U.S. patent application number 09/844080 was filed with the patent office on 2002-03-07 for particle or cell analyzer and method.
This patent application is currently assigned to GUAVA TECHNOLOGIES, INC.. Invention is credited to Goix, Philippe J., Lingane, Paul J., Phi-Wilson, Janette T..
Application Number | 20020028434 09/844080 |
Document ID | / |
Family ID | 26924172 |
Filed Date | 2002-03-07 |
United States Patent
Application |
20020028434 |
Kind Code |
A1 |
Goix, Philippe J. ; et
al. |
March 7, 2002 |
Particle or cell analyzer and method
Abstract
A particle analyzer in which tagged particles to be analyzed are
drawn through a suspended capillary tube where a predetermined
volume in the capillary tube is illuminated. The illumination
scattered by said particles is detected by a detector to count all
particles. The fluorescent illumination emitted by tagged particles
is detected and the output signals from the fluorescent detectors
and scatter detector are processed to provide an analysis of the
particles.
Inventors: |
Goix, Philippe J.; (Oakland,
CA) ; Lingane, Paul J.; (Redwood City, CA) ;
Phi-Wilson, Janette T.; (Los Altos Hills, CA) |
Correspondence
Address: |
Aldo J. Test
FLEHR HOHBACH TEST ALBRITTON & HERBERT LLP
Four Embarcadero Center, Suite 3400
San Francisco
CA
94111-4187
US
|
Assignee: |
GUAVA TECHNOLOGIES, INC.
|
Family ID: |
26924172 |
Appl. No.: |
09/844080 |
Filed: |
April 26, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60230380 |
Sep 6, 2000 |
|
|
|
Current U.S.
Class: |
435/4 ; 356/300;
356/39 |
Current CPC
Class: |
G01N 15/1404 20130101;
G01N 21/64 20130101 |
Class at
Publication: |
435/4 ; 356/39;
356/300 |
International
Class: |
C12Q 001/00; G01N
033/48; G01J 003/00 |
Claims
What is claimed is:
1. A particle analyzing apparatus for analyzing a sample
comprising: an elongated capillary channel having a predetermined
internal cross-sectional area, a pump connected to one end of the
capillary channel for drawing sample into the other end of the
capillary channel and through the capillary channel to cause
particles to flow along said capillary channel, a light source for
illuminating a predetermined analyzing volume of sample in the
capillary channel, at least one detector for detecting fluorescent
light emitted by particles in said volume excited by the
illumination impinging upon particles in said predetermined
volume.
2. A particle analyzing apparatus as in claim 1 in which said
capillary channel is in a capillary tube.
3. A particle analyzing apparatus as in claim 2 in which the
capillary channel is cylindrical.
4. A particle analyzing apparatus as in claim 2 in which the
capillary channel is rectangular.
5. A particle analyzing apparatus as in claim 1 including a
detector for detecting all particles flowing along said capillary
tube.
6. A particle analyzing apparatus for analyzing a sample as in
claim 5 in which said particle detector detects light scattered by
particles in said predetermined analyzing volume.
7. A particle analyzing apparatus as in claim 5 in which said
detector detects a change in impedance caused by said flowing
particles.
8. A particle analyzing apparatus for analyzing a sample as in
claim 1 in which said detector for detecting fluorescent light
includes a lens for intercepting fluorescent light, and a slit
located at the focus of the lens for blocking unwanted light from
said detector.
9. A particle analyzing apparatus for analyzing a sample as in
claim 8 including a particle detector for detecting light scattered
by the particles in said analyzing volume.
10. A particle analyzing apparatus for analyzing a sample as in
claim 6 or 9 in which said particle detector includes a beam
blocker for blocking direct light whereby said detector receives
only scattered light.
11. A particle analyzing apparatus for analyzing a sample as in
claims 6 or 9 in which the particle detector is an off-axis
detector.
