U.S. patent application number 16/110285 was filed with the patent office on 2018-12-20 for particle detection using reflective surface.
The applicant listed for this patent is CAPTL LLC. Invention is credited to J. Paul Robinson, Masanobu Yamamoto.
Application Number | 20180364159 16/110285 |
Document ID | / |
Family ID | 57007697 |
Filed Date | 2018-12-20 |
United States Patent
Application |
20180364159 |
Kind Code |
A1 |
Yamamoto; Masanobu ; et
al. |
December 20, 2018 |
Particle Detection Using Reflective Surface
Abstract
An example assembly includes a target holder that retains a
target in a detection region. Areflective surface reflects at least
part of a focused spot of light to provide resultant light. An
irradiation system irradiates at least part of the detection region
with the focused spot of light. A motion system causes motion of
the focused spot of light relative to the reflective surface. A
detection system detects the resultant light. An example device,
e.g., a lab-on-chip, includes a substrate, a sample inlet, and a
reflective grating. The grating is retains a fluidic sample in a
detection region fluidically connected to the sample inlet. The
detection region is operatively arranged with respect to the
reflective grating so that at least a portion of light passing
through the detection region towards the reflective grating also
passes through the detection region after reflecting off the
reflective grating.
Inventors: |
Yamamoto; Masanobu; (West
Lafayette, IN) ; Robinson; J. Paul; (West Lafayette,
IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CAPTL LLC |
West Lafayette |
IN |
US |
|
|
Family ID: |
57007697 |
Appl. No.: |
16/110285 |
Filed: |
August 23, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15564117 |
Oct 3, 2017 |
10060850 |
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PCT/US16/25650 |
Apr 1, 2016 |
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16110285 |
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62142818 |
Apr 3, 2015 |
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62211452 |
Aug 28, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2015/1006 20130101;
B01L 2300/168 20130101; G01N 21/645 20130101; G01N 15/147 20130101;
G01N 21/47 20130101; B01L 3/502715 20130101; G01N 21/55 20130101;
B01L 2400/0409 20130101; G01N 2021/6439 20130101; G01N 2021/6463
20130101; G01N 21/4788 20130101; B01L 2400/049 20130101; B01L
2300/0654 20130101; G01N 15/1429 20130101; B01L 2400/0406 20130101;
G01N 15/1484 20130101; G01N 15/1425 20130101; G01N 15/1434
20130101; B01L 2300/0806 20130101 |
International
Class: |
G01N 21/47 20060101
G01N021/47; B01L 3/00 20060101 B01L003/00 |
Claims
1. An assembly, comprising: a target holder configured to retain a
target in a detection region, wherein the target holder comprises a
reflective surface configured to reflect at least part of a focused
spot of light to provide resultant light; an irradiation system
configured to irradiate at least part of the detection region with
the focused spot of light; a motion system configured to cause
motion of the focused spot of light relative to the reflective
surface; and a detection system configured to detect the resultant
light.
2. The assembly according to claim 1, further comprising a
sample-delivery system configured to apply a fluidic sample
comprising the target to the at least part of the detection
region.
3. The assembly according to claim 2, wherein: the reflective
surface comprises a reflective grating; the irradiation system is
further configured to provide the focused spot of light, which
comprises light of a predetermined wavelength; the irradiation
system comprises a lens having a predetermined numerical aperture
and being arranged to focus the light of the predetermined
wavelength to provide the focused spot of light; the reflective
grating has a pitch exceeding a reference pitch substantially equal
to a result of dividing the predetermined wavelength by twice the
numerical aperture; the fluidic sample comprises a fluid having a
predetermined refractive index; and the reflective grating includes
at least one groove having a depth substantially between a first
reference value and a second reference value, the first reference
value being the result of dividing the predetermined wavelength by
the product of sixteen and the refractive index and the second
reference value being substantially three times the first reference
value.
4. The assembly according to any one of claims 1-3, wherein: the
detection system comprises at least one split photodetector
configured to provide a plurality of detection signals; and the
assembly further comprises a data-processing system configured to
determine, based at least in part on at least two of the plurality
of detection signals, at least one of a sum signal, a transverse
push-pull signal, or a radial push-pull signal.
5. The assembly according to claim 4, wherein: the irradiation
system is further configured to provide the focused spot of light,
which comprises light of a predetermined wavelength; and the
detection system further comprises at least one optical detector
configured to detect light of a different wavelength than the
predetermined wavelength and to provide a detection signal
corresponding to the light of the different wavelength.
6. The assembly according to claim 5, further comprising a gating
system configured to provide a particle signal associated with a
temporal overlap between at least one of the plurality of detection
signals and the detection signal corresponding to the light of the
different wavelength.
7. The assembly according to any one of claims 1-6, wherein: the
reflective surface comprises a reflective grating having at least
one groove; and the motion system is further configured to cause
relative motion of the focused spot of light substantially across
the at least one groove.
8. A device, comprising: a substrate; a sample inlet associated
with the substrate; and a reflective grating associated with the
substrate, wherein: the reflective grating is configured to retain
a fluidic sample in a detection region; the detection region is
fluidically connected to the sample inlet; and the detection region
is operatively arranged with respect to the reflective grating so
that at least a portion of light passing through the detection
region towards the reflective grating also passes through the
detection region after reflecting off the reflective grating.
9. The device according to claim 8, further including a cover
spaced apart from the reflective grating and arranged so that the
detection region is at least partly between the reflective grating
and the cover, wherein the cover is at least partly transparent to
the at least a portion of the light.
10. The device according to either claim 8 or 9, further comprising
a reservoir fluidically connected to the detection region and
spaced apart from the sample inlet.
11. The device according to any one of claims 8-10, wherein the
reflective grating comprises at least one groove, and the at least
one groove comprises at least two of: a spiral groove portion, a
straight groove portion, or a circular groove portion.
12. The device according to any one of claims 8-11, wherein: the
sample inlet is configured to receive a provided sample; and the
device further comprises a filter element operatively arranged to
filter the provided sample to provide the fluidic sample.
13. The device according to any one of claims 8-11, wherein the
substrate is a disc-format substrate.
14. The device according to any one of claims 8-11, wherein: the
sample inlet is one of a plurality of spaced-apart sample inlets of
the device; the detection region is one of a plurality of detection
regions of the device; and individual detection regions of the
plurality of detection regions are fluidically connected to at
least one of the plurality of sample inlets.
15. A method, comprising: disposing a fluidic sample comprising a
target over a reflective surface; irradiating the reflective
surface using a spot of light; and detecting resultant light from
the reflective surface.
16. The method according to claim 15, further comprising causing
the fluidic sample to flow through a flow channel across the
reflective surface and causing the spot of light to move with
respect to the reflective surface at least partly across the flow
channel.
17. The method according to either claim 15 or 16, wherein the
reflective surface is associated with a disc; and wherein the
method further comprises: rotating the disc, wherein the disc moves
with respect to the spot of light during the rotating and the
reflective surface comprises at least one track; irradiating the
reflective surface during the rotating; and causing the spot of
light to move to follow the track of the reflective surface.
18. The method according to any one of claims 15-17, further
comprising: applying a dye to the fluidic sample before the
disposing; and detecting light emitted by the dye in response to
the irradiating using the spot of light.
19. The method according to any one of claims 15-18, wherein the
target comprises at least two portions having respective, different
refractive indices.
20. The method according to claim 19, wherein the fluidic sample
has a refractive index different from each of the refractive
indices of the target.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of, and claims priority
to and the benefit of, U.S. patent application Ser. No. 15/564,117,
filed Oct. 3, 2017, and entitled "Particle Detection Using
Reflective Surface" (filed under Atty. Docket No. C096-0008US),
which is a national-stage application of International Application
No. PCT/US2016/025650, filed Apr. 1, 2016(filed under Atty. Docket
No. C096-0008PCT), which claims priority to, and the benefit of,
bith U.S. Provisional Patent Application Ser. No. 62/142,818, filed
Apr. 3, 2015, and entitled "Particle Detection Using Reflective
Phase Grating" (filed under Atty. Docket No. C096-0008USP1), and
U.S. Provisional Patent Application Ser. No. 62/211,452, filed Aug.
28, 2015, and entitled "Photon Detection" (filed under Atty. Docket
No. C096-0009USP1), the entirety of each of which is incorporated
herein by reference.
BACKGROUND
[0002] Various techniques are used for investigation of microscopic
particles. For example, electron micrographs can be taken of
nanostructures in vacuum. Brownian motion due to microparticles in
a suspension can be detected. Flow cytometers can be used to detect
light scattered by nanoparticles. However, these techniques are not
suitable for, e.g., clinical diagnostic testing.
BRIEF DESCRIPTION OF THE DRAWINGS
[0003] Features and advantages of various aspects will become more
apparent when taken in conjunction with the following description
and drawings. Identical reference numerals have been used, where
possible, to designate identical features that are common to the
figures. The attached drawings are for purposes of illustration and
are not necessarily to scale.
[0004] FIG. 1 shows an elevational cross-section of an example
lab-on-chip device (LOC) and related components.
[0005] FIG. 2 shows an example LOC in a disc format having a spiral
track.
[0006] FIG. 3 shows an example LOC in a disc format having a spiral
track.
[0007] FIG. 4 shows an example plan view of grooves on an example
LOC.
[0008] FIG. 5 shows an example plan view of grooves on an example
LOC.
[0009] FIG. 6 shows components of an example LOC in a disc format
and configured, e.g., for analysis of blood samples.
[0010] FIG. 7 shows a perspective and partial cross-sections of
components of an example LOC, and example groove patterns.
[0011] FIG. 8 shows a plan-wise cross-section of an example
LOC.
[0012] FIG. 9 shows an elevational cross-section along the line
IX-IX in FIG. 8.
[0013] FIG. 10 shows an annotated graphical representation of a
photograph of an example LOC in a disc format.
[0014] FIG. 11 shows a schematic plan view of an example reflective
grating, and example measurement techniques.
[0015] FIG. 12 shows a perspective of components of an example
optical detection system, e.g., configured for detecting or
measuring targets such as microvesicles.
[0016] FIG. 13 shows a schematic of an example optical detection
system.
[0017] FIG. 14 shows a block diagram of an example
target-measurement system.
[0018] FIG. 15 shows components of an example measurement system,
including sample carrier and optical detection components.
[0019] FIG. 16 shows a components of an example measurement system,
including sample carrier and optical detection components.
[0020] FIG. 17 is a circuit diagram of components of an example
amplifier.
[0021] FIG. 18 is a block diagram showing components of an example
measurement system, including data-processing components useful
with various examples.
[0022] FIG. 19 shows components of an example measurement system,
including gating components.
[0023] FIG. 20 shows simulated tracking signals and example gating
components of an example measurement system.
[0024] FIG. 21 shows measured and representative example tracking
signals, and representative example gating signals.
[0025] FIG. 22 is a schematic of an example target-measurement
system.
[0026] FIG. 23 is a schematic elevational cross-section of an
example LOC.
[0027] FIG. 24 is a flow diagram of an example process for
analyzing a sample.
[0028] FIG. 25 is a flow diagram of an example process for
detecting a target, e.g., in a sample.
[0029] FIG. 26 is a flow diagram of an example process for
detecting a target.
[0030] FIG. 27 is a flow diagram of an example process for
detecting a target.
[0031] FIG. 28 is a flow diagram of an example process for
analyzing a sample.
[0032] FIG. 29 shows measured data of 100 nm gold
nanoparticles.
[0033] FIG. 30 shows measured data of 100 nm gold
nanoparticles.
[0034] FIG. 31 shows measured data of particles in a groove of a
reflective grating.
[0035] FIG. 32 shows measured data of particles on a land of a
reflective grating.
[0036] FIG. 33 shows measured data of particles on substantially
flat reflective surface ("mirror").
[0037] FIG. 34 is an example plot of modulation amplitude by
optical path difference.
DETAILED DESCRIPTION
Overview
[0038] In the fields related to life sciences such as genetics,
immunology, molecular biology, and environmental science, optical
techniques are widely used to analyze microparticulate samples such
as living cells, yeast, and bacteria. For example, particles or
cells from 500 nm up to 50 .mu.m can generally be measured in flow
cytometry. In some examples, a label made of a fluorescent
substance is attached to the surface of a cell to be analyzed.
Next, laser light is radiated towards a predetermined position to
irradiate the cell. Then, forward-scattered light and
side-scattered light, which are generated due to the size and
structure of each cell, and fluorescence, which is generated by
excitation due to the light irradiation, are observed. In the case
of observing fluorescence from a cell, a configuration for spectral
analysis of the fluorescence condensed in a direction other than an
irradiation path of excitation light is widely used to avoid
adverse effects of transmitted or scattered excitation light.
Fluorescent substances to be attached or combined for each type of
cells are known. Accordingly, the wavelength and intensity of the
fluorescence are observed and the intensity component to be
superimposed is compensated to thereby identify the type of each
cell.
[0039] There is a need for detecting and measuring sub-structures
within cells or produced by cells. For example, cells can produce
or receive "vesicles" or "microvesicles," lipid bilayers
surrounding proteins, DNA, RNA, mRNA, miRNA, proteins, cytotoxins,
waste from metabolic processes, or other components of a cell or
substances that can be found within a cell. Vesicles can be, e.g.,
4 nm-8 .mu.m, or 100 nm-1 .mu.m, in mean diameter. Some example
vesicles include protein-enriched micro-vesicles. By
"protein-enriched micro-vesicle" is meant a fusogenic structure
that includes an amount of one or more target proteins in a lipid
bilayer envelope. As used herein, the term "fusogenic" refers to
the property of the micro-vesicle which provides for the fusion of
the membrane of the micro-vesicles to the membrane of the target
cell. Fusogenic micro-vesicles are capable of fusion with the lipid
bilayer membrane of a target cell to deliver their contents, e.g.,
the target protein(s), into the cell. As used herein, a "target"
can be a microvesicle or other microparticulate structure to be
measured. As used herein, a "sample" can be a solid, liquid, or
gaseous aggregate of one or more targets. A sample can additionally
include non-target substances. For example, a blood sample can
include microvesicle targets and plasma; the plasma in this example
is a non-target substance. In other examples, the sample can
include water and the targets can be suspended in the water. In
still other examples, the sample can include a resist, solvent or
other chemical used in semiconductor fabrication, or other
chemical, and the target can include a nanoparticle or particulate
used in industrial applications. Some examples permit measuring
targets, e.g., nanoparticles, having sizes below the diffraction
limit of prior flow-cytometry systems.
[0040] Various aspects relate to a quantitative microvesicle (MV)
measurement platform. Some examples use a focused light spot, e.g.,
a scanning laser spot, on a reflective surface. The reflective
surface can include a grating, e.g., a reflective grating. The
terms "optical" and "light" are not limited to the visible range
of, e.g., 400 nm-700 nm unless otherwise expressly specified. The
term "reflective" can describe, but does not require, 100%
reflection in any particular wavelength band.
[0041] Some example targets can include microvesicles, which can be
intracellular or extracellular. Extracellular vesicles (EVs) can
include, e.g., substantially spherical EVs having diameters between
30 nm and 1000 nm; tubular or other substantially elongated EVs
between 1 .mu.m and 5 .mu.m in size; or EVs between 4 nm and 500 nm
in size. Example targets can include high-density lipoprotein (HDL)
EVs of .about.10 nm in size or low-density lipoprotein (LDL) EVs of
.about.22 nm in size. Other targets can include large fragments up
to about 8 .mu.m in size.
