellated
organisms using the velocimetry mode of the EQELS device. The EQELS
device can also determine the microbe's electrophoretic mobility.
This information can be compared with data in an EQELS database for
further confirmation of the identity of the particle.
[0347] If indicated, EQELS will then be used to help determine the
appropriate antimicrobial (antibiotic, antiviral, etc) agent. A
know concentration of antimicrobial agent will be added to the
specimen. Binding of the agent to the microbe will be determined by
a change in the microbe's electrophoretic mobility. Further, if the
drug kills the microbe, its surface charge density changes
resulting in a rather large change in the killed microbe's
electrophoretic mobility.
Example 6: Drug Efficacy: Effectiveness of Cell Inhibition
Specific Example of Aspirin or/and P2Y12 Inhibition of Platelet
Activation
[0348] Platelet inhibition in arterial thrombophilic diseases is
considered the standard of care. Unfortunately, in some patients,
conventional drugs do not inhibit the patient's platelets.
Approximately 25% of ASA-treated and 29% of Plavix-treated patients
do not respond to treatment. Currently, there is no accepted assay
to identify the resistant/refractory patients.
[0349] The assay described herein can use particle sizing, based on
the following concept, to identify resistant/refractory patients.
The goal of treatment with anti-platelet drugs is to inhibit
activation. When the platelet is activated, new surface epitopes
appear. So, in this embodiment, the patient's platelets are first
obtained as platelet rich plasma (PRP). A baseline measurement of
the particle size and/or distribution is then made. Next, an
aliquot of the patient PRP can be mixed with a platelet activator
(agonist). If the drug works, no activation occurs. If it does not
work, the platelet will activate. The assay can then probe the
platelet surface for the appearance of new epitopes like CD62, CD41
and the like. The surface can be probed with a ligand (antibody or
otherwise) that is conjugated to a particle. If binding occurs, the
probe particle will bind with the activated platelet, and a
particle with a different (larger) particle size (i.e., the
activated platelet+conjugated-probe particle) will appear. If the
drug works, no change in the particle size distribution will
occur.
Example 7: Lipid Droplets as Drug Delivery Vehicles
[0350] Certain sized particles do not perfuse capillaries well.
Since a typical capillary is approximately 2-3 microns in diameter,
particles larger that that size must have a surface to volume ratio
that will permit the distortion of the particle so that is can
enter the capillary system; similar to a red blood cell. In this
Example, particle sizing is used to identify particles which fall
into a range that will not be perfused, and result in potential
malaise or death to the patient. Further, the surface
characteristics of some particles lead to instability, that may
cause particle aggregation or fragmentation. Similar problems are
well recognized in colloidal chemistry.
[0351] In this embodiment, drugs like Ambisome.TM., Daunosome.TM.,
Doxil.TM., or other liposomal drug delivery vehicles, can be
screened for a safe particle size distribution prior to infusion.
The screen can identify particles by particle size and
distribution, so if relatively large particles, corresponding to
agglomerated liposomal or other particles are observed, then the
sample can be rejected before being infused. Alternatively, the
sample can be subjected to conditions which de-agglomerate the
particles (for example, ultrasound and the like), and the sample
re-tested. These assays can also be used to optimize the solution,
and particle surface optimization for identifying the best lead
compound(s).
Example 8: Small Molecule Distribution Assays. Specific Examples of
vWF or Serum Plasma
[0352] vWF is a polydispersed molecule with a molecular weight
distribution range of 500,000 to 20 million or even higher. Thus,
there is a substantial difference in the molecular size
distribution. Current technology requires a minimum of several days
to complete an analysis.
[0353] In this embodiment, gold beads are conjugated with an
anti-vWF antibody in a manner that would bind one bead to one vWF
multimer. Since the mass density of the gold bead results in more
efficient light scattering, vWF molecules bound to the gold beads
will be visible to the focused scattering sizing and counting
device. Thus, the presence of, and in some embodiments, the amount
of, vWF can be determined.
[0354] Accordingly, although the invention has been described
herein with reference to various illustrative aspects, features and
embodiments, it will be recognized that the invention is not thus
limited, but rather extends to and encompasses other variations,
modifications and alternative embodiments, such as will suggest
themselves to those of ordinary skill in the art, based on the
disclosure herein. The claims hereafter set forth therefore are
intended to be broadly construed and interpreted as including all
such variations, modifications and alternative embodiments within
their spirit and scope.
* * * * *