12. A particle analyzing apparatus for analyzing a sample
comprising a capillary tube having a predetermined internal
cross-section having one end suspended, a pump connected to one end
of the capillary tube for drawing sample into the tube through the
suspended end of the capillary tube, a source of light, an optical
system for receiving and focusing the light from said source to
form and direct a thin rectangular beam through said capillary tube
to define an analyzing volume in said capillary tube, said
capillary tube selected to have a diameter which causes
substantially all particles to be singulated as they pass through
said analyzing volume, at least one detector for detecting those
particles in which fluorescence is excited by said beam, and a
detector for detecting all particles which pass through said
analyzing volume.
13. A particle analyzing apparatus as in claim 12 including a
plurality of detectors for detecting particles which fluoresces at
different wavelengths.
14. A particle analyzing apparatus as in claim 12 in which said
particle detector detects light scattered by said particles.
15. A particle analyzing apparatus as in claim 12 in which said
particle detector detects the electrical impedance of said
particles.
16. A particle analyzing apparatus as in claim 12 in which the
capillary tube internal cross-section is rectangular.
17. A particle analyzing apparatus as in claim 12 in which the
capillary tube internal cross-section is cylindrical.
18. A particle analyzing apparatus as in claim 12 in which the
optical system is configured to form a thin, wide rectangular beam
at the capillary to illuminate a small analyzing volume defined by
the thickness of the beam in the direction of flow of said fluid
and by the internal walls of the capillary in the other
direction.
19. A particle analyzing apparatus as in claim 14 including a beam
blocker for blocking the direct transmission of illumination from
said beam to the particle detector.
20. A particle analyzing apparatus comprising: a capillary tube of
predetermined internal cross-section selected such as to singulate
particles flowing therethrough, a tee, a pump connected to one arm
of said tee, a capillary conduit extending from another arm of said
tee and connected to one end of the capillary tube, a discharge
conduit and a valve connected to the other arm of said tee, whereby
when the valve is closed, the pump can draw fluid from the other
end of the capillary through said capillary, and when the valve is
opened the pump can discharge fluid sample previously drawn through
the capillary, a light source for illuminating a predetermined
analyzing volume of fluid in said capillary tube, at least one
detector for detecting fluorescent light emitted by particles
flowing through said volume in response to said illumination, and a
detector for detecting particles which flow through said analyzing
volume.
21. A particle analyzing apparatus as in claim 19 in which said at
least one detector means for detecting fluorescent light emitted by
particles in said volume includes: means for gathering fluorescent
light emitted by particles in said volume, a beam splitter for
receiving said gathered light and reflecting light above a
predetermined wavelength and passing light below said predetermined
wavelength, a first detector for receiving the transmitted light
and providing a first output signal for particles tagged to emit
light below said predetermined wavelength, a second detector for
receiving the reflected light and providing a second output signal
for particles tagged to emit light above said predetermined
wavelength.
22. A particle analyzing apparatus as in claim 21 in which said
predetermined wavelength is 620 nm, said light below said
predetermined wavelength is 580 nm, and above said predetermined
wavelength is 675 nm.
23. A particle analyzing apparatus as in claim 22 including a
filter interposed between the beam splitter and each detector for
passing light at 580 nm and 675 nm, respectively.
24. A particle analyzing apparatus as in claim 21 in which said
means for gathering the fluorescent light emitted by particles
comprises: a lens for collecting the light, and a slit positioned
in front of said at least one detector to define the analyzing
volume observed by said detector.
25. A particle counting apparatus as in claim 20 or 21 in which the
diameter of said discharge conduit is substantially larger than the
diameter of the capillary tube.
26. Method of detecting particles in a sample fluid which comprises
the steps of: providing a capillary tube of predetermined internal
cross-section with a suspended end, drawing the sample fluid
through the suspended end of said capillary tube and through the
capillary tube to cause substantial singulation of the particles,
focusing a beam of light onto the capillary tube to illuminate
particles in an analyzing volume bounded by the walls in one
direction and the beam size in the other direction, and detecting
fluorescent light emitted by tagged particles in response to the
illumination of said volume, and providing an output signal.
27. The method of claim 26 including the additional step of
detecting light scattered by said particles as they travel through
said volume and providing an output signal.
28. The method of claim 26 including the additional step of
processing said output signals and identifying the particles and
their concentration.