[0042] In some examples, microvesicles can be present in human
blood plasma in concentrations from 10.sup.4-10.sup.12 per mL. In
some examples, over 80% of vesicles in a sample can have diameters
less than 100 nm. Vesicles can have an index of refraction n, e.g.,
of about 1.40, compared to silica at n.apprxeq.1.45 and polystyrene
at n.apprxeq.1.61. In some prior flow-cytometry schemes,
microvesicles such as liposomes having diameters .apprxeq.100 nm
can be difficult to distinguish from threshold noise in the
system.
[0043] A quantitative measurement platform, e.g., for
microvesicles, can provide nanoparticle detection on reflective
phase grating implemented using a focused scanning laser spot on a
reflective phase grating in a microfluidic channel. Various
examples of such measurement platforms, systems, and techniques are
discussed below with reference to FIGS. 2-31. Various examples of
simulated and measured data are described below with reference to
FIGS. 20, 21, and 29-33. Various example techniques and systems
described herein can provide quantitative measurements of
particles, e.g., 10 nm-1000 nm in mean diameter. 10 nm is beyond
the resolution of many prior optical detection systems. Some prior
schemes for high-resolution imaging or particle detection include
electron microscopes, Brownian-motion detection of particles in
suspension (e.g., NANOSIGHT), and flow cytometry. However, although
some electron microscopes may be able to observe 10 nm targets,
electron microscopes are unable to observe living cells or cell
components such as vesicles since electron microscopy involves
drying the sample and placing it in a vacuum chamber. Moreover,
scattering measurements from flow cytometers often cannot detect
particles smaller than the diffraction limit at a particular
wavelength, e.g., smaller than about 500 nm.
[0044] Various aspects herein permit measuring targets, e.g., small
particles or structures such as vesicles or nanoparticles. Various
aspects permit performing such measurements without damaging living
cells or microorganisms that are being measured or the area
producing the targets being measured, e.g., a living cell. Various
aspects can provide quantitative measurements of targets. Various
aspects can be used in a clinical environment as a disposable chip
or disc (individually or collectively a "lab on chip" or LOC,
whether in chip format or otherwise) including sample
preparation/processing/target measurement/disposal, e.g., without
the need for free liquid reagents or suspensions. Various aspects
use fluorescence to further measure properties of specific targets.
Example targets can be substantially equal to, e.g., 100 nm, 70 nm,
50 nm, or 30 nm in diameter. Targets having other diameters can
also be measured.
[0045] Various aspects include diffraction detection of targets on
reflective phase grating structures. Such structures can provide
double phase modulation of light traveling through the targets.
Various phase gratings provide a microscopic reference scale of
sinusoidal signal characteristics.
[0046] Some examples herein provide quantitative measurement method
of micro vesicles or any kind of nanoparticles, e.g., in solution.
Some examples herein permit clinical testing, e.g., using closed
system in a disposable chip or disc format. Example LOCs can
include sample preparation, processing, and disposal in a single
LOC. Some examples herein permit identifying the origin of detected
microvesicles, e.g., using fluorescent tagging.
[0047] Some examples provide detection of a particle on a
reflective surface, e.g., a substantially flat reflective surface
such as a mirror, by detecting light absorption or refraction due
to a target. Some examples provide diffraction detection of a
particle on a reflective phase grating structure, which structure
can double the phase modulation sensitivity of a detection system.
The phase grating can provide a microscopic reference scale against
which targets can be measured. Some examples use a phase grating
having a height or depth, e.g., substantially equal to .lamda./8n,
e.g., 40 nm, or substantially within the range
.lamda./16n-3.lamda./16n, where .lamda. is a wavelength of incident
optical radiation and n is a refractive index of the medium, e.g.,
a fluidic sample. This configuration can provide higher amplitude
or improved signal-to-noise ratio (SNR) of push-pull signals. In
some examples, .lamda..apprxeq.405 nm, NA.apprxeq.0.85, and a cover
having a thickness of about 0.1 mm is placed over the sample. In
some examples, relatively shorter wavelengths or relatively higher
numerical apertures (NAs) can be used to detect relatively smaller
particles. In some examples, .lamda..gtoreq..about.405 nm can be
used with polymer disposable LOCs.
[0048] In some examples, a reflective phase grating can be used and
a focused scanning laser spot can be applied. In some examples, an
LOC can have a disc or rectangular style (e.g., slide glass)
format.
Illustrative Examples
[0049] FIG. 1 shows example of a lab-on-chip device (LOC). The LOC
is an example of a sample carrier, a device for retaining or
transporting a sample for measurement as described herein. In some
examples, as graphically represented by the double-headed "light"
arrow, a target in a detection region is irradiated by a focused
spot of light. The focused spot of light can have a range of
irradiation wavelengths, of which the peak power, or mean, median,
or other representative wavelength can be an irradiation
wavelength. Resultant light is detected concurrently with or
subsequent to irradiation. Resultant light can include, but is not
limited to, light reflected by the target, light emitted by the
target, or light reflected or diffracted by a reflective surface,
e.g., a reflective grating, in the detection region. References
herein to detecting or measuring resultant light can include
producing signals, e.g., analog or digital electrical signals,
representing at least some of the resultant light, unless expressly
indicated.
[0050] In some examples, dimension A can be .about.1.1 mm,
dimension B can be .about.1 .mu.m or .about.2 .mu.m, dimension C
can be .about.100 .mu.m, or dimension D can be .about.500 .mu.m. In
some examples, a reflective surface, e.g., a reflective grating,
can be directly overcoated with a layer, e.g., a clearcoat
protective layer. Targets, e.g., in a fluid sample, such as a
liquid sample, can be deposited onto the reflective layer or the
clearcoat layer for measurement.
[0051] The fluid sample can be added through a sample inlet
("INLET"), e.g., after removing a seal ("SEAL"). The inlet can be
associated with a substrate of the LOC, e.g., can be arranged in,
on, over, or through the substrate. The inlet can include or be
associated with a filter ("FILTER"). The inlet can convey at least
some of the fluid sample, e.g., in the direction indicated by the
open arrow, to a fluid channel ("CHANNEL") configured to convey at
least some of the fluid sample towards (e.g., over) the reflective
surface. The reflective surface can be associated with the
substrate of the LOC, e.g., can be arranged on, in, or over the
substrate. The sample inlet can be fluidically connected to the
channel. As used herein, components that are "fluidically
connected" are structured and arranged to permit a relevant fluid
to flow between them. Fluidically connected components can be
directly connected, or can be connected via intermediaries (e.g.,
as inlet area 806 and 810, FIG. 8 are fluidically connected via
fluid channel 808, as discussed below).
[0052] Table 1 lists attributes of sample carriers according to
various examples herein. This disclosure expressly contemplates
each possible combination of attributes formed by selecting one
attribute value from each row of Table 1. Sample carriers can
include, e.g., disc, slide, or chip formats, as described
herein.
TABLE-US-00001 TABLE 1 Category Attributes Reflective Surface
"Mirror" (substantially flat); grating Cover between Present;
absent optics and sample Cover material Glass; plastic Target
carrier vacuum; air; dried formerly-liquid sample; stationary
liquid; flowing fluid Target type Homogeneous refractive index;
heterogeneous refractive index Material between Present; absent
reflective surface and target Format Disc; slide; chip
[0053] In addition, this disclosure expressly contemplates
detecting resultant light, e.g., light reflected by or emitted from
or over a sample carrier having any combination of attributes
listed in Table 1. This disclosure expressly contemplates detecting
such light using detection systems such as those listed in Table 2,
e.g., using respective systems according to each possible
combination of one attribute value per row. That is, any selection
of one attribute per row of Table 1 can be combined with any
selection of one attribute per row of Table 2. In Table 2,
"focusing" refers to adjusting spot position substantially normal
to a sample carrier, and tracking refers to adjusting spot position
substantially laterally with respect to a sample carrier, e.g., to
track a groove such as those shown in FIGS. 2-7.
TABLE-US-00002 TABLE 2 Category Attributes Emission Ultraviolet;
visible; infrared wavelength FWHM Specific values for, e.g.,
Gaussian or flat-top profiles (see, e.g., Table 3) Numerical
Specific value (see, e.g., Table 3) aperture Type Laser; collimated
beam Focusing Present; absent Tracking Present; absent Detection
Substantially the same as an emission wavelength; wavelength
substantially different from an emission wavelength or from any
emission wavelengths Motion Stationary irradiation spot; moving
irradiation spot
[0054] Examples of specific attribute values are shown in Table 3.
This disclosure expressly contemplates using any of the attribute
values shown in Table 3, or any combination thereof, with any
combination of corresponding attributes listed in Table 1 or Table
2. In some examples, the emission wavelength can be, e.g., an
irradiation wavelength as described herein. The emission wavelength
can be selected to excite fluorescent emissions from a target,
though this is not required. Fluorescence is described herein with
reference to, e.g., FIG. 13-15, 19, or 26.
TABLE-US-00003 TABLE 3 Category Attributes Cover material
Polycarbonate; glass Target carrier Flowing liquid; flowing gas
Material between A spin-coating over a reflective grating; a glass
or reflective surface plastic slide having the reflective material
on the and target opposite side from the surface that receives the
sample. Format A microscope slide having a reflective coating; a
fluidic chip including a flow cell, flow chamber, or other flow
channel. Emission 785 nm; 650 nm; 405 nm wavelength FWHM 1040 nm;
557 nm; 245 nm Numerical 0.45; 0.60; 0.85 aperture Focusing Using a
lens to focus a laser beam on a flow channel Detection The
wavelength of fluorescence of a dye added to wavelength the sample
to indicate presence of targets Motion A substantially stationary
irradiation spot used with a flowing fluidic sample; a moving
irradiation spot scanned, e.g., raster-scanned or scanned linearly,
across a flow channel.
[0055] FIG. 2 shows an example disc (the spellings "disk" and
"disc" are interchangeable herein) with a hole in the center. A
spiral track, e.g., a land or groove, is shown dotted and has a
fixed pitch p across the surface of the disc. "Lands" include
non-recessed or raised areas; "grooves" include recessed or lowered
areas (or non-raised areas, in examples including raised lands).
Shown superimposed on the spiral in heavy lines is a cross-section.
In the illustrated example, the track follows grooves that are
separated by lands of the same widths as the grooves. In this
example, the track does not have pits and lands burned along the
length of the track as a data-bearing BLU-RAY disc does. In other
examples, the track includes some data-bearing areas. In some
examples, a single disc carries data-bearing tracks and
non-data-bearing tracks. Various examples of reflective phase
gratings are discussed and shown herein, e.g., the groove in FIG.
2. However, the orientation(s) of grooves or lands in a reflective
phase grating and the orientation(s) of laser spot travel (if any)
are not constrained. In some examples, the laser spot moves
substantially radially rather than substantially tangentially or
substantially along a spiral. As used herein, the term "disc" does
not require that the disc be perfectly circular. For example, a
disc-format LOC can include a disc trimmed to fit within a
rectangle, as for business-card sized CD-ROMs having two shorter,
rounded edges and two longer, straight edges.
[0056] FIG. 3 shows an example disc in which the track does not
extend across the full radius of the disc. The track covers a
portion of the disc between an inner radius and an outer radius.
Multiple tracks can be arranged in different radial zones of the
disc. Multiple tracks can be interleaved, e.g., starting from the
same radius but 180.degree. around the disc from each other.
[0057] LOCs, e.g., in disc format, can be manufactured having
desired numbers of tracks, track pitches, radii, interleaving,
groove width, land width, or orientation, using CD, DVD, or BLU-RAY
duplication technology. The pitch of the tracks can be selected
based on the size of the targets to be detected. A single LOC can
include multiple regions, spaced apart or overlapped. Each region
can have a respective groove pitch, depth, or pattern, the same as
or different from at the pitch, depth, or pattern of at least one
other region, respectively. For example, a disc-format LOC can
include two spaced-apart concentric spiral grooves, one having a
relatively narrower pitch (e.g., for detecting relatively smaller
particles) and one having a relatively wider pitch (e.g., for
detecting relatively larger particles).
[0058] In some examples, the negative pattern of the lands and
grooves of the track can be transferred to a stamper, e.g., made of
nickel. The stamper can be positioned with respect to an injection
mold so that polycarbonate (PC) or another material
injection-molded in that mold takes on the desired pattern of lands
and grooves. The molded polycarbonate can then be sputter-coated,
e.g., with aluminum or silver, e.g., .about.18 nm, .about.20 nm,
.about.50 nm, or .about.100 nm thick, to form a reflective surface
that can be read by a laser. Compared to a relatively thinner
reflective surface, a relatively thicker reflective surface can,
e.g., have increased reflectivity, exhibit reduced autofluorescence
(AFL); or experience reduced thermal damage during measurement.
Increased reflectivity or reduced AFL can increase measurement
sensitivity, and reduced thermal damage can increase lifetime of a
sample carrier. In some examples, the reflective layer can be,
e.g., overcoated with a transparent thin-film hard-coat. In some
examples, the reflective layer can comprise, e.g., anodized Al or
another anodized substance.
[0059] In some examples, phase transition mastering (PTM) can be
used to manufacture the stamper. PTM involves developing the
desired pattern in an inorganic photoresist over a silicon wafer.
The wafer can then be etched to prepare the stamper.
[0060] In some examples, laser writing such as used in dye-based
recordable DVDs can be used to manufacture discs. The desired
pattern of grooves can be burned into a dye layer of such a disc
using a laser.
[0061] Above-described manufacturing techniques for discs can be
used to prepare spiral tracks or other shapes or groove or pit
patterns in non-circular substrates, e.g., substantially
quadrilateral (e.g., rectangular) shapes used for lab-on-chip
devices (LOCs). These devices can be scanned with focused laser
light, as described herein. LOCs, e.g., in disc or chip format or
other formats described herein, can be reusable or disposable. For
example, a single reflective grating can be repeatedly washed and
reused for each of multiple samples in succession, or multiple
gratings can be used for respective samples tested in succession.
As used herein, the term "focused spot" and similar terms refer to
a spot produced by an irradiation system configured to focus the
spot to a desired extent on a surface. The term "focused spot" does
not constrain the tolerances of components of the irradiation
system or other systems herein.
[0062] One or more portions of a disc or other LOC can have
respective groove patterns (e.g., spiral, straight, concentric
circles, or other curves). Different portions can have different
patterns or the same pattern, in any combination. In an example, at
least two portions of a disc-shaped LOC have spiral groove patterns
with different pitch values (p). This can permit effectively
detecting targets in different size ranges. A LOC (disc or
otherwise) can have one or more sample inlets, vacuum outlets
(e.g., ports or channels to which vacuum can be applied to move
sample), or one or more detection regions. Example LOCs according
to various aspects are described herein with reference to in FIGS.
1-10 and 16. For example, the grooves can have a depth of 40 nm and
a pitch of, e.g., .about.500 nm (e.g., 250 nm or 250.5 nm width for
the grooves or the lands).
[0063] FIG. 4 shows an example plan view of spiral grooves, e.g.,
forming a reflective phase grating, e.g., on an LOC such as a
rectangular LOC. As in FIG. 2, the groove profile is represented by
the cross-section in heavy lines.
[0064] FIG. 5 shows an example plan view of straight grooves, e.g.,
forming a reflective phase grating, e.g., on an LOC such as a
rectangular LOC. As in FIG. 2, the groove profile is represented by
the cross-section in heavy lines.