29. A particle analyzing apparatus for analyzing a sample
comprising: an elongated capillary tube having a predetermined
internal cross-section, a pump connected to one end of the
capillary tube for drawing sample into the other end of the
capillary tube and through the capillary tube to cause particles to
flow along said capillary tube, a light source for illuminating a
predetermined analyzing volume of sample in the capillary tube,
means for gathering a fluorescent light emitted by said particles
excited by illumination impinging on the particles, and at least
one detector for receiving said gathered fluorescent light.
30. A particle analyzing apparatus as in claim 29 in which said
gathering means comprises a fiber optic waveguide for guiding the
light into the detector.
31. A particle analyzing apparatus as in claim 20 in which said at
least one detector means for detecting fluorescent light emitted by
particles in said volume includes a plurality of detectors
configured to detect fluorescent light emitted at a plurality of
different wavelengths.
32. A particle analyzing apparatus as in claim 31 in which said
means for illuminating the predetermined volume illuminates the
volume with light of a plurality of different wavelengths.
33. A particle analyzing apparatus as in claim 29 or 30 in which
said light source comprises means for projecting a thin rectangular
beam through the capillary.
Description
Related Applications
[0001] This application claims priority to provisional application
Ser. No. 60/230,380 filed Sep. 6, 2000.
BRIEF DESCRIPTION OF THE INVENTION
[0002] This invention relates generally to a particle or cell
analyzer and method, and more particularly to a particle or cell
analyzer and method in which the sample solution containing the
particles or cells is drawn through a capillary for presentation to
a shaped light beam.
BACKGROUND OF THE INVENTION
[0003] The detection and analysis of individual particles or cells
is important in medical and biological research. It is particularly
important to be able to measure characteristics of particles such
as concentration, number, viability, identification and size.
Individual particles or cells as herein defined include, for
example, bacteria, viruses, DNA fragments, cells, molecules and
constituents of whole blood.
[0004] Typically, such characteristics of particles are measured
using flow cytometers. In flow cytometers, particles which are
either intrinsically fluorescent or are labeled with a fluorescent
marker or label, are hydrodynamically focused within a sheath fluid
and caused to flow past a beam of radiant energy which excites the
particles or labels to cause generation of fluorescent light. One
or more photodetectors detect the fluorescent light emitted by the
particles or labels at selected wavelengths as they flow through
the light beam, and generates output signals representative of the
particles. In most cytometers, a photodetector is also used to
measure forward scatter of the light to generate signals indicative
of the presence and size of all of the particles.
[0005] U.S. Pat. No. 5,547,849 describes a scanning imaging
cytometer wherein an unprocessed biological fluid sample is reacted
with a fluorescently labeled binding agent. The reacted sample
undergoes minimal processing before it is enclosed in a capillary
tube of predetermined size. The capillary tube with the enclosed
sample is optically scanned and the fluorescent excitation is
recorded from a plurality of columnar regions along the capillary
tube. Each columnar region is generally defined by the spot size of
the excitation beam and the depth dimension of the capillary tube.
A spacial filter of sufficient pinhole diameter is selected to
allow simultaneous volumetric detection of all fluorescent targets
in each columnar region. The cellular components or particles are
identified as is their concentration.
OBJECTS AND SUMMARY OF THE INVENTION
[0006] It is an object of the present invention to provide a
particle analyzer and method having high particle selectivity.
[0007] It is another object of the present invention to provide a
compact, high-sensitivity particle analyzer.
[0008] It is still another object of the present invention to
provide a portable particle analyzer and method for use in
immunology, microbiology, cell biology, hematology and cell
analysis.
[0009] It is a further object of the present invention to provide a
simple-to-use, less expensive, particle analyzing apparatus for
counting particles in small volumes of sample fluids and
determining their characteristics.
[0010] It is still another object of the present invention to
provide a particle analyzer and method for analyzing low volumes of
low-density sample fluids.
[0011] The foregoing and other objects of the invention are
achieved by a particle analyzing apparatus which analyzes particles
in a sample fluid flowing through a capillary tube which has a
suspended sampling end for insertion into a sample fluid, and a
pump coupled to the other end for drawing the sample fluid and
particles through the capillary. An illumination source is provided
for projecting a beam of light through a predetermined volume of
the capillary to impinge upon the particles that flow through that
volume. At least one detector is disposed to receive fluorescent
light emitted by excited fluorescing particles and provide an
output pulse for each fluorescing particle, and another detector
senses the passage of all particles which flow through the volume
and provides an output signal, whereby the output signals from the
detectors can be used to characterize the particles.
[0012] A method of analyzing samples containing particles, which
includes drawing the sample through a capillary volume where the
particles are illuminated by a light source, and scattered light
and fluorescent light from labeled particles excited by the light
source is detected to provide output signals which are processed to
provide an analysis of the sample.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] The foregoing and other objects of the invention will be
more clearly understood from the following detailed description
when read in conjunction with the accompanying drawings in
which:
[0014] FIG. 1 schematically shows a particle analyzer in accordance
with the present invention.
[0015] FIG. 2 is a top plan view showing the optical components
shown in FIG. 1 mounted on a support shelf.
[0016] FIG. 3 is a front elevational view partly in section of FIG.
2.
[0017] FIG. 4 is a side elevational view of the beam-forming
optical system.
[0018] FIG. 5 is a top plan view of the beam-forming optical system
of FIG. 4.
[0019] FIG. 6 shows the sample fluid flow and pumping system.
[0020] FIG. 7 is a perspective view of a portion of a capillary
used in connection with one embodiment of the present
invention.
[0021] FIG. 8 is a perspective view of a portion of a capillary
tube used in connection with another embodiment of the present
invention.
[0022] FIG. 9 schematically shows a control and data acquisition
system associated with the particle analyzer.
[0023] FIG. 10 is a timing and data acquisition diagram
illustrating operation of the particle analyzer.
[0024] FIG. 11 is a schematic view of a four-color particle
analyzer.
[0025] FIG. 12 schematically illustrates an analyzer having
multiple analyzing stations along the capillary tube.
[0026] FIG. 13 shows an impedance detector for detecting particles
as they flow past a detection region.
[0027] FIG. 14 schematically shows a circuit suitable for
correlating signals from an impedance cell sensor with
photomultiplier output signals
[0028] FIG. 15 shows a particle analyzer in accordance with another
embodiment of the invention.
Description of Preferred Embodiment(s)
[0029] Referring to FIG. 1, there is schematically illustrated a
particle analyzer in accordance with one embodiment of the present
invention. As used herein, "particles" means particles or cells,
for example, bacteria, viruses, DNA fragments, blood cells,
molecules and constituents of whole blood. A capillary tube 11 has
a suspended end 12 adapted to be immersed into a sample solution 13
retained in a cuvet or vial 14. It will be apparent that, although
a square capillary is illustrated, the capillary may be cylindrical
or of other shape, such as a microchannel. Sample fluid is drawn
into the end of the capillary as shown by the arrow 16. As will be
presently described, the fluid or liquid sample is drawn through
the capillary by a calibrated pump connected to the other end of
the capillary. The size or bore of the capillary tube 11 is
selected such that the particles 18 are singulated as they pass a
viewing or analyzing volume 19. A light source, preferably a laser,
21 emits light 22 of selected wavelength. The light is received by
an optical focusing system 23 which focuses said light and forms
and directs a beam 24 to the capillary where it passes through the
analyzing volume 19. The optical focusing system is configured to
form a flat, thin rectangular beam which impinges on the capillary
tube 11. The thickness of the flat beam and the walls of the
capillary define the analyzing volume. In order to count all
particles which traverse the detection volume, that is particles
which are tagged to fluoresce and untagged particles, scattered
light is detected. In one embodiment, a beam blocker 26 is
positioned to intercept the beam after it passes through the
capillary tube 11. Light scattered by a particle that flows through
the beam is directed onto a detector 27 by lens 28. The detector
provides an output signal such as the one illustrated by the peak
29, when a particle passes through the beam and scatters the light.
The size of the peak is dependent upon the size of the particle,
and the occurrence of the peak indicates that a particle in the
volume 19 (fluorescent or non-fluorescent) has traversed the thin
beam of light. Another approach is to employ an off-axis detector,
such as illustrated in FIG. 15, to measure the scattered light. In
such event, a beam blocker is not required. There is also described
below an impedance method of detecting particles.