[0065] FIG. 6 shows an example disc and filter system configured
for separation of plasma containing microparticles, e.g.,
microvesicles. The illustrated filter system can alternatively be
used with other configurations of LOC or reflective grating, e.g.,
glass slides. Samples can flow through channels in the LOC, e.g.,
due to vacuum pressure via the illustrated vacuum channel, due to
capillary force (e.g., in air, in vacuum, or in another
atmosphere), or due to other forces.
[0066] Any number of sample inlets, vacuum channels, or detection
regions (e.g., measurement zones) can be used. Vacuum can also or
alternatively be applied via vacuum ports. Samples or vacuum can be
applied before or during relative motion of the laser spot and
disc. Capillary channels, e.g., micropillar channels, or other
features can be used to draw sample into the detection regions.
[0067] Micropillars, inlets, channels, ports, and other features
shown can be formed using stampers in an injection mold, e.g., as
for the reflective phase grating, or can be formed using etching,
machining, or other techniques. Multiple layers of a disc or other
LOC can be prepared separately and affixed, or layers can be built
up or formed together or one after another, in any combination.
[0068] Any number of the, e.g., eight samples can be applied
substantially simultaneously, or at different times. Any number of
samples can be measured in a single rotation of the disc or other
single laser pass across the reflective phase grating, or in
multiple passes, in any combination.
[0069] FIG. 7 shows another example of an LOC 700. In some
examples, LOC 700 includes multiple fluid tanks, e.g., four tanks
702, 704, 706, and 708 (or three tanks 702, 704, and 708, or any
combination of any number of the four tanks). For example, tank 702
can include a 20 .mu.L tank to which sample, e.g., blood, is added,
e.g., through the top, end, or side of LOC 700 or tank 702. Tank
704 can include cell-separation or other filtering features, e.g.,
micropillars. An example micropillar arrangement is shown in plan
in inset 710, with arrows indicating the direction of sample flow.
In some examples, tank 706 can hold dye(s) or other substances to
enhance optical detection of targets. Tank 708 can be, e.g., a
reservoir or waste tank. In some examples, tank 708 cna include an
outlet 712, e.g., configured to connect to a vacuum source.
[0070] In detection region 714, targets (illustrated as dots) can
be irradiated. Resultant light from detection region 714 can be
detected. Detection region 714 can include grooves 716, graphically
represented as lines.
[0071] In various examples, a disc or other LOC can have lands L
between grooves 716 of depth Dg. Targets can be in the grooves or
on the lands. In some examples, the groove depth Dg is
substantially equal to .lamda./8n to increase magnitude of the
tangential push-pull (PP) signals from the phase grating. In some
examples, the track pitchp is set to at least twice the size (e.g.,
mean diameter) of targets to be detected. In some nonlimiting
example configurations of grooves 716, an individual groove 716 can
be straight or curved, or can have both straight segments and
curved segments. The grooves 716 can be connected, e.g., as part of
a spiral, or can be separate. An individual groove 716 can have a
substantially constant cross-sectional profile (e.g., depth, width,
and area), but this is not required.
[0072] FIG. 8 shows a plan-wise cross-section of an example LOC 800
having a substrate 802, e.g., including glass or polymer. A sample
inlet 804, e.g., a hole in a cover over the substrate, is
fluidically connected to inlet area 806. Inlet area 806 is
fluidically connected to fluid channel 808, which can be or include
a detection region in some examples. Fluid channel 808 is
fluidically connected to reservoir 810. In some examples, fluid can
flow from sample inlet 804, through inlet area 806, fluid channel
808, and into reservoir 810, e.g., under capillary pressure.
[0073] FIG. 9 shows an elevational cross-section of example LOC 800
along the line IX-IX in FIG. 8. Sample inlet 804 in this example is
a through hole formed in a substrate 902, e.g., glass such as
non-alkali glass. The substrate 902 can have a thickness of, e.g.,
0.7 mm. Through-hole sample inlet 804 in this example also passes
through a reflective layer 904 and oxide layer 906. Oxide layer 906
can represent, e.g., an electrically insulating layer. Reflective
layer 904 can, e.g., comprise aluminum or another metal. Reflective
layer 904 can have a thickness of, e.g., .about.18 nm, .about.20
nm, .about.100 nm, or .about.200 nm. The illustrated reflective
layer 904 is substantially flat; however, this is not limiting. A
reflective grating such as described herein with reference to FIGS.
1-7 can be used as or in place of at least part of the reflective
layer 904. Oxide layer 906 can, e.g., comprise SiO.sub.2. Oxide
layer 906 can have a thickness of, e.g., 15 nm. Spacers 908
separate the oxide layer 906 from a cover 910, thereby defining a
channel 912. Spacers 908 can comprise, e.g., resin. Spacers 908 can
have a thickness of, e.g., 1 .mu.m. Cover 910 can comprise, e.g.,
glass, such as non-alkali glass. Cover 910 can have a thickness of,
e.g., 0.1 mm. Targets in channel 912 can be irradiated through
cover 910, and resultant light can pass through cover 910 back to a
detection system, as represented by the double-headed "LIGHT"
arrow.
[0074] In FIGS. 8 and 9, in some nonlimiting examples, the
illustrated dimensions can be as shown in Table 4. In addition,
sample inlet 804 can have a diameter of 0.5 mm. Sample inlet 804 is
shown tapered in FIG. 9, but can alternatively have a substantially
constant diameter.
TABLE-US-00004 TABLE 4 Label Example Value (mm) A 25.0 B 12.5 C 5.5
D 7.0 E 6.0 F 6.5 G 8.0 H 10.0 I 1.0 J 6.0 K 4.0 L .gtoreq.0.5 M
0.5
[0075] Various examples permit measuring a small number of
microparticles, e.g., one microparticle at a time. Some examples
use a flow cell and a small spot to illuminate one particle at a
time in the spot. For example, a flow cell having dimension I
(channel 808 width) of approximately 1 .mu.m can provide a
microvesicle concentration of .about.10 .mu.m.times.10 .mu.m area
per particle. A 0.4-.mu.m spot can be used. These parameters can
provide individual measurement of one particle in a spot at a time.
By contrast, some prior flow cells and optics illuminate 800-1000
microvesicles at the same time. This can make it difficult to
distinguish individual particles and fluorescence thereof. Some
prior schemes dilute samples .about.1000.times., but this can
reduce microvesicle concentration to 1/100 .mu.m.sup.3, making id
difficult to measure the microvesicles. Some examples herein do not
have these limitations. Some examples can use a channel width
(e.g., dimension I) of 2 .mu.m or 2 mm.
[0076] FIG. 10 shows an annotated graphical representation of a
photograph of an example LOC 1000 that was prepared in a disc
format. An optical disc was prepared from a customized master
having a track pitch p=500 nm. The master was prepared with phase
transition mastering (PTM). A nickel stamper was manufactured and
the test disc was injection molded and stamped with the master.
Injection molding of transparent polycarbonate was performed, then
the polycarbonate was cooled, then aluminum was sputtered over the
polycarbonate.
[0077] In the depicted constructed example, the disc had a single
spiral groove 1002 recessed below the majority of the surface of
the disc. The groove was arranged between an inner radius of 38 mm
and an outer radius of 42 mm. The portions of the disc not bearing
a groove are referred to as a "mirror" herein. The term "mirror"
does not require that the referenced reflective surface be
perfectly flat or planar. Concave or convex mirrors can
additionally or alternatively be used, provided that the reflective
surface is substantially flat over areas at least twice the mean
size of the targets to be measured. In some examples, the
reflective surface can comprise at least a section curved in order
to affect the particle distribution on the reflective surface in a
desired manner. The groove can be arranged relatively farther from
the objective lens during measurement; the land can be relatively
closer to the objective lens. The relative widths of the lands and
grooves, e.g., shown in the cross-section in FIG. 2, can be
substantially equal or can be different. Examples using equal land
and groove widths can provide improved push-pull signal SNR.
Regions 1004 ("Bead drop") are discussed below.
[0078] FIG. 11 shows example measurement techniques. An LOC 1100
(or other substrate) is irradiated by a spot, e.g., a laser spot.
The laser spot can scan or move relative to the LOC, e.g., via
motion of the spot or via motion of the LOC such as rotation of a
disc-format LOC, other LOC, or other substrate. As used herein,
descriptions of LOC embodiments can apply to disc or other LOC
configurations. A radial direction R and a groove pitch p are
shown. The laser spot can track at a fixed radial position or a
fixed radial rate ("no tracking"), or can be controlled to follow a
land ("on-land" tracking) or a groove ("in-groove" tracking). In
the illustrated example, the spot follows the groove. In some
examples, e.g., of a rectangular LOC such as one shaped like a
standard glass microscope slide, the spot can be scanned, e.g.,
rasterized, across the LOC, e.g., using galvos or polygon scanners.
In some examples, e.g., of a disc-format LOC, the disc can be
rotated, e.g., while moving the spot radially to track along a
groove or land.
[0079] A four-way split photodetector 1102 (or two two-way split
photodetectors orthogonal to each other, or another arrangement of
quadrant photodetectors, e.g., a split photodiode or photodiodes)
detects laser light reflected off the disc in four quadrants,
A1-A4. The sum of the detected light, e.g., a sum signal or
aggregate signal, is referred to herein as the "HF" signal.
Considering the direction of travel of the laser spot with respect
to the track as a "frontward" direction, the difference between
frontward and rearward halves of a split photodiode (e.g.,
A1+A3-(A2+A4)) is referred to as a tangential push-pull ("TPP")
signal. The difference between left and right halves of a split
photodiode when viewed facing frontward (e.g., A1+A2-(A3+A4)) is
referred to as a radial push-pull ("RPP") signal. The radial
push-pull signal indicates how closely the spot is following the
track and can be used as an input to a control loop that adjusts
the radial spot position to follow the track. A tracking error
signal ("TES") can be determined as (A1+A2-(A3+A4))/(A1+A2+A3+A4)
(RPP divided by HF). HF and push-pull ("PP") signals can both be
used to detect targets. Signal-measurement techniques and track
structures described in the Kuroda, Nakajima, and Nakayama patent
documents submitted herewith and noted above can be used in
conjunction with measurement hardware and techniques herein in
various examples. In some examples, the HF signal is modulated by
particles of a sufficiently large size (depending on irradiation
wavelength, irradiation spot size, and grating pitch/reflectivity).
The HF signal can be processed to detect larger cells and
particles. Example HF and PP signals are shown in FIGS. 20, 21, and
29-33.
[0080] As used herein, TPP and RPP signals are not limited to
disc-format LOCs. For example, for X-Y scanning over a chip-format
LOC, TPP signals can be along the grooves and RPP signals can be
across the grooves, or vice versa. For X-v scanning, i.e., scanning
the spot in an X direction different from (e.g., substantially
perpendicular to) a flow direction (v) of a liquid sample, TPP
signals can be in the X direction and RPP signals in the v
direction, or vice versa. Alternatively, TPP and RPP signals can be
at an angle to grooves or flow directions, e.g., because of
interactions between X scan velocity and v flow velocity in an X-v
scanning configuration.
[0081] In some examples in which targets are in grooves on the LOC,
in-groove tracking can be used. In some examples, on-land tracking
can be used to detect large particles that may be located on lands,
e.g., because they are too big to fit in the grooves. Measurements
can be taken with both in-groove and on-land tracking and the
results combined, in some examples.
[0082] FIG. 12 shows a perspective of example optical apparatus
1200, e.g., including a BLU-RAY optical pick up. A portion of a
disc 1202 is shown in phantom for clarity of explanation. In the
illustrated example, optical radiation from a laser diode strikes
disc 1202. The laser light, or other light radiated onto or into
the disc or other LOC (e.g., an LOC including a reflective surface
such as a reflective phase grating), is referred to herein as
"incident light." In some examples, the incident light can pass
into or through a flow chamber, e.g., to irradiate samples or
targets in the flow chamber.
[0083] Light reflected or refracted from the disc, or light emitted
from samples, targets, dyes such as fluorescent dyes, or other
substances in or over the disc, is referred to herein as "resultant
light." In flow-chamber configurations, resultant light can include
light transmitted through the flow chamber. Resultant light can
include forward-scattered (FS) light and side-scattered (SS) light.
FS and SS can have substantially the same wavelength as the light
source does. Resultant light can also include fluorescent light,
e.g., emitted by substances in or over the irradiated surface (or
within the flow chamber) such as labeled antibodies. Resultant
light can be, e.g., substantially directional (e.g., transmitted
light of the laser light or substantially omnidirectional (e.g.,
fluorescence).
[0084] It is not required that all of the laser light be incident
on any particular target (e.g., a microvesicle). For example,
useful information can be gathered while scanning the irradiation
spot over the membrane of a microvesicle, even if some of the
irradiation spot is not striking the microvesicle.
[0085] In various aspects, light provided by a source other than a
laser is used instead of or in addition to the laser light. The
light source can be any source that can be focused to produce an
irradiation spot, e.g., a diffraction-limited spot, a spot having a
FWHM<2 .mu.m, or a spot smaller than the target to be
irradiated. Example sources can include, e.g., a lamp positioned at
the focus of a parabolic reflector, or a light-emitting diode (LED)
focused through a lens.
[0086] In an example, transmitted light or forward-scattered light
can be coherent light that is affected by scattering, refraction,
absorption, rotation of the plane of polarization, or other effects
on the light due to the irradiation of the laser light onto the
targets. In an example, the fluorescence or side-scattered light
can be incoherent light. Coherent side-scatter and back-scatter
light can also be detected.
[0087] In some configurations, targets are detected using outputs
from the photodetectors. In the illustrated example, at least some
resultant light passes back towards mirror 1204, e.g., a
beam-folding mirror. In some configurations, the coating of the
mirror 1204 is selected to transmit some light from fluorescent
dyes in or bonded to targets. Such light can be collected by the
fiber optic cable or other collection optics. In some
configurations, the coating of the mirror 1204 is selected to
reflect some light having a wavelength similar to that of the
incident light. The illustrated polarization beamsplitter or
another beamsplitter can be used to direct the resultant light
having the wavelength similar to that of the incident light to,
e.g., split photodetectors. In some examples, the linear velocity
of a target over the objective lens is .about.1 m/s. In some
examples not shown, a flow cell is used that carries targets at,
e.g., .about.1 m/s.
[0088] In some examples using incident light at .about.405 nm from
the laser diode, the mirror 1204 is .about.100% reflective at 405
nm. In some examples, in order to get fluorescence (fluorescent)
light through mirror 1204, a coating with R405.apprxeq.100%
(reflectance at 405 nm) and T>405 nm.apprxeq.100% (transmission
at wavelengths longer than 405 nm) is applied to mirror 1204.
[0089] FIG. 13 shows a schematic of an example optical detection
system. A laser diode (LD) provides optical radiation through a
polarization beamsplitter (PBS). The radiation passes through a
quarter-wave plate (.lamda./4) and is reflected by a 405
nm-reflective beamsplitter (405R-BS). The radiation is focused by
an objective lens ("Obj.Lens") onto an LOC, in this example a disc
rotated by a motor. The lens and associated components, or the
motor and disc, can be translated left-to-right in the figure,
e.g., to track grooves. The resultant light from the LOC passes
back to the 405R-BS. Fluorescent or other resultant light at a
substantially different wavelength from the incident light passes
through the 405R-BS to an optical fiber. Resultant light
substantially at the same wavelength as the incident light passes
through the .lamda./4 plate, is reflected by the PBS to a
photodetector, e.g., an Optoelectronic Integrated Circuit (OEIC)
such as an OEIC including a split photodiode and readout
electronics. PMTs (photomultiplier tubes) or other optical sensors
can also or alternatively be used, e.g., a PMT, avalanche
photodiode (APD), or multi-pixel photon counter (MPPC). Other
optical sensors can also or alternatively be used.