[0030] If the particles are intrinsically fluorescent, or if the
particles have been tagged with a fluorescent dye, they will emit
light 31 at a characteristic wavelength as they pass through the
volume defined by thin beam of light 24 which excites fluorescence.
The fluorescent light is detected at an angle with respect to the
beam axis so that no direct beam light is detected. In the
embodiment of FIGS. 1-3, a collector lens 32 receives the
fluorescent light from the particles and focuses it at detectors 36
and 37. We have found that initially we included slits 33 or 34
oriented in the direction of the thin beam to block any stray
light. However, we have found that if the beam is properly focused
into a thin flat beam, stray light is not a problem. This greatly
simplifies assembly of the analyzer, since there is no need to
carefully align the slits. The light impinges onto a dichroic beam
splitter 38 which passes light of selected wavelengths through
filter 39 to detector 36, and deflects light of other selected
wavelengths through filter 41 to photodetector 37. For example, the
dichroic beam splitter reflects light having wavelengths less than
620 nm, and transmits light having a greater wavelength. The
filters 39 and 41 are selected to pass the wavelengths
corresponding to the fluorescence wavelength expected from the
fluorescing particles. In one example, the filters 39 and 41 were
selected to pass light at 580 nm and 675 nm, respectively. This
permitted identification and counting of particles which had been
tagged with fluorescent material which emits at these wavelengths
in response to the optical beam. The outputs of the photodetectors
are pulses such as those schematically illustrated at 42 and 43,
FIG. 1.
[0031] FIGS. 2 and 3 show the components of a particle analyzer in
accordance with the above-described embodiment mounted on a support
plate 51. The support plate 51 carries an optical block 52 adapted
to receive and support the suspended capillary tube 11. Capillary
tube 11 includes a hub 53, FIGS. 3 and 6, which is received in a
well 54 to retain and position the capillary in the optical block.
The capillary 11 is positioned in the optical block 52 by threading
it through a narrow slot (not shown) and held in position by
nylon-tipped set screws inserted in threaded holes 56 and 57. As it
is inserted through the block, the capillary tube can be viewed
through the viewing port 58. The end of the capillary tube is
suspended and extends downwardly for insertion into a vial or cuvet
14 which contains the sample fluid or specimen. It is apparent that
the capillary can be positioned and suspended by other supporting
arrangements.
[0032] In one embodiment, a rotatable vial support member having
two arms 59 is rotatably and slidably received by a guide post 60
secured to the base. A vial holder 61 is disposed at the end of
each arm. In operation, the support is moved downwardly along the
post 59, rotated to bring a vial under the capillary, and moved
upwardly whereby the end of the capillary is immersed in the sample
fluid. As the sample is being analyzed, another vial with another
sample can be placed in the other holder whereby it can be brought
into cooperative relationship with the capillary tip as soon as the
analysis of the prior sample has been completed.
[0033] The housing 23 for the laser and optical focusing system
which forms the beam 24 is carried on mounting block 62. The
optical system is shown in FIGS. 4 and 5. It receives collimated
light 22 from the laser 21, and generates the light beam 24, which
impinges upon the capillary tube 1l. The optical system may
include, for example, a first plano-convex lens 63, a second
plano-convex lens 64 and a cylindrical lens 66. The action of the
lens assembly is to form a sheet-like thin rectangular beam which
in one example was 20 .mu.m in thickness along the longitudinal
direction of the capillary, and 400 .mu.m broad in the
perpendicular direction, whereby a rectangular volume of sample was
illuminated. The arrows 67 and 68, FIGS. 4 and 5, show the thin and
broad configuration of the beam, respectively.
[0034] The photodetector 27 is mounted on the block 52 and
supported axially with respect to the axis of the beam 24. The beam
blocker bar 26 is mounted in the block 52 and intercepts and blocks
out the direct beam after it passes through the capillary 11. The
scattered light which passes around beam-blocking bar 26 is focused
onto the detector 27 by a lens 28. Thus, the scattered light will
provide an output signal for any tagged or untagged particle
flowing past the observation volume 19, thus providing a total
particle count. The output of the detector is then representative
of the passage of a particle or cluster of particles and the size
of the particle or cluster of particles. As will be explained
below, this, taken together with the fluorescent signal, enables
analysis of the sample. If the detector 27 is located off-axis, it
will only receive scattered light and there is no need for a
beam-blocking bar. Furthermore, this would be less sensitive to
stray light in the forward direction which carries broadband laser
noise which can mask out low level particle signals.
[0035] As described above, light emitted by fluorescence from
intrinsically fluorescent particles, or particles which have been
tagged with a fluorescent dye or material, is detected at an angle
with respect to the beam axis. Referring to FIG. 2, the condenser
lens 31 is carried by the block 52. The lens 31 receives the
fluorescent light and focuses it at the detectors 36 and 37, which
may be photomultipliers, charge-coupled diodes (CCDs), or other
photodetectors. More particularly, the fluorescent light from the
lens 31 impinges upon a dichroic beam splitter 38 which splits the
beam into two wavelengths, one which passes through the beam
splitter and one which is deflected by the dichroic beam splitter
38. Filters 39 and 41 filter the transmitted and reflected light to
pass only light at the wavelength of the fluorescence of the
particles to reject light at other wavelengths. If slits 33 and 34
are present, they reject any stray light from regions outside of
the volume 19 defined by the thin rectangular beam 24. However, as
discussed above, slits may not be required because the effect of
stray light is minimized. The photo-multipliers or other
photodetectors each provide an output signal representing the
intensity of light at the filtered wavelength. As described above,
the dichroic beam splitter reflects light having wavelengths less
than 620 nm, and transmits light having greater wavelengths. The
filter 39 passes light at 580 nm, while the filter 41 passes light
at 675 nm. This permits analysis of particles which have been
tagged with fluorescent substances which emit light at 580 nm and
675 nm to be individually counted. The output of the
photo-multipliers are pulses 42 and 43, one for each particle
emitting light at the particular wavelength, such as those
schematically illustrated in FIG. 1. It is apparent that the
wavelengths selected for the filters depends upon the fluorescent
wavelength of the marker or label affixed to the particles.
[0036] In order to identify and count the particles in the fluid in
a volumetric manner, the volume of fluid must be correlated with
the number of particles detected in a given volume. In the present
invention, the fluid sample is drawn through the capillary tube at
a constant rate by an electrically operated calibrated pump or
syringe 71, FIG. 6. The pump may be any other type of pump which
can draw known volume samples through the capillary. The pump is
connected to the capillary tube by a conduit or tube 72. This
permits changing capillaries 11 to substitute a clean capillary or
a capillary having a different diameter which may be needed for
various types and sizes of particles or cells. As illustrated, the
pump comprises a syringe pump in which sample fluid is drawn into
the capillary by moving the plunger 73. The pump 71 is also
connected to a waste or drain conduit 74 which includes a valve 76.
When the valve is closed, the pump draws sample from the vial or
cuvet through the capillary tube 11 past the detection volume 19.
After an analysis has been completed, the valve 76 is opened,
whereby reversal of direction of the plunger 73 causes fluid to
flow through conduit 74 into the waste container 77. In accordance
with a feature of the present invention, the diameter of the waste
tube 74 is selected to be many times, 10 or more than that of the
capillary, whereby substantially all of the fluid from the syringe
is discharged into the waste. For example, if there is a factor of
ten ratio in diameter, only 1/10,000 of the fluid will travel back
through the capillary, a negligible amount.
[0037] The pump is designed such that a predetermined movement of
the plunger 73 will draw a known volume of sample through the
capillary tube. The pump can be calibrated for each capillary by
drawing a fluid into the pump by moving the plunger a known
distance and then discharging the fluid and measuring the volume of
the discharged fluid. Thereafter, for a given movement of the
plunger, the volume of sample which flows through the analyzing
volume is known. The volume can either be determined by measuring
the movement of the plunger or measuring the time the plunger is
moved if it is calibrated as a function of time. Although a syringe
pump is described, other types of pumps which can draw known
volumes of fluid through the capillary can be used.