[0090] In some examples, the detection system and the LOC are
arranged to provide a gap between the objective lens and the flow
channel. In some examples, the gap is .lamda./NA.sup.2, e.g., 0.56
.mu.m for BLU-RAY optics. In some examples, the gap is .ltoreq.5
.mu.m, or .ltoreq.2 .mu.m, or 1 .mu.m. Gaps<5 .mu.m can permit
moving fluid in a channel via capillary action. In some examples,
the gap is <c.lamda./NA.sup.2, for
.about.4.ltoreq.c.ltoreq..about.8, or between .about.5 .mu.m and
.about.10 .mu.m. In some examples, the channel height is .ltoreq.5
.mu.m, or .ltoreq.2 .mu.m, or 1 .mu.m.
[0091] FIG. 14 shows an example target-measurement system 1400.
Details of various illustrated components are discussed herein with
reference to FIGS. 13 and 15-19.
[0092] "Error" represents computation of Focus & Tracking Error
Signals, e.g., as used in CD-ROM, DVD, or BLU-RAY discs. Error
signals are provided to a servo drive ("servo"), which can operate
a voice coil or other actuator to adjust position or orientation of
one or more lenses or other optical components to maintain focus
and tracking. Various aspects include separating a detected signal
into servo bandwidth signals and detected particle signals. This
can be done, e.g., using split photodetectors or using filters on
specific temporal-frequency ranges. Examples of temporal-frequency
ranges are shown as annotations on illustrated signal paths.
[0093] In some examples, the servo drive can receive control
signals ("CTL") and can adjust the focus depth of the spot in
response to the control signals. This can permit focusing the spot
on the reflective surface or on targets, e.g., the surfaces or
centers of targets. This can also permit adjusting focus to more
effectively detect targets either on-land or in-groove.
[0094] In some examples, the servo drive can respond to the control
signals to adjust the focal plane, e.g., within a 0.5 .mu.m range.
This can permit scanning a volume, e.g., of a groove or flow
channel. Volume scanning can in turn permit reducing
double-counting of targets, or increasing accuracy of target
scanning when the targets are moving.
[0095] Actuators (omitted for brevity) can rotate ("(DISC)") or
translate ("(SLIDE)") the LOC or other sample carrier, in
conjunction with or independently of motion of the irradiation
spot, to permit scanning the spot across a sample as discussed
herein.
[0096] Inset 1402 shows details of an LOC and detection optics. The
illustrated LOC is in a disc format. Disc rotation is around the
depicted axis vertically in the plane of figure. Left to right in
the figure is radially on the disc. In some examples, the
irradiation wavelength .lamda.=405 nm, the lens has a numerical
aperture (NA) of 0.85, and the focused spot size is 0.39 .mu.m
(1/e.sup.2). The illustrated reflective grating is arranged over a
polymer substrate and has lands and grooves at a pitch P
(groove-to-groove or land-to-land) of 500 nm and a groove depth D
of 38 nm. Targets are illustrated as hatched ovals, and can
include, e.g., Micro vesicles in a medium, such as blood
plasma.
[0097] In some examples, resultant light is detected by one or more
OEIC(s), PMT(s), or other optical detectors. In some examples,
e.g., as described herein with reference to FIGS. 19-21, a PMT can
be configured to detect fluorescent emissions from target(s), and
the output of the PMT can be provided to a discriminator or photon
counter.
[0098] The illustrated example includes a focused scanning laser
spot on reflective grating and single photon detection. However, in
some examples, a substantially flat reflective surface is used
instead of the illustrated land/groove (L/G) structure.
[0099] FIG. 15 shows example detection optics 1500, e.g., for
microvesicles, and an example of a sample carrier 1502. In the
illustrated example, a laser (e.g., .lamda.=405 nm) illuminates a
reflective grating (e.g., of an LOC) through an objective lens. As
shown, a target (e.g., a microvesicle or other microparticle,
depicted as an oval) is arranged over the reflective grating. An
antibody (depicted as a star) is bound to the target. In this
example, the antibody is tagged with a fluorescent dye. When the
illumination strikes the dye, the die fluoresces. Additionally, the
reflective grating and the target interact to modulate, scatter,
reflect, or refract some of the incident light.
[0100] In the illustrated example, reflected light from the grating
at the incident wavelength is passed through a pellicle,
polarization, or other beamsplitter to a detector array (e.g.,
split photodetector 1102 to produce high-frequency (HF), radial
push-pull (RPP), tangential push-pull (TPP), or focus error
signals. Light not at the incident wavelength, e.g., emitted by
fluorescent dyes on antibodies bound to targets in the sample,
passes through mirror 1504 and is collected by a lens and coupled
into an optical fiber. Minor 1504, in some examples, is configured
to reflect light (e.g., substantially all light) at the incident
wavelength (here, 405 nm) and to transmit light (e.g.,
substantially all light) at wavelengths longer than the incident
wavelength. In order to collect the fluorescent light to the fiber,
a collimator lens can be used, e.g., a collimating aspherical lens
similar to that used to collimate laser light. In some examples,
the 405 nm irradiation spot is a diffraction limited spot. In some
examples, aberration can be present in the fluorescent-collection
optics and the fluorescent light can couple to the fiber having a
core diameter, e.g., .about.0.1 mm-.about.0.6 mm in diameter. The
fiber carries the light to a photomultiplier tube (PMT) or other
optical detector. In some examples, the fluorescence signal is
detected by detection of single photons without averaging. In some
examples, detected photons are averaged with an FWHM time constant
.tau..apprxeq.DFWHM/v. DFWHM is the diameter, e.g., in meters or
microns, at FWHM. In some examples, high-NA optics are used to
improve collection efficiency. In some examples, optics having low
auto-fluorescence are used to reduce noise photons. Examples herein
can provide high-speed single photon detection with low dark
counts.
[0101] In some examples, anti-body reagent(s) or marker(s) are
applied to specific micro vesicles. Then, fluorescence is detected
from coupled micro vesicles (MVs). Compared to a large live cell
(e.g., 10 .mu.m diameter), MVs have a much smaller particle surface
and volume. Assuming a sphere shape, example ratios are given in
Table 5.
TABLE-US-00005 TABLE 5 Particle Diameter Surface Volume Diameter
Ratio Ratio Ratio 10 .mu.m 1.0 1.0 1.0 1 .mu.m 1/10 1/100 1/1,000
0.1 .mu.m 1/100 1/10,000 1/1,000,000 0.01 .mu.m 1/1,000 1/1,000,000
1/1,000,000,000
[0102] For example, if 1,000,000 conjugated anti-bodies bind on a
10 .mu.m size, 0.1 .mu.m-size cell, 1,000,000.times.1/10,000=100
dye molecules bind to a 0.1 .mu.m microvesicle, in a nonlimiting
example.
[0103] Under irradiation, e.g., laser light, dye molecules emit
photon(s) by electron excitation and decay. Photon ratio, QE
(quantum efficiency), is emitted photons divided by excited
photons. Photon energy is E=1.24/.lamda. (eV). For some dye
molecules, once the molecule is excited and the photon emitted, the
dye molecule is quenched (photo bleaching). Therefore, some
examples provide exposure to fresh dye molecules over time to
improve system response. Some examples do this using a flying spot,
e.g., by scanning irradiation across a sample containing dyed
antibodies conjugated to targets. Some examples include rotating a
substrate holding the samples to move samples and irradiation with
respect to each other. Some examples including moving a substrate
linearly. Some examples include moving targets, e.g., using a flow
of carrier fluid. In some examples, in order to excite a target
(e.g., a microvesicle or other micro particle) with sufficient
intensity and without overlapping other nearby targets, a focused
small laser spot is used. In some examples, a flow cell is used.
Moving an irradiation spot with respect to a target (e.g., with
respect to a sample or reflective surface bearing the sample) can
permit reducing the effect of photo-bleaching, improving detection
effectiveness.
[0104] FIG. 16 shows another example configuration of detection of
microparticles on a reflective grating. The illustrated
configuration includes a 1.1 mm-thick polycarbonate (PC) substrate
overcoated with an .about.18 nm-thick Al reflective layer (or
.about.20 nm, .about.100 nm, >100 nm, etc.). A 100 .mu.m-thick
cover, e.g., also PC, is spaced apart from the substrate to form a
channel or other sample chamber. The channel can be filled with air
or another fluid, e.g., a gas or liquid. The base of the grooves is
230 .mu.m from the far side of the cover in this nonlimiting
example. The groove pitch P can be, e.g., 500 nm, and the grooves
can be 38 nm deep.
[0105] FIG. 17 shows an example transimpedance amplifier useful
with various examples. The illustrated example amplifier has a 70
MHz bandwidth (BW) and can output analog signals to an A/D
converter input. In some examples, tested micro-photomultiplier
tubes (.mu.PMTs) with an example I-V amp achieved 100 MHz BW with 6
ns pulse width.
[0106] In some examples, a gating signal is produced based at least
on the HF and TPP signals. Further details are discussed herein
with reference to FIGS. 19-21. In some examples, a gating function
is used to distinguish signal photons from thermal-noise photons or
other photons produced by substances other than target dyes or
other chromophores capable of associate with targets.
[0107] Various aspects, e.g., some aspects described herein with
reference to FIG. 1-7, 10-16, 18, 19, 23, 24, or 26-28, include at
least some of a reflective phase grating arranged in or adjacent to
a microfluidic channel, a sample-delivery system, an irradiation
system, and a detection system. Using a reflective grating provides
effective optics and doubles phase detection compared to some prior
schemes. Using a reflective grating also permits using low-cost
manufacturing techniques developed for CD, DVD, and BLU-RAY discs
(providing, e.g., 3 s per disc manufacturing time). The irradiation
system can include one or more lenses or scanners to provide a
focused scanning spot, e.g., a focused scanning laser spot. The
detection system can include on or more photomultiplier tubes
(PMTs) or other optical detectors. PMTs can be used, e.g., to
detect fluorescence from targets or from dyes (e.g., containing
fluorochromes) in, on, or associated with targets. The
sample-delivery system can include a filter, e.g., to separate red
blood cells from plasma so that targets in the plasma, e.g.,
microvesicles, can be measured apart from the red blood cells.
[0108] In various aspects, targets such as nanoparticles are
detected as HF and PP signal perturbations caused by optical path
differences such as different optical path length. In various
aspects, the detected HF pulse width and tangential PP signal
modulation are used to determine tangential particle size. In
various aspects, radial PP signal modulation is used to determine
radial particle size. Various aspects use radial PP together with
HF or tangential PP (or both) to provide 2D data of targets.
[0109] Various aspects include a substrate including a microfluidic
channel on, over, or adjacent to a reflective phase grating. The
microfluidic channel can have a height (away from the grating),
e.g., of <5 .mu.m. These aspects can be embodied in, e.g., LOC
structures. LOCs can include rigid, semi-rigid, or flexible
substrates.
[0110] Various aspects, e.g., of LOCs, include disposable chips
having sample-processing structures such as cell-separation
structures, staining structures, filtering structures, measurement
structures, and waste-retention structures. Such aspects can be
used, e.g., in clinical applications. For example, LOCs can be used
to perform high-volume analysis of samples from many patients in a
hospital, or many customers of an analysis lab.
[0111] Various aspects include an optical assembly including a
tracking system for focusing and tracking of a focused spot such as
a laser spot, e.g., a focused laser spot, on a reflective grating.
The tracking system can operate in rotation, e.g., for disc-format
LOCs, or in traverse (e.g., X-Y axis), e.g., for slide-glass-shaped
LOCs.
[0112] In some examples, microvesicles or other targets adhere to
lands or grooves of a disc or LOC, e.g., due to electric field
interactions, van der Waal's forces, surface tension and wetting of
the surface of the disc, or other microscale interactions.
Reflective surfaces, e.g., reflective gratings, can have surface
materials, finishes, electrical or magnetic characteristics, or
other properties configured to retain a sample, e.g., a fluidic or
non-fluidic sample, at least partly in a detection zone such as
that illustrated in FIG. 1.
[0113] Various aspects include acts to enhance refractive index
difference between particles and medium. For example, a sugar
solution can be selected to have a different refractive index than
the targets of interest. In some examples, air is used as a medium
so n.apprxeq.1. In some examples, a polymer medium is used and the
wavelength of light is selected to achieve a desired n.sub.80 of
the polymer. In some examples, water or another aqueous medium is
used and solutes are added to adjust refractive index n or
dielectric constant k of the medium. For example, a 10% glucose
solution can have a refractive index of 1.3477, a 20% glucose
solution 1.3635, or a 60% glucose solution 1.4394. In some
examples, the refractive index of the medium can be varied and
measurements can be taken at various medium n values, e.g.,
continuously or in discrete steps. A refractive index of the medium
can be selected, e.g., from those tested, that provides a selected
signal-to-noise ratio or other metric of measurement quality.
[0114] In some examples, statistics of the measured data are
gathered to, e.g., extrapolate from measurements of fewer than all
of the particles. For example, average particle size can be
computed from measurements of a selected number of particles.
[0115] Various aspects include data acquisition and analysis
software for detecting variations in the light reflected off a
reflective phase grating.
[0116] Various aspects include structures or techniques for
fluorochrome staining of microvesicles or other targets. These
aspects can be used to identify the origins, compositions, or
identities of particles, or to distinguish particles from
background noise such as electrical noise. Electrical noise can
include, e.g., shot noise in the photodetectors measuring light
reflected by the phase grating.
[0117] Various aspects include scanning of phase gratings, focused
laser spots, or both. Scanning can be performed by mechanical
scanning devices such as disc rotation systems or X-Y stages, or by
optical scanning devices such as galvanometer motors ("galvos") or
beam deflectors.
[0118] Various aspects include one or more of the following
properties: (a) Detection optics: .lamda.=405 nm, NA=0.85, cover
thickness=0.1 mm, focused spot size=0.39 .mu.m(1/e2), p>240 nm,
or any combination of those; (b) Reflective phase grating groove
depth of .lamda./8n; (c) Reflective-grating pitch p>.lamda./2NA;
(d) A disc structure having a spiral groove in which successive
turns are separated by the pitch p; (e) p=1.6 .mu.m, p=0.74 .mu.m,
or p=0.32 .mu.m; (f) Phase grating depth .about.40 nm and pitch
p=500 nm; (g) Phase-grating land-groove aspect ratio is 50:50.
Other ranges can be used, e.g., 60:40 or 40:60; (h) A disc
structure in which the reflective phase grating is arranged around
a center of the disc between about 38 mm from the center and about
42 mm from the center; (i) A detection region with no grooves,
e.g., a mirror or substantially flat metal surface of a disc or
other LOC; (j) Servo bandwidth <50 kHz and particle signal
bandwidth >100 kHz; (k) Data acquisition has sufficient
bandwidth (BW) and resolution. E.g., at 1.0 m/s scanning velocity,
a 10 nm particle can be detected as a 10 ns-wide pulse. Measurement
hardware having a bandwidth of 200 MHz (5 ns) can be used in this
example. In some examples, signal rise time can be computed as
0.35/BW. Signals with a 5 ns rise time can therefore be measured
with hardware having at least 70 MHz BW; (l) Detecting fluorescence
of micro particles with a conjugated anti-body; (m) Detecting
fluorescence of microparticles stained with a dye. For example,
proteins can be stained with FITC, lipids with Nile red, or DNA
with Hoechst 33342. Other dyes can be selected for use depending on
the excitation wavelength; (n) Excitation by a focused flying laser
spot with smaller than 2 .mu.m FWHM; (o) Detecting single photon
pulses without averaging, e.g., less than 20 ns width; (p)
Detecting photon pulses with time constant .about..tau.=DFWHM/v;
(q) Distinguishing signal & noise photons by gating with
HF/Push-Pull signals from particles, e.g., HF and TPP; (r)
Investigating micro particle fluorescence by single photon
spectroscopy; (s) Measuring microvesicles; (t) Detecting
fluorescence and providing imaging for micro and nano
particles.