[0038] Preferably, the capillary tubes are of rectangular
configuration. FIGS. 7 and 8 show a capillary tube that includes an
opaque coating 81 which is removed over an area 82, FIG. 7, or 83,
FIG. 8. In the embodiment of FIG. 7, the beam projects through the
window 82 which has a rectangular configuration to accept of the
beam 24. In FIG. 8, the slit masks the walls of the capillary tube
and prevents diffraction of light by the walls. A combination of
the two masks would confine the detected light to that emitted by a
particle traveling through the capillary to block out any stray
light.
[0039] An example of the operation of the apparatus to analyze a
sample containing particles which do not fluoresce, and particles
which intrinsically fluoresce or are marked or tagged to fluoresce,
at two different wavelengths, for example 580 nm and 675 nm, is now
provided with reference to FIGS. 9 and 10.
[0040] With the syringe pump plunger 73 extended to empty the pump,
the sample vial containing the particles is positioned to immerse
the end of the capillary 11 in the sample. The sample is then drawn
through the capillary by applying a control signal from the
controller 121 to start the pump 71. The controller receives the
command from processor 122. The pump 71 is driven at a constant
rate whereby the volume of sample passing through the analyzing
volume 19 can be measured by timing the counting period. After
sample has been drawn from the vial for a predetermined time to
assure that the new sample has reached the volume 19, the processor
begins to process the output 29 from the scatter photodetector and
the outputs 42 and 43 from the photodetectors and does so for a
predetermined time which will represent a known volume of sample
passing through the analyzing volume. The processing time is
schematically illustrated in FIG. 10A by the curve 123. FIG. 10B
illustrates the output pulses 28 from the scatter detector. It is
seen that there are individual particles which provide a trace 124
and a cluster of particles which provides a trace 126. FIG. 10C
shows traces 126 for particles which fluoresce at a first
wavelength, for example 675 nm. Of note is the fact that the
cluster 126 includes three such particles. FIG. 10D shows traces
127 for particles which fluoresce at another wavelength, for
example 750 nm. Of note is the fact that there is also one such
particle 128 in the cluster 126. The processor can call for a
number of analyzing cycles. Finally, when an analysis is completed,
the processor instructs the controller to open the valve 76 and
reverse the pump to discharge the analyzed sample into the waste
77. A new sample cuvet can then be installed and a new sample
analyzed. The processor can be configured to average the counts
over a number of cycles and to process the counts to provide
outputs representing the concentration of the various particles,
the number of particles, etc. Using suitable labels or markers one
can conduct viability assays and antibody screening assays or
monitor apoptosis.
[0041] Although the apparatus has been described for a two-color
analysis it can easily be modified for four-color analysis. This is
schematically shown in FIG. 11. The input light beam 24 impinges
upon the analyzing volume 19. The photodetector 27 and associated
lens 28 provide the scatter signal. The fluorescent light 31 is
focused by lens 32 to pass through three dichroic beam splitters
81, 82 and 83 which reflect light at three different wavelengths
through filters 86, 87 and 88 onto photodetectors 91, 92 and 93.
The light at the fourth wavelength, passed by the three dichroic
beam splitters 81, 82 and 83 passes through filter 94 onto
photodetector 96. Thus, up to four different particles which
intrinsically fluoresce or are labeled to fluoresce at four
different wavelengths can be analyzed by choosing the proper
reflecting wavelengths for the dichroic beam splitter and the
filters.
[0042] FIG. 12 schematically shows a system using a plurality of
light sources (not shown) projecting light beams 106, 107 and 108
to analyzing volumes 111, 112 and 113, which are at a predetermined
distance apart. The scattered light indicated by arrows 114, 116,
117 is detected by individual detecting systems of the type
described. The fluorescent light represented by arrows 118, 119 and
121 is detected by individual analyzing systems of the type
described above. This arrangement permits analyzing particles which
have been tagged with different labels by selecting the wavelength
of the light source to excite different fluorescent tags or
markers. Alternatively, the plurality of light beams may project
onto a single analyzing volume and the individual analyzing systems
receive the different fluorescent wavelengths.