[0119] In some examples, one focusing point is sufficient since the
number of microvesicles is relatively high. In other examples,
multiple focusing points are used (e.g., in-groove and
on-land).
[0120] In view of the foregoing, various aspects provide effective
detection or size measurement of small particles. A technical
effect of various aspects is to measure physical properties of a
sample, e.g., a sample of blood or another bodily fluid.
[0121] FIG. 18 is a high-level diagram showing the components of an
example data-processing system 1801 for analyzing data and
performing other analyses described herein, and related components.
The system 1801 includes a processor 1886, a peripheral system
1820, a user interface system 1830, and a data storage system 1840.
The peripheral system 1820, the user interface system 1830 and the
data storage system 1840 are communicatively connected to the
processor 1886. Processor 1886 can be communicatively connected to
network 1850 (shown in phantom), e.g., the Internet or a leased
line, as discussed below. At least one of the systems shown in FIG.
12-15, 17, or 19 can include at least one of systems 1886, 1820,
1830, 1840, and can connect to one or more network(s) 1850.
Processor 1886, and other processing devices described herein, can
each include one or more microprocessors, microcontrollers,
field-programmable gate arrays (FPGAs), application-specific
integrated circuits (ASICs), programmable logic devices (PLDs),
programmable logic arrays (PLAs), programmable array logic devices
(PALs), or digital signal processors (DSPs).
[0122] Processor 1886 can implement processes of various aspects
described herein. Processor 1886 and related components can, e.g.,
carry out processes for receiving a sample on a substrate having a
reflective phase grating thereon or thereover, irradiating the
sample with a focused spot of light while traversing over the
substrate, detecting variations in reflected light, and determining
properties of a target (e.g., presence, size, orientation) using
the detected variations. Processor 1886 and related components can,
e.g., carry out processes for making discs or LOCs by controlling
mastering equipment to make stampers, mold discs or LOCs using
stampers, sputter discs or other LOCs, or apply cover materials to
discs or LOCs.
[0123] Processor 1886 can be or include one or more device(s) for
automatically operating on data, e.g., a central processing unit
(CPU), microcontroller (MCU), desktop computer, laptop computer,
mainframe computer, personal digital assistant, digital camera,
cellular phone, smartphone, or any other device for processing
data, managing data, or handling data, whether implemented with
electrical, magnetic, optical, biological components, or
otherwise.
[0124] The phrase "communicatively connected" includes any type of
connection, wired or wireless, for communicating data between
devices or processors. These devices or processors can be located
in physical proximity or not. For example, subsystems such as
peripheral system 1820, user interface system 1830, and data
storage system 1840 are shown separately from the processor 1886
but can be stored completely or partially within the processor
1886.
[0125] The peripheral system 1820 can include or be communicatively
connected with one or more devices configured or otherwise adapted
to provide digital content records to the processor 1886 or to take
action in response to processor 1886. For example, the peripheral
system 1820 can include digital still cameras, digital video
cameras, cellular phones, or other data processors. The processor
1886, upon receipt of digital content records from a device in the
peripheral system 1820, can store such digital content records in
the data storage system 1840.
[0126] In the example shown, peripheral system 1820 is connected to
a drive, a sensor, a laser, and a sample-delivery system. The drive
spins the disc (in other configurations, the drive can scan a laser
across a stationary LOC), e.g., as done to spin BLU-RAY discs. The
sample-delivery system applies to the disc or other LOC fluid
samples to be measured for the presence or properties of targets.
For example, the sample-delivery system can include an automated
pipette or other metering system for applying a selected amount of
sample. The laser irradiates the disc or other LOC and the sensor
measures reflected light (illustrated as coming from a
beamsplitter, though other configurations can be used, e.g., a
laser at an angle to the substrate other than normal). Processor
1886 and peripheral system 1820 coordinate these components to
measure the samples and target(s) therein. Other examples of
components that can be operated by processor 1886 are shown in FIG.
12-15, 17, or 19. Various examples of LOCs that can be used with
components shown in FIG. 18 are shown in FIGS. 1-11, 16, and
23.
[0127] In some examples, processor 1886 can provide control
signals, e.g., as discussed above with reference to FIG. 14, to
adjust a focal position of the spot of light with respect to the
reflective surface. For example, processor 1886 can provide the
control signals to rasterize the spot of light in three dimensions,
to repeatedly scan the spot of light substantially normal to at
least a portion of the reflective surface (e.g., normal N in FIG.
1), or to focus the spot of light, e.g., to detect particles within
a groove (e.g., longer focal distance) or to detect particles on a
land (e.g., shorter focal length).
[0128] The user interface system 1830 can convey information in
either direction, or in both directions, between a user 1838 (or
other entity) and the processor 1886 or other components of system
1801. The user interface system 1830 can include a mouse, a
keyboard, another computer (connected, e.g., via a network or a
null-modem cable), or any device or combination of devices from
which data is input to the processor 1886. The user interface
system 1830 also can include a display device, a
processor-accessible memory, or any device or combination of
devices to which data is output by the processor 1886. The user
interface system 1830 and the data storage system 1840 can share a
processor-accessible memory.
[0129] In various aspects, processor 1886 includes or is connected
to communication interface 1815 that is coupled via network link
1816 (shown in phantom) to network 1850. For example, communication
interface 1815 can include an integrated services digital network
(ISDN) terminal adapter or a modem to communicate data via a
telephone line; a network interface to communicate data via a
local-area network (LAN), e.g., an Ethernet LAN, or wide-area
network (WAN); or a radio to communicate data via a wireless link,
e.g., WIFI or GSM. Communication interface 1815 sends and receives
electrical, electromagnetic, or optical signals that carry digital
or analog data streams representing various types of information
across network link 1816 to network 1850. Network link 1816 can be
connected to network 1850 via a switch, gateway, hub, router, or
other networking device.
[0130] In various aspects, system 1801 can communicate, e.g., via
network 1850, with a data processing system 1802, which can include
the same types of components as system 1801 but is not required to
be identical thereto. Systems 1801, 1802 are communicatively
connected via the network 1850. Each system 1801, 1802 executes
computer program instructions to, e.g., carry out measurements as
described herein.
[0131] Processor 1886 can send messages and receive data, including
program code, through network 1850, network link 1816 and
communication interface 1815. For example, a server can store
requested code for an application program (e.g., a JAVA applet) on
a tangible non-volatile computer-readable storage medium to which
it is connected. The server can retrieve the code from the medium
and transmit it through network 1850 to communication interface
1815. The received code can be executed by processor 1886 as it is
received, or stored in data storage system 1840 for later
execution.
[0132] Data storage system 1840 can include or be communicatively
connected with one or more processor-accessible memories configured
or otherwise adapted to store information. The memories can be,
e.g., within a chassis or as parts of a distributed system. The
phrase "processor-accessible memory" is intended to include any
data storage device to or from which processor 1886 can transfer
data (using appropriate components of peripheral system 1820),
whether volatile or nonvolatile; removable or fixed; electronic,
magnetic, optical, chemical, mechanical, or otherwise. Example
processor-accessible memories include but are not limited to:
registers, floppy disks, hard disks, tapes, bar codes, Compact
Discs, DVDs, read-only memories (ROM), erasable programmable
read-only memories (EPROM, EEPROM, or Flash), and random-access
memories (RAMs). One of the processor-accessible memories in the
data storage system 1840 can be a tangible non-transitory
computer-readable storage medium, i.e., a non-transitory device or
article of manufacture that participates in storing instructions
that can be provided to processor 1886 for execution.
[0133] In an example, data storage system 1840 includes code memory
1841, e.g., a RAM, and disk 1843, e.g., a tangible
computer-readable rotational storage device or medium such as a
hard drive. Computer program instructions are read into code memory
1841 from disk 1843. Processor 1886 then executes one or more
sequences of the computer program instructions loaded into code
memory 1841, as a result performing process steps described herein.
In this way, processor 1886 carries out a computer implemented
process. For example, steps of methods described herein, blocks of
the flowchart illustrations (e.g., FIGS. 24-28) or block diagrams
herein, and combinations of those, can be implemented by computer
program instructions. Code memory 1841 can also store data, or can
store only code.
[0134] Various aspects described herein may be embodied as systems
or methods. Accordingly, various aspects herein may take the form
of an entirely hardware aspect, an entirely software aspect
(including firmware, resident software, micro-code, etc.), or an
aspect combining software and hardware aspects These aspects can
all generally be referred to herein as a "service," "circuit,"
"circuitry," "module," or "system."
[0135] Furthermore, various aspects herein may be embodied as
computer program products including computer readable program code
("program code") stored on a computer readable medium, e.g., a
tangible non-transitory computer storage medium or a communication
medium. A computer storage medium can include tangible storage
units such as volatile memory, nonvolatile memory, or other
persistent or auxiliary computer storage media, removable and
non-removable computer storage media implemented in any method or
technology for storage of information such as computer-readable
instructions, data structures, program modules, or other data. A
computer storage medium can be manufactured as is conventional for
such articles, e.g., by pressing a CD-ROM or electronically writing
data into a Flash memory. In contrast to computer storage media,
communication media may embody computer-readable instructions, data
structures, program modules, or other data in a modulated data
signal, such as a carrier wave or other transmission mechanism. As
defined herein, computer storage media do not include communication
media. That is, computer storage media do not include
communications media consisting solely of a modulated data signal,
a carrier wave, or a propagated signal, per se.
[0136] The program code includes computer program instructions that
can be loaded into processor 1886 (and possibly also other
processors), and that, when loaded into processor 1886, cause
functions, acts, or operational steps of various aspects herein to
be performed by processor 1886 (or other processor). Computer
program code for carrying out operations for various aspects
described herein may be written in any combination of one or more
programming language(s), and can be loaded from disk 1843 into code
memory 1841 for execution. The program code may execute, e.g.,
entirely on processor 1886, partly on processor 1886 and partly on
a remote computer connected to network 1850, or entirely on the
remote computer.
[0137] FIG. 19 shows a block diagram of an example measurement
system 1900 incorporating gating logic. Resultant light is divided
between the split photodetector (light substantially at the
incident .lamda.) and the fluorescence detector (other than e.g.,
as described above with reference to FIG. 12. Tracking signals from
the split photodetector are used by the focus/tracking control
block to determine drive for a servo to maintain tracking or
focus.
[0138] HF and TPP signals are produced and provided to a
comparator. The comparator provides a gating function, e.g., by
thresholding or detecting edges of, e.g., the HF or TPP signals
from the split photodiode. Various examples of comparators are
discussed below with reference to FIG. 20. The HF, TPP, and RPP
signals can also be digitized by an A/D converter and provided,
e.g., for particle analysis.
[0139] A signal from a fluorescence detector (FLD), e.g., a
.mu.PMT, can be passed through a discriminator. The discriminator
can remove noise, e.g., thermal noise. In some examples, the
discriminator removes pulses having a pulse height (e.g., measured
in volts) smaller in magnitude than a selected lower level of
discrimination (LLD). In some examples, the discriminator removes
pulses having a pulse height larger in magnitude than a selected
upper level of discrimination (ULD). The resulting pulses 1902 are
gated by the gating function ("Gate"), e.g., by passing pulses only
when the gating function 1904 has a high logic value or other
gate-open value. The gated pulses 1906 are counted. The counts of
pulses can then be provided for fluorescence analysis. In some
examples (omitted for brevity), gating can be additionally or
alternatively based in RPP signals.
[0140] FIG. 20 shows simulated HF, TPP and RPP signals. In the
example plots, a particle as centered at zero on the X-axis. Units
on both axes or arbitrary. The axes can be, e.g., microns on the
X-axis and volts on the Y-axis. The curves show respective,
different particle sizes from 10 nm-100 nm.
[0141] On the left-hand, HF plot, the dotted line shows a
threshold. In some examples, a gating signal corresponding to the
HF signal has a true logic value (e.g., a 1 value) when the HF
signal is below the threshold, as represented by the dashed line.
In other examples, the gating signal corresponding to the HF signal
has a true logic value when the HF signal is above the threshold,
within a specified range, or outside a specified range. In an
example, the gate is open (true logic level) during the 240 ns FWHM
of a laser pulse.
[0142] On the center, TPP plot, the TPP signal has a negative
excursion when beginning to cross the particle and a positive
excursion when leaving the particle. The dotted lines show
thresholds for the excursions. In the illustrated example, the
logic signal (dashed) corresponding to the TPP signal has a true
logic value between the low-going crossing of the threshold in the
negative excursion and the low-going crossing of the threshold in
the positive excursion.
[0143] In some examples, the gate is open (photons are counted)
when the HF logic signal has a true logic value, when the TPP logic
signal has a true logic value, or when both the HF and TPP logic
signals have true logic values. These examples are illustrated in
the box labeled "Comparator."
[0144] In some examples, the size of a particle is determined from
the height and width of the HF pulse, e.g., by measuring levels of
the HF pulse, ramp rates of the HF pulse, or FWHM of the HF pulse.
For example, D-FWHM or the duration of the true logic level on the
HF logic signal can indicate particle size. In some examples, the
size of a particle is determined from the duration of the true
logic level on the TPP logic signal, or from the heights or widths
of the excursions on the TPP signal. In some examples, the lateral
position of the particle in the groove or on the land is determined
using the pulse height or ripple of the RPP signal.
[0145] FIG. 21 shows examples of photon pulses and gating. Curve
2102 shows measured photon pulses. Curve 2104 (shown only
partially) shows comparator outputs. Curve 2106 shows a measured HF
signal. Curve 2108 shows a measured TPP signal. Plot 2110 shows
simulated example photon pulses. Plot 2112 shows simulated example
gating signals. Plot 2114 shows pulses resulting from the gating of
plot 2110 with the gating signal of plot 2112. As shown in plot
2114, gating can permit discarding noise-photon pulses and
retaining signal-photon pulses.
[0146] FIG. 22 shows an example target-measurement system 2200.
System 2200 can represent systems described herein with reference
to FIG. 1, 6, or 11-20. A reflective surface 2202, e.g., over a
substrate 2204 of a sample carrier such as an LOC, is configured to
retain a target 2206 (shown in phantom), e.g., a microscale target,
in a detection region 2208 and to reflect at least part of a
focused spot 2210 of light to provide resultant light 2212, e.g.,
reflected light, diffracted light, or other resultant light. An
irradiation system 2214 is configured to irradiate at least part of
the detection region 2208 with the focused spot 2210. The
illustrated angle of incidence of focused spot 2210 is for clarity
of explanation and is not limiting. A motion system 2216 is
configured to cause motion of the focused spot 2210 relative to the
reflective surface 2202. Although illustrated as causing motion of
substrate 2204, this is not limiting. Motion system 2216 can
additionally or alternatively cause motion of focused spot 2210
with respect to reflective surface 2202, a sample thereon, or
target 2206. Examples are discussed above, e.g., with reference to
FIG. 1,7-16,18, or 19.
[0147] A detection system 2218 is configured to detect the
resultant light 2212, e.g., as described herein with reference to
FIGS. 12-19. Examples of LOCs useful with target-measurement system
2200 are described herein with reference to FIG. 1-11,14-16, or 23.
Examples of irradiation system 2214, motion system 2216, or
detection system 2218 are described herein with reference to FIGS.
11-20.
[0148] In some examples, a sample-delivery system 2220 is
configured to apply a sample comprising the target 2206 to at least
a portion of the detection region. Examples of sample-delivery
systems 2220 and other components useful with various examples of
system 2200 are described herein with reference to FIG. 1,6-9, or
18. In some examples, a gating system 2222 is configured to provide
a particle signal associated with a temporal overlap between at
least one of the plurality of detection signals and the second
detection signal. Examples of gating system 2222 are described
herein with reference to FIGS. 19-21.
[0149] In some examples, a target holder 2224 can be configured to
retain target 2206 in detection region 2208. Target holder 2224 can
comprise reflective surface 2202 configured to reflect at least
part of the focused spot 2210 of light to provide the resultant
light 2212. In some examples, target holder 2224 can comprise one
or more LOCs as described herein, e.g., with reference to FIGS.
1-11. In some examples, reflective surface 2202 can be separate
from, e.g., spaced apart from, target holder 2224. For example,
target holder 2224 can include transparent structures to retain
target 2206, e.g., in a fluidic sample, over a mirror or reflective
grating (reflective surface 2202), when viewed along an irradiation
direction of the spot 2210 of light.
[0150] System 2200 can be an example of a target-measurement
assembly, as can other systems described herein, e.g., with
reference to FIG. 1,13-16,18, or 19. As used herein, the term
"assembly" does not require that the components of the assembly be
physically mounted to each other. An assembly, e.g., system 2200,
can include multiple components configured to interoperate. In some
examples, an assembly can comprise an LOC and a measurement
platform. The LOC can comprise reflective surface 2202 and
substrate 2204. The measurement platform can comprise at least some
of irradiation system 2214, motion system 2216, detection system
2218, sample-delivery system 2220, or gating system 2222. The
measurement platform can be configured to receive the LOC and
perform operations, e.g., described herein with reference to FIGS.
24-28.
[0151] FIG. 23 shows an example device 2300, e.g., a sample carrier
such as an LOC. Device 2300 can represent devices described herein
with reference to FIG. 1-11,14-16, or 18-20. Device 2300 can
additionally or alternatively represent target holder 2224, FIG.
22. Device 2300 includes a substrate 2302 and a sample inlet 2304
arranged on, in, or over the substrate 2302. A reflective grating
2306 or other reflective surface is arranged on, in, or over the
substrate 2302. The reflective grating 2306 is configured to retain
a fluidic sample 2308 (shown in phantom) in a detection region
2310, e.g., in or of a fluidic channel 2312. The detection region
2310 is fluidically connected to the sample inlet 2304. The
detection region 2310 is operatively arranged with respect to the
reflective grating 2306 so that at least some light passing through
the detection region 2310 towards the reflective grating 2306
(e.g., light of the focused spot 2210, shown in phantom) also
passes through the detection region 2310 after reflecting or
diffracting off the reflective grating 2306 (e.g., resultant light
2212, shown in phantom). For example, the detection region 2310 can
be arranged between the reflective grating 2306 and an irradiation
system 2214, FIG. 22. In some examples, device 2300 can include a
cover 2314, e.g., as described herein with reference to FIG. 1 or
9; a reservoir, e.g., as FIG. 8, at least one groove, e.g., as FIG.
2-7, 10, or 11; a filter, e.g., as FIG. 1; or multiple sample
inlets or vacuum ports, e.g., as FIG. 6.
Illustrative Processes and Further Illustrative Examples
[0152] FIG. 24 shows a flowchart 2400 of example methods of
measuring targets. Steps of FIG. 24 or other methods described
herein, e.g., in FIGS. 25-28, can be performed in any order except
when otherwise specified, or when data from an earlier step is used
in a later step. For clarity of explanation, reference is herein
made to various components shown in FIGS. 1-23 that can carry out
or participate in the steps of the example method. It should be
noted, however, that other components can be used; that is, example
method(s) shown in FIGS. 24-28 are not limited to being carried out
by the identified components. Methods described herein can be
carried out by structures on an LOC, in conjunction with
measurement systems described herein. Methods herein can be used,
e.g., with liquid biopsy samples of human patients, or with samples
of semiconductor-fabrication chemicals.
[0153] At block 2402, a sample can be received, e.g., a whole-blood
sample or chemical sample. The sample can be received on an LOC or
other sample carrier, e.g., as described herein with reference to
FIG. 1-10 or 23. In some examples, block 2402 can include receiving
the sample having a refractive index different from the refractive
index of targets in the sample. In some examples, the refractive
indices of the sample and the targets differ by An. In some
examples, |.DELTA.n| can be .about.0.05, or .about.0.1, or
.about.0.05-.about.0.1, or greater than .about.0.1, or greater than
.about.0.2. Various examples can improve detection, as sensitivity
is proportional to .DELTA.n.times.d in some examples. In some
examples, targets have more than one refractive index, e.g., as
discussed below with reference to microvesicles. An can be computed
with reference to the refractive index of at least one of an
exterior, a membrane, or an interior of a target, or with respect
to an average refractive index of the target as a whole. The sample
can be selected to have a refractive index different from any of
these refractive indices by any of the An values above. In some
examples, respective refractive indices of at least one of the
substrate, the reflective layer, or the cover can be selected to
differ from at least one of the respective refractive indices of at
least one of a target, a component of a target, or a sample by any
of the An values listed above.
[0154] At block 2404, filtering can be performed. For example,
cells can be filtered out of a whole-blood sample, or contaminants
can be filtered out of any sample. In some examples, an LOC can
include a filter element operatively arranged to filter a provided
sample to provide the fluidic sample, e.g., as shown in FIG. 1 or
7. The filter element can include a filter, e.g., an open structure
such as an open-cell foam or a woven fabric or plastic mesh, a
micropillar matrix such as shown in FIG. 7, or any combination of
any of those.
[0155] At block 2406, staining can be performed. For example, a dye
or antibody such as described herein with reference to FIG. 15 can
be added to the sample. Examples are described herein with
reference to FIGS. 7, 15, and 19.
[0156] At block 2408, measurement can be performed. For example,
the sample can be irradiated and resultant light detected, e.g., as
described herein with reference to FIG. 1 or 11-20.
[0157] At block 2410, at least some of the sample can be processed
as waste. For example, at least some of the sample can be removed
from the LOC by a vacuum or other pump, retained in a reservoir or
absorbent material of the LOC, or otherwise treated for storage or
disposal. Examples of vacuum ports, reservoirs, or other
waste-processing structures or techniques are described herein with
reference to FIGS. 6-8.
[0158] FIG. 25 is a flow diagram of an example process 2500 for
detecting a target. Block 2408 can include blocks 2504 or 2506.
[0159] At block 2502, a fluidic sample can be disposed over (e.g.,
on or above) a reflective surface. The reflective surface can
include, e.g., a mirror or reflective grating. The fluidic sample
can include a target, e.g., one or more microvesicles or
microparticles. Block 2502 can include at least one of blocks 2402,
2404, or 2406, or can be performed between any one of blocks 2402,
2404, or 2406 and block 2408.
[0160] At block 2504, the reflective surface can be irradiated
using a spot of light, e.g., a focused spot. In some examples, at
least some of the sample over the reflective surface can be
irradiated using the spot of light. Examples are discussed above,
e.g., with reference to FIGS. 1 and 11-23.
[0161] At block 2506, resultant light from the reflective surface
can be detected. In some examples, the resultant light can include
light reflected or diffracted by the reflective surface, e.g.,
light diffracted by a reflective grating. Examples are discussed
above, e.g., with reference to FIGS. 1 and 11-23.
[0162] FIG. 26 is a flow diagram of an example process 2600 for
detecting a target, e.g., in a sample. Some examples use blocks
2602 and 2604.
[0163] At block 2602, a dye can be applied to the fluidic sample
before the disposing (block 2502). Examples are discussed above,
e.g., with reference to block 2406.
[0164] At block 2604, light emitted by the dye can be detected. The
light, e.g., fluorescence, can be emitted in response to the
irradiating using the spot of light (block 2504). Examples are
discussed above, e.g., with reference to FIG. 13-15 or 19.
[0165] At block 2606, relative motion can be caused of the spot of
light and the fluidic sample. In some examples, block 2606 can
include block 2608. In some examples, block 2606 can include blocks
2610 and 2612. Various examples of relative motion are described
herein with reference to FIG. 1-16, 18, 19, 22, or 23, or Tables 2,
3, or 6.
[0166] At block 2608, in some examples, the fluidic sample can be
pumped or otherwise caused to flow through a flow channel across
the reflective surface. The spot can be caused to move with respect
to the reflective surface at least partly across the flow channel.
For example, the spot can be scanned, e.g., substantially
perpendicular to the direction of flow, across the flow channel
concurrently with the flowing of the sample. Various examples of
flow are described herein with reference to FIG. 1, 6-10, 18, 22,
or 23, or Tables 2, 3, or 6.
[0167] At block 2610, in some examples, a disc comprising the
reflective surface can be rotated, e.g., as described herein with
reference to at least FIG. 13. The disc can move with respect to
the spot of light during the rotating. In some examples, block 2504
can include irradiating the reflective surface during the
rotating.
[0168] At block 2612, in some examples, the spot of light can be
caused to move to follow a track of the reflective surface, e.g.,
during the rotating. Examples are discussed above, e.g., with
reference to FIGS. 2-4, 11, 14, and 19.
[0169] FIG. 27 is a flow diagram of an example process 2700 for
detecting a target. Processing can begin with block 2702 or block
2704.
[0170] At block 2702, a reflective phase grating can be constructed
or arranged in, on, or over a substrate. For example, a disc-format
LOC can be constructed using PTM plus metallization, as described
above.
[0171] At block 2704, a sample, e.g., a fluidic sample such as a
liquid sample, can be applied to the reflective phase grating (or
to another reflective surface, and likewise throughout). Examples
are discussed above, e.g., with reference to blocks 2402-2406,
2502, or 2602.
[0172] At block 2706, the reflective phase grating can be
irradiated, e.g., using a focused spot of light such as a laser
spot. Examples are discussed above, e.g., with reference to blocks
2408 or 2504.
[0173] At block 2708, resultant light can be measured. Block 2708
can be followed by block 2706, block 2710 or block 2712. This can
permit scanning, e.g., across the reflective surface or in focal
depth, as described herein.
[0174] At block 2710, the spot of light can be moved with respect
to the sample, e.g., with respect to the reflective grating.
Examples are discussed above, e.g., with reference to blocks
2606-2612, FIG. 1-16, 18, 19, 22, or 23, or Tables 2, 3, or 6.
[0175] At block 2712, the detected resultant light can be processed
to detect targets or to measure properties of the targets. For
example, HF and PP signals coinciding in time, e.g., as shown in
FIGS. 30 and 31, discussed below, can indicate presence of a
target. For example, the duration of a PP signal in time can
indicate the size of a target. Examples are discussed herein, e.g.,
with reference to blocks 2408, 2506, or 2604, or FIG. 7, 11, 15,
17, 19-21, or 28-33.
[0176] FIG. 28 shows a flowchart 2800 of example methods of
analyzing a sample.
[0177] At block 2802, particles of selected size range(s) can be
removed from a sample. For example, samples can be centrifuged,
filtered, fractionated, or otherwise divided into portions based on
particle size. For example, large particles can be removed from a
sample to permit analysis of small particles remaining in the
sample. Examples are discussed above, e.g., with reference to FIG.
1 or 7. Block 2802 can be followed by block 2806.
[0178] At block 2804, a reflective phase grating can be constructed
or arranged in, on, or over a substrate. For example, grooves can
be etched into a slide, or a BLU-RAY or other disc-format LOC can
be prepared using stamping with a PTM master as described herein,
e.g., with reference to FIG. 3.
[0179] At block 2806, a sample can be applied to, disposed over, or
received at the reflective phase grating. This can be done, e.g.,
using a micropipette. Examples are discussed above, e.g., with
reference to blocks 2402-2406, 2502, or 2704.
[0180] At block 2808, the reflective phase grating can be
irradiated, e.g., using a laser, lamp, LED, or other light source.
Focusing or collimating optics can additionally be used. Examples
are discussed above, e.g., with reference to blocks 2408, 2504, or
2706.
[0181] At block 2810, presence-related signals, e.g., representing
or otherwise based at least in part on resultant light, can be
determined, e.g., by detecting or measuring resultant light.
Presence-related signals can include HF, RPP, TPP, or other signals
useful for determining whether or when a target is being
irradiated. In some examples, before, concurrently with, or in
addition to measuring fluorescence, non-fluorescence or other
additional signal(s) are measured to determine, e.g., presence,
size, shape, or volume of a target or other particle being
irradiated. In some examples, the number of particles or the sizes
and shapes of individual particles are measured, and fluorescence
data from some of the particles are measured, e.g., to determine
the origins of the particles. Examples are discussed above, e.g.,
with reference to blocks 2408, 2506,2604, or 2708.
[0182] In some examples, block 2810 can include measuring HF or PP
signals in a reference area. The reference area can include, e.g.,
a mirror (non-grooved) area of a disc or LOC, or an area with
grooves. The reference area can be substantially free of samples or
targets. Block 2810 can include measuring the HF or PP signals in
the detection region and determining a variation of the measured
signal from the previously-measured signal in the reference area.
In some examples, variations between a reference signal and a
corresponding signal measured in a sample indicate the presence of
targets, EVs, or other microparticles in the sample, e.g., as
discussed herein with reference to FIGS. 11-27.
[0183] At block 2812, emitted fluorescence or other reflected,
transmitted, emitted, or scattered light can be measured, e.g.,
using a .mu.PMT or other sensors described herein. This can be
done, e.g., when or during a period in which the presence-related
signals indicate a target is being irradiated. This can reduce
mis-identification as target-related of photons not from a target.
Examples are discussed above, e.g., with reference to block 2604 or
FIG. 15.
[0184] At block 2814, detection signals from a .mu.PMT or other
sensors can be gated. For example, gating can be performed as
discussed above with reference to FIGS. 19-21. Although shown as
part of block 2812 for purposes of explanation, functions performed
at block 2814 can be performed before or after functions performed
at block 2812. For example, gating can be performed in the analog
domain on .mu.PMT or amplifier (FIG. 17) outputs, or can be
performed in the digital domain on digitized representations of
those outputs.
[0185] At block 2816, the location of irradiation ("irradiation
spot") can be moved, i.e., relative motion of an irradiation spot
and the reflective phase grating can be caused. For example, a
laser can be redirected, e.g., rasterized across the sample, or the
sample can be moved, e.g., by the spinning of a disc over a laser
or other irradiation source substantially fixed in tangential
position with respect to the axis of rotation of the disc. A
spot-traversal system can cause such relative motion, and can
include one or more of the following: rasterizers; polygons or
other movable mirrors; mirror or micromirror arrays; fixed mirrors;
rotational, linear, XY, or XYZ stages or drives; lenses; or
gratings. Examples are discussed above, e.g., with reference to
FIG. 22 or blocks 2606-2612 or 2710.
[0186] At block 2818, data of the detected fluorescence can be
processed to detect targets or measure properties of targets, e.g.,
as described herein. For example, detected photon signals can be
gated, e.g., as described herein. Example gating can be performed
based at least in part on HF and TPP signals, e.g., as in FIGS.