[0043] Rather than sensing particles by light scattered by the
particles, a change in electrical current can detect the particles
as they travel past spaced electrodes disposed on opposite sides of
the flow path. Referring to FIG. 13, a capillary 11 is shown with
spaced electrodes 123 and 124 which extend into the capillary 11.
The electrodes are spaced along the capillary from the analyzing
volume 19. As the cell or particle flows between the electrodes,
the electrically conductive working fluid is displaced and the
resulting change in current (impedance) can be detected. This
method avoids any laser noise problem. It is usually most
convenient mechanically to place the electrodes along the fluid
flow path either before or after the point where the laser beam 24
impinges onto the capillary. This creates a timing problem in that
the impedance detector will detect a cell at a different point in
time than the fluorescence detector, and it is possible that a
second cell near the first may create a signal in the fluorescence
detector at the same time as the first cell creates a signal in the
impedance channel. This necessitates the use of a delay element to
shift one signal in time with respect to the other by an amount
equal to the distance between the two detectors divided by the flow
rate, so that the two signals from one cell become congruent. This
delay element may be implemented in hardware with a delay line or
circuit. FIG. 14 shows the output signal 126 from the impedance
cell sensor and the fluorescent signal 42 or 43 (FIG. 1) with a
delay 127 in the photomultiplier signal, whereby the signals are
correlated. This can also be implemented in software by sampling
the signal from each detector into its own data stream and then
shifting one data stream with respect to the other. A further
feature of this arrangement is that, if the physical distance
between the two detectors is known, then the actual flow rate can
be deduced by finding the delay that corresponds to the best
correlation between the two channels; this might be helpful when
trying to identify a clogged capillary.
[0044] As explained above, we have discovered that because the
illumination traversing the capillary is in the form of a thin
rectangular beam the detection volume is accurately defined by the
thickness of the beam and the walls of the capillary 11. With this
in mind, we conducted experiments in which the slits 33 and 34 were
eliminated. We found that the results obtained in tests of
variously labeled particles were comparable to those obtained with
slits. Referring to FIG. 17, an embodiment of the invention making
use of this discovery is schematically illustrated. The particle
detector includes a light source, for example a laser, whose output
is optically focused by the optics 23 to form a thin, flat beam 24
as described above. The beam traverses the capillary 11 to define
the detection volume 19. The scatter detector includes an off-axis
detector assembly including a collection lens 26a and a detector
27a. As much as possible of the emitted fluorescent light from the
tagged cells or particles is gathered or intercepted by an off-axis
detector assembly. It can be gathered by a condenser lens as
illustrated in FIG. 1. However, in the present embodiment, it is
collected by a light guide 141 which receives the light 142 and
conveys it to the beam splitter 143. The light beam is directed to
optical filters 144 and 146 and directly to detectors 147 and 148.
The output signals from the detectors and the scatter signals are
processed to provide particle counts, cell viability, antibody
screening, etc.
[0045] There has been provided a simple-to-use particle analyzing
apparatus for characterizing particles such as determining their
count, viability, concentration and identification. The analyzing
apparatus detects particles in a sample fluid flowing through a
capillary tube which has a sampling end for insertion into a sample
fluid, and a pump coupled to the other end for drawing sample
through the capillary. A light source is provided for projecting a
beam of light through a predetermined analyzing volume of the
capillary tube to excite fluorescence in particles that flow
through the volume. At least one detector is disposed to receive
the fluorescent light from excited particles and another detector
is disposed to provide a signal representing all particles which
flow through the analyzing volume. The output of said detectors
provides signals which can be processed to provide the
characteristics of the particles.
[0046] The foregoing descriptions of specific embodiments of the
present invention have been presented for purposes of illustration
and description. They are not intended to be exhaustive or to limit
the invention to the precise forms disclosed, and obviously many
modifications and variations are possible in light of the above
teaching. The embodiments were chosen and described in order to
best explain the principles of the invention and its practical
application, to thereby enable others skilled in the art to best
use the invention and various embodiments with various
modifications as are suited to the particular use contemplated. It
is intended that the scope of the invention be defined by the
claims appended hereto and their equivalents.
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