19-21. Pulse heights can be measured and recorded, e.g., in
histogram bins, or pulses can be counted. Examples are discussed
above, e.g., with reference to FIG. 1,11,13-15,20, or 21.
[0187] In some examples, at block 2818, measured, estimated, or
computed refractive indices of at least one of the medium (e.g.,
blood plasma or air), the target, or a portion of the target can be
used in computing sizes of the targets. Various aspects use
fluorescent dyes or a pre-check to distinguish targets from, e.g.,
surface defects, dust particles, non-target air bubbles, or other
microstructures that are not targets to be measured. In some
examples, the pre-check includes measuring data before applying
targets to the reflective phase grating, e.g., to detect, e.g.,
nanometer-scale defects in the substrate.
[0188] Targets can be detected using a rotating reflective grating
and an irradiation spot. Targets can also be detected using a
focused scanning spot in combination with a flow cell, e.g., as
described herein with reference to FIG. 1-6 or 10-16. In some
examples, the pitch of the phase grating can be set to
approximately twice the size of the targets to be detected. In some
examples, the refractive indices of the sample and the target can
be used to determine the particle size from the pulse width or
height of the HF or PP signals.
[0189] Some example parameters for various formats of LOCs or
measurement thereof are given in Table 6, below. Illustrative axes
are r (radial position on a disc), .theta. (angular position on a
disc, X/Y (Cartesian coordinates on a disc or chip), or v (position
along a flow axis of a flowing sample).
TABLE-US-00006 TABLE 6 Parameter Disc Format Chip Format Scanning
Axes r-.theta. r-.theta., X-Y, or X-v (v = flow axis) Shape disc
disc, rectangle, or flow cell Pattern 1 Pit, Track, Phase Scale or
Minor Mark Pattern 2 Spiral Spiral, Concentric, Parallel, None
Signal Dimension Fix on plane Three-dimension in gap channel Signal
Source 1 Embossed Particle in carrier fluid Depth (liquid/gas)
Signal Source 2 Surface Volume within focus depth Cover Refractive
Uniform RI Multiple refractive indices (RIs), Index e.g., three RIs
Main signal HF TPP + FL Cover thickness 1.2 mm (CD), 0.12 mm
(microscope slide) or 0.6 mm (DVD), other formats 0.1 mm
(BLU-RAY)
[0190] In some nonlimiting examples, an LOC as described herein
does not include data-storage structures such as pits, lands,
embedded dyes, or other structures used to record information on
optical discs. In some examples, an LOC as described herein
comprises a mirror or other substantially non-pitted or non-grooved
reflective surface. In some examples, the size of the focused spot
is calibrated to provide a scale for the detected particles (e.g.,
spot width in meters can be related to scan speed in m/s and pulse
width in seconds to determine target size). In some examples, a
reflective phase grating is configured with pitch and groove depth
(or land height) to provide gain under desired optical
configurations. In some examples, the focused spot is smaller than
the width of a groove or is smaller than the width of a land.
[0191] Some examples include one or more of the following: (a)
reflective grating or optics are scaled for particle size, e.g.,
the pitch, irradiation system, or lens are selected to maintain
p.gtoreq..lamda./2NA as described herein; (b) reflective grating
has a 1:1 aspect ratio L:G; (c) groove depth is .lamda./8n, where n
is the refractive index of the fluid sample containing the targets;
(d) grating pitch is decided by particle size and spot size; (e)
spot size is <2 .mu.m FWHM; (f) channel gap is approximately
equal to the focus depth; (g) channel gap is less than eight times
the focus depth; (h) a mirror use used and the size of the focused
spot provides scale information for determining particle size; (i)
the spot is scanned over the sample, e.g., to count particles/unit
volume; (j) scanning is configured as at least one of r-.theta.,
X-Y, or X-v; or (k) the targets are .gtoreq.10 nm.
Illustrative Results and Further Illustrative Examples
[0192] In some examples, drops of liquid were placed on the disc
discussed above with reference to FIG. 10. The drops were added to
regions 1004. The drops were permitted to dry for 3-8 min, forming
a drop area of 5-6 mm.sup.2. The drops included 170 nm polystyrene
beads. A concentration of .about.3.times.10.sup.9mL was tested.
[0193] Some tested example configurations used disc-format LOCs
such as the LOC shown in FIG. 10. In some examples, a traverse
signal was captured with the disc rotating but with no tracking.
The focus servo was active. A traverse signal was captured with the
irradiation spot scanning radially at a fixed speed).
[0194] In a tested example, at 1 m/s scanning velocity, a pulse
width of a measured pulse was substantially equal to FWHM spot
size: 240 nm, or 240 ns at 1 m/s, using a BLU-RAY laser spot of
.lamda.=405 nm. The measurement time per particle was .about.0.25
.mu.s/particle. In some examples, the irradiation spot has a
diameter <2 .mu.m FWHM.
[0195] FIG. 29 shows measured data of 100 nm gold (Au)
nanoparticles detected at a spot-scanning speed of 1.4 m/s. The
scanning speed of 1.4 m/s was the linear velocity of the spot with
respect to the disc. Experiments were performed with nanoparticles
in a liquid sample. Gold and polymer particles were tested. Gold
100 nm particles showed clear pulse signals with 30% modulation
amplitude and particles as small as 30 nm showed detectable signal.
In this configuration, laser beam illuminates individual particles
at appropriate concentration.
[0196] FIG. 30 shows measured data of 100 nm gold (Au)
nanoparticles detected at a spot-scanning speed of 1.4 m/s. As
shown, the nanoparticle produced a detectable signal having a width
of approximately 200 ns.
[0197] Further experiments were performed to evaluate particle
detection in a liquid environment. A 2 .mu.m gap microchannel was
formed on a phase grating surface of a disc and sealed by 100 um
cover glass with a 500 um diameter sample inlet made in a 1.1
mm-thick polymer supporting substrate. A similar structure is
discussed herein with reference to FIG. 1. The sample liquid filled
the micron-gap well by capillary action in an air atmosphere.
100-nm gold particles in water were detectable by modulation, and
spatial resolution was confirmed for k-dominant gold particles.
[0198] FIG. 31 shows measured data of a tested example. Waveforms
are shown for particles detected in grooves of a reflective phase
grating. A laser wavelength .lamda.=405 nm, a lens with numerical
aperture NA=0.85, and a disc with a cover thickness of 0.1 mm were
used. Other .lamda., NA, and cover-thickness values can also or
alternatively be used. Lower .lamda. or higher NA values can permit
detecting smaller targets than higher .lamda. or lower NA
values.
[0199] In FIG. 31, and also in FIGS. 32 and 33, HF and radial PP
signals are shown. The bottom half is a magnification of the
indicated portion on the top half The dashed box shows signals that
were measured of a target. The amplitude of modulation of the
measured signal, compared to (e.g., divided by) the peak-to-peak
excursion of that signal, is correlated with the optical path
length Anxd of the target, where An is the refractive-index
difference between the particle and the medium around it, and d is
the size of the particle (e.g., mean diameter). In some examples,
An is measured with reference to the polymer material of the disc.
In some examples, the medium is selected to have a different
refractive index than the disc, the particles, or both. In some
example, the substrate has n=1.50, the targets have n=1.53, and the
sample has n=1.58 (.DELTA.n=0.05).
[0200] FIG. 32 shows measured data of a tested example. Waveforms
are shown for particles detected on lands of a reflective phase
grating.
[0201] FIG. 33 shows measured data of a tested example. Waveforms
are shown for particles detected on a reflective surface
substantially without a grating ("mirror").
[0202] FIG. 34 shows an example plot correlating optical path
distance change (Anxd) with modulation amplitude (percent). In some
examples, the pulse width of an HF or TPP pulse times the scanning
velocity of the spot with respect to the target can be an estimate
of the tangential size of particle. The RPP pulse height can be
correlated with radial size, or the TPP pulse height can be
correlated with tangential size. In an example of a 100 nm particle
having n=1.592, a PP signal of 0.85V with a modulation
.DELTA.PP/PP=28% gives a .DELTA.n.times.d of 8 nm, so d=8 nm,
.DELTA.n=8 nm/0.05, giving an estimate, to a first order, of 160
nm. In some examples, for HF-signal noise .about.50 mV,
.DELTA.n>=.about.0.035 can be measured, e.g., particles as small
as 6 nm in some examples.
[0203] In some examples, RPP and TPP pulses can be detected for a
particular target (e.g., temporally overlapping RPP and TPP
pulses). The extents of the particle in radial and tangential
directions can be determined. A two-dimensional image of the target
can be determined from the extents. These determinations can be
performed for multiple targets, and a two-dimensional image of the
targets with respect to each other can be determined from the
extents of the particles and the relative timings of the signals,
which are correlated with the relative positions of the
targets.
[0204] In some examples, PP signals have high gain when the groove
depth of a reflective grating is .lamda./8n. In some examples, HF
signals have high gain when the groove depth of a reflective
grating is .lamda./4n. In some examples, a grating has a groove
depth of .lamda./8n or .lamda./4n. In some examples, a detection
system and reflective phase grating are configured for use with
.lamda.=405 nm and n=1.33, e.g., for aqueous samples. Accordingly,
groove depth can be, e.g., .lamda./8n=38.0 nm or .lamda./4n=76.1
nm. The PP signal can be measured to detect double-pass phase shift
due to the presence of a target. The optical path difference due to
target can be, e.g., .DELTA..phi.=.DELTA.n.times.thickness.times.2.
The modulation depth by a target can this be, or be proportional
to, sin (.pi./2.times..DELTA..phi./(.lamda./8)).
[0205] In some examples, a microvesicle has a diameter of
.about.100 nm. The microvesicle can include a lipid bilayer surface
with a thickness of .about.10 nm and n=1.48 surrounding an aqueous
core with n=1.38.+-.0.02. The microvesicle target can be included
in a water sample with n=1.333. In some examples, reflective
surface 2202 (e.g., the surface of a reflective phase grating) can
include Al with n=0.879 and k=2.984, at a thickness of, e.g.,
.about.50 nm-100 nm. Cover 2314 can include glass or polymer with
n=.about.1.46-.about.1.65 and a thickness of 100 .mu.m.+-.3
.mu.m.
[0206] Some experiments detected a 170 nm coated bead arranged in a
pressure-sensitive adhesive (PSA) layer. Some experiments detected
a 170 nm polystyrene (PS) bead. Some experiments detected a 100 nm
bead in air. Some experiments detected a 70 nm polymer nanoparticle
in air. Some experiments detected 50 nm Au nanoparticles or 30 nm
Au nanoparticles. Some of these experiments used only the HF signal
to detect the nanoparticles. In some examples, HF pulse height was
correlated with particle size.
[0207] Some experiments had a noise limit of approximately 30 nm,
corresponding to 34 mV of noise and 200 mV of signal for a 170 nm
bead. Some experiments had a noise limit of .about.10 nm,
corresponding to a 5.5 V(pk-pk) TPP signal and 0.22 V(pk-pk) of
noise for a 250 nm target. In some experiments, TPP signals or RPP
signals were detected.
[0208] A reflective grating and focused scanning spot together
provided effective detection of targets, e.g., vesicles or
nanoparticles. In some experiments, the number of particles was
countable by the number of pulses, e.g., of the HF signal. In some
experiments, particle size was correlated with the pulse width and
modulation depth of the HF and PP signals.
Example Clauses
[0209] A: A measurement system comprising a reflective phase
grating, a light source, a motion system configured to induce
relative motion of the light source and the phase grating, and a
detection system configured to detect light reflected by the phase
grating.
[0210] B: The system according to paragraph A, further including a
cover spaced apart from the reflective phase grating.
[0211] C: The system according to paragraph A or B, further
including a sample inlet.
[0212] D: The system according to paragraph C, further including a
vacuum port.
[0213] E: The system according to paragraph D, wherein the vacuum
port and the sample inlet are arranged on opposite sides of a
measurement area, e.g., a detection region, including the
reflective phase grating.
[0214] F: The system according to any of paragraphs A-E, further
including a vacuum port.
[0215] G: A measurement device comprising a substrate and a
reflective phase grating arranged on, in, or over the
substrate.
[0216] H: The device according to paragraph G, further including a
cover spaced apart from the reflective phase grating.
[0217] I: The device according to paragraph G or H, further
including a sample inlet.
[0218] J: The device according to paragraph I, further including a
vacuum port.
[0219] K: The device according to paragraph J, wherein the vacuum
port and the sample inlet are arranged on opposite sides of a
measurement are, e.g., a detection region, including the reflective
phase grating.
[0220] L: The device according to any of paragraphs G-K, further
including a vacuum port.
[0221] M: A method of measuring, comprising irradiating a
reflective phase grating and measuring reflected light.
[0222] N: The method according to paragraph M, further including
disposing a sample over the reflective phase grating or receiving a
sample at the reflective phase grating.
[0223] O: The method according to paragraph M or N, further
including causing relative motion of an irradiation spot and the
reflective phase grating.
[0224] P: A measurement system, comprising: a spot-traversal system
for causing relative motion of a sample and an irradiation spot,
wherein the sample includes one or more fluorescent markers having
respective fluorescence wavelengths; and an optical detection
system configured to detect fluorescent light from at least some of
the fluorescent markers irradiated by the irradiation spot.
[0225] Q: The system according to paragraph P, wherein the
fluorescent markers include fluorescent-dyed anti-bodies configured
to conjugate with molecules of interest in the sample, or include
fluorescent tags configured to conjugate with the molecules of
interest.
[0226] R: The system according to paragraph P or Q, wherein the
irradiation spot includes a focused laser spot with a full-width at
half maximum diameter smaller than fourteen microns or fourteen
hundred nanometers.
[0227] S: The system according to any of paragraphs P-R, the
optical detection system configured to detect light at each of a
plurality of wavelengths and the system further including a
spectral discriminator arranged between the sample and the optical
detection system, the spectral discriminator configured to provide
light at wavelength(s) of the plurality of wavelengths to the
optical detection system.
[0228] T: The system according to paragraph S, wherein the spectral
discriminator includes at least one of a dichroic mirror, a
grating, a prism, a filter, or a polychromator.
[0229] U: The system according to any of paragraphs P-T, wherein
the optical detection system is configured to detect single photon
pulses without averaging.
[0230] V: The system according to any of paragraphs P-U, further
including a tracking system configured to provide one or more
tracking signals based at least in part on resultant light
substantially at a wavelength of the irradiation spot, wherein the
optical detection system is configured to gate detected photon
signals based at least in part on one(s) of the tracking
signal(s)
[0231] W: The system according to paragraph V, wherein the gating
is based at least in part on a high-frequency (HF) tracking signal,
a tangential push-pull (TPP) signal, or both.
[0232] X: The system according to any of paragraphs P-W, further
comprising a reflective phase grating over which the sample is
arranged.
[0233] Y: The system according to paragraph X, further including a
cover spaced apart from the reflective phase grating.
[0234] Z: The system according to any of paragraphs P-Y, further
including a sample inlet.
[0235] AA: The system according to any preceding system paragraph,
further including a vacuum port.
[0236] AB: The system according to paragraph AA, wherein the vacuum
port and the sample inlet are arranged on opposite sides of a
measurement area including the reflective phase grating.
[0237] AC: A method of measuring, comprising irradiating a
reflective phase grating measuring resultant light in a selected
wavelength range, and gating the measured light based at least in
part upon a tracking signal.
[0238] AD: The method according to paragraph AC, further including
disposing a sample over the reflective phase grating or receiving a
sample at the reflective phase grating.
[0239] AE: The method according to paragraph AC or AD, further
including causing relative motion of an irradiation spot and the
reflective phase grating.
[0240] AF: An assembly, comprising: a target holder configured to
retain a target in a detection region, wherein the target holder
comprises a reflective surface configured to reflect at least part
of a focused spot of light to provide resultant light; an
irradiation system configured to irradiate at least part of the
detection region with the focused spot of light; a motion system
configured to cause motion of the focused spot of light relative to
the reflective surface; and a detection system configured to detect
the resultant light.
[0241] AG: The assembly according to paragraph AF, wherein: the
irradiation system is configured to provide the focused spot of
light, which comprises light of a predetermined wavelength; the
irradiation system comprises a lens having a predetermined
numerical aperture and arranged to focus the light of the
predetermined wavelength to provide the focused spot of light; and
the reflective grating has a pitch exceeding a reference pitch
substantially equal to a result of dividing the predetermined
wavelength by twice the predetermined numerical aperture.
[0242] AH: The assembly according to paragraph AF or AG, wherein:
the irradiation system is configured to provide the focused spot of
light, which comprises light of a predetermined wavelength; and the
detection system comprises at least one optical detector configured
to detect light of a different wavelength than the predetermined
wavelength.
[0243] AI: The assembly according to any of paragraphs AF-AH,
further comprising a sample-delivery system configured to apply a
sample comprising the target to the at least part of the detection
region.
[0244] AJ: The assembly according to any of paragraphs AF-AI,
further comprising a sample-delivery system configured to apply a
fluidic sample comprising the target to the at least part of the
detection region.
[0245] AK: The assembly according to paragraph AJ, wherein: the
reflective surface comprises a reflective grating; the irradiation
system is further configured to provide the focused spot of light,
which comprises light of a predetermined wavelength; the
irradiation system comprises a lens having a predetermined
numerical aperture and being arranged to focus the light of the
predetermined wavelength to provide the focused spot of light; the
reflective grating has a pitch exceeding a reference pitch
substantially equal to a result of dividing the predetermined
wavelength by twice the numerical aperture; the fluidic sample
comprises a fluid having a predetermined refractive index; and the
reflective grating includes at least one groove having a depth
substantially between a first reference value and a second
reference value, the first reference value being the result of
dividing the predetermined wavelength by the product of sixteen and
the refractive index and the second reference value being
substantially three times the first reference value.
[0246] AL: The assembly according to paragraph AJ or AK, wherein
the sample-delivery system is configured to apply the fluidic
sample, which comprises a liquid.
[0247] AM: The assembly according to any of paragraphs AJ-AL,
further comprising a flow system, e.g., a vacuum pump or fluid
pump, configured to move the fluidic sample through the detection
region.
[0248] AN: The assembly according to any of paragraphs AJ-AM,
wherein the fluidic sample comprises a fluid having a predetermined
refractive index and the irradiation system is configured to
provide the focused spot of light comprising light of a
predetermined wavelength.
[0249] AO: The assembly according to paragraph AN, wherein the
reflective grating includes at least one groove having a depth
substantially equal to a result of dividing the predetermined
wavelength by eight times the predetermined refractive index.
[0250] AP: The assembly according to paragraph AN or AO, wherein:
the assembly further comprises a fluid channel at least partly
overlapping with the at least part of the detection region; and the
irradiation system is configured to provide the light substantially
in an irradiation direction.
[0251] AQ: The assembly according to paragraph AP, wherein the
channel has a height substantially along the irradiation direction
substantially equal to a result of dividing the predetermined
wavelength by eight times the predetermined refractive index.
[0252] AR: The assembly according to paragraph AP or AQ, wherein
the channel has a height substantially along the irradiation
direction substantially between a first reference value and a
second reference value, the first reference value being the result
of dividing the predetermined wavelength by the product of sixteen
and the predetermined refractive index and the third reference
value being substantially three times the first reference
value.
[0253] AS: The assembly according to any of paragraphs AJ-AR,
wherein the flow system is configured to cause the fluidic sample
to move through the at least part of the detection region at least
substantially in a flow direction, and the motion system is
configured to concurrently cause relative motion of the focused
spot of light in a second direction different from (e.g.,
substantially perpendicular to) the first direction.
[0254] AT: The assembly according to any of paragraphs AF-AS,
wherein: the detection system comprises at least one split
photodetector configured to provide a plurality of detection
signals; and the assembly further comprises a data-processing
system configured to determine, based at least in part on at least
two of the plurality of detection signals, at least one of a sum
signal, a transverse push-pull signal, or a radial push-pull
signal.
[0255] AU: The assembly according to paragraph AT, further
comprising a gating system configured to provide a particle signal
associated with a temporal overlap between a first signal of the
plurality of detection signals and a second, different signal of
the plurality of detection signals.
[0256] AV: The assembly according to paragraph AU, wherein the
first signal comprises the sum signal and the second signal
comprises the transverse push-pull signal.
[0257] AW: The assembly according to any of paragraphs AT-AV,
wherein: the irradiation system is further configured to provide
the focused spot of light, which comprises light of a predetermined
wavelength; and the detection system further comprises at least one
optical detector configured to detect light of a different
wavelength than the predetermined wavelength and to provide a
detection signal corresponding to the light of the different
wavelength.
[0258] AX: The assembly according to paragraph AW, further
comprising a gating system configured to provide a particle signal
associated with a temporal overlap between at least one of the
plurality of detection signals and the detection signal
corresponding to the light of the different wavelength.
[0259] AY: The assembly according to paragraph AX, further
comprising a counter configured to count discrete occurrences of
the particle signal.
[0260] AZ: The assembly according to any of paragraphs AW-AY,
wherein the different wavelength is a wavelength longer than the
predetermined wavelength.
[0261] BA: The assembly according to any of paragraphs AF-AZ,
wherein: the reflective surface comprises a reflective grating
having at least one groove; and the motion system is further
configured to cause relative motion of the focused spot of light
substantially across the at least one groove.
[0262] BB: The assembly according to any of paragraphs AF-BA,
wherein the reflective surface comprises a reflective grating
having at least one groove.
[0263] BC: The assembly according to paragraph BB, wherein the
motion system is further configured to cause relative motion of the
focused spot of light substantially along the at least one
groove.
[0264] BD: The assembly according to paragraph BC, wherein the
motion system is further configured to rotate the reflective
surface to define a tangential direction of the rotating reflective
surface, and to concurrently translate the focused spot of light
substantially in a direction different from the tangential
direction to track the focused spot of light substantially along
the at least one groove.
[0265] BE: The assembly according to any of paragraphs AF-BD,
wherein the motion system is further configured to cause relative
motion of the focused spot of light in two different (e.g.,
substantially perpendicular) directions over the reflective
surface.
[0266] BF: The assembly according to any of paragraphs AF-BE,
wherein the motion system is further configured to cause motion of
the reflective surface in a first direction and to concurrently
cause relative motion of the focused spot of light in a second
direction different from (e.g., substantially perpendicular to) the
first direction.
[0267] BG: The assembly according to any of paragraphs AF-BF,
wherein the motion system is further configured to cause motion of
the reflective surface and to concurrently maintain a focus of the
focused spot of light with respect to the reflective surface.
[0268] BH: The assembly according to any of paragraphs AF-BG,
wherein the reflective surface comprises a substantially flat
reflective surface.
[0269] BI: The assembly according to any of paragraphs AF-BH,
wherein the motion system is further configured to adjust a focal
position of the focused spot of light along an axis substantially
normal to at least a portion of the reflective surface.
[0270] BJ: A device, comprising: a substrate; a sample inlet
associated with the substrate; and a reflective grating associated
with the substrate, wherein: the reflective grating is configured
to retain a fluidic sample in a detection region; the detection
region is fluidically connected to the sample inlet; and the
detection region is operatively arranged with respect to the
reflective grating so that at least a portion of light passing
through the detection region towards the reflective grating also
passes through the detection region after reflecting off the
reflective grating.
[0271] BK: The device according to paragraph BJ, wherein the
fluidic sample comprises a liquid.
[0272] BL: The device according to paragraph BJ or BK, further
including an outlet fluidically connected to the detection region
and spaced apart from the sample inlet.
[0273] BM: The device according to any of paragraphs BJ-BL, further
including a cover spaced apart from the reflective grating and
arranged so that the detection region is at least partly between
the reflective grating and the cover, wherein the cover is at least
partly transparent to the at least a portion of the light.
[0274] BN: The device according to any of paragraphs BJ-BM, further
comprising a reservoir fluidically connected to the detection
region and spaced apart from the sample inlet.
[0275] BO: The device according to paragraph BN, wherein the
reservoir has a volume exceeding a sum of a volume of the sample
inlet and a volume of the detection region.
[0276] BP: The device according to any of paragraphs BJ-BO, wherein
the reflective grating comprises at least one groove, and the at
least one groove comprises at least two of: a spiral groove
portion, a straight groove portion, or a circular groove
portion.
[0277] BQ: The device according to paragraph BP, wherein the at
least one groove comprises a first groove and a second groove, the
first groove comprises at least a first one of the spiral groove
portion, the straight groove portion, and the circular groove
portion, and the second groove comprises at least a second,
different one of the spiral groove portion, the straight groove
portion, and the circular groove portion.
[0278] BR: The device according to any of paragraphs BJ-BQ,
wherein: the sample inlet is configured to receive a provided
sample; and the device further comprises a filter element
operatively arranged to filter the provided sample to provide the
fluidic sample.
[0279] BS: The device according to any of paragraphs BJ-BR, wherein
the substrate is a disc-format substrate.
[0280] BT: The device according to any of paragraphs BJ-BS, wherein
the sample inlet is one of a plurality of spaced-apart sample
inlets of the device; the detection region is one of a plurality of
detection regions of the device; and the device further comprises
one or more spaced-apart vacuum ports; wherein individual detection
regions of the plurality of detection regions are fluidically
connected to at least one of the one or more vacuum ports and are
fluidically connected to at least one of the plurality of sample
inlets.
[0281] BU: The device according to paragraph BT, further comprising
a vacuum channel fluidically connected to at least one of the one
or more spaced-apart vacuum ports.
[0282] BV: The device according to paragraph BT or BU, wherein at
least one of the vacuum ports is arranged on an opposite side of
the detection region from at least one of the sample inlets.
[0283] BW: The device according to any of paragraphs BJ-BV,
wherein: the sample inlet is one of a plurality of spaced-apart
sample inlets of the device; the detection region is one of a
plurality of detection regions of the device; and individual
detection regions of the plurality of detection regions are
fluidically connected to at least one of the plurality of sample
inlets.
[0284] BX: The device according to paragraph BW, further comprising
at least one reservoir, wherein individual detection regions of the
plurality of detection regions are fluidically connected to at
least one of the at least one reservoir.
[0285] BY: A method, comprising: disposing a fluidic sample
comprising a target over a reflective surface; irradiating the
reflective surface using a spot of light; and detecting resultant
light from the reflective surface.
[0286] BZ: The method according to paragraph BY, further comprising
causing the fluidic sample to flow through a flow channel across
the reflective surface and causing the spot of light to move with
respect to the reflective surface at least partly across the flow
channel.
[0287] CA: The method according to paragraph BY or BZ, wherein the
fluidic sample comprises a liquid.
[0288] CB: The method according to any of paragraphs BY-CA, further
including causing relative motion of the spot of light and at least
a portion of the fluidic sample.
[0289] CC: The method according to paragraph CB, wherein the
causing relative motion comprises causing the spot of light to move
with respect to the reflective surface.
[0290] CD: The method according to paragraph CC, wherein the
causing relative motion comprises moving the reflective surface
with respect to the spot of light.
[0291] CE: The method according to any of paragraphs CB-CD, wherein
the causing relative motion comprises causing the at least a
portion of the fluidic sample to flow across the reflective surface
substantially in a flow direction.
[0292] CF: The method according to paragraph CE, wherein the
causing relative motion further comprises causing motion of the
spot of light relative to the reflective surface (e.g,.
concurrently with the causing the at least a portion of the fluidic
sample to flow across the reflective surface).
[0293] CG: The method according to paragraph CF, further comprising
causing relative motion of the spot of light in a direction
substantially different from (e.g., substantially perpendicular to)
the flow direction.
[0294] CH: The method according to any of paragraphs BY-CG, further
comprising drying the fluidic sample after the disposing and before
the irradiating.
[0295] CI: The method according to any of paragraphs BY-CH, wherein
the reflective surface comprises a reflective grating.
[0296] CJ: The method according to any of paragraphs BY-CI, wherein
the reflective surface comprises a substantially flat surface.
[0297] CK: The method according to any of paragraphs BY-CJ, wherein
the reflective surface is associated with a disc; and wherein the
method further comprises: rotating the disc, wherein the disc moves
with respect to the spot of light during the rotating and the
reflective surface comprises at least one track; irradiating the
reflective surface during the rotating; and causing the spot of
light to move to follow the track of the reflective surface.
[0298] CL: The method according to paragraph CK, further comprising
maintaining a focus of the spot of light with respect to the
reflective surface concurrently with the rotating.
[0299] CM: The method according to any of paragraphs BY-CL, further
comprising: applying a dye to the fluidic sample before the
disposing; and detecting light emitted by the dye in response to
the irradiating using the spot of light.
[0300] CN: The method according to any of paragraphs BY-CM, wherein
the target comprises at least two portions having respective,
different refractive indices.
[0301] CO: The method according to paragraph CN, wherein the
fluidic sample has a refractive index different from each of the
refractive indices of the target.
[0302] CP: The method according to any of paragraphs BY-CO, further
comprising adjusting a focal position of the spot of light with
respect to the reflective surface.
[0303] CX: The method according to any of paragraphs BY-CP, further
comprising adjusting a focal position of the spot of light with
respect to the reflective surface.
[0304] CY: A computer-readable medium, e.g., a computer storage
medium, having thereon computer-executable instructions, the
computer-executable instructions upon execution configuring a
computer to perform operations as recited in any of paragraphs
BY-CX.
[0305] CZ: A device comprising: a processor; and a
computer-readable medium, e.g., a computer storage medium, having
thereon computer-executable instructions, the computer-executable
instructions upon execution by the processor configuring the device
to perform operations as recited in any of paragraphs BY-CX.
[0306] DA: A system comprising: means for processing; and means for
storing having thereon computer-executable instructions, the
computer-executable instructions including means to configure the
system to carry out a method as recited in any of paragraphs
BY-CX.
[0307] DB: An assembly, comprising: a target holder configured to
retain a target in a detection region; a reflective surface
configured to reflect at least part of a focused spot of light to
provide resultant light; an irradiation system configured to
irradiate at least part of the detection region with the focused
spot of light; a motion system configured to cause motion of the
focused spot of light relative to the reflective surface; and a
detection system configured to detect the resultant light.
[0308] DC: The assembly according to paragraph DB, further
comprising at least one of the features listed in any of paragraphs
AG-BI.
[0309] DD: An assembly according to any of paragraphs AF-BI, DB, or
DC, wherein the target holder comprises a device as recited in any
of paragraphs BJ-BX.
Conclusion
[0310] In view of the foregoing, various aspects provide effective
detection or size measurement of small particles. A technical
effect of various aspects is to measure physical properties of a
sample, e.g., a sample of blood or another bodily fluid. Some
examples use in-focus spots to provide a small spot size, which can
improve detection accuracy and can permit detecting small
particles. Some examples of in-focus spots permit using a phase
grating to improve detection sensitivity. The claims are not
limited to implementations that address any of the example use
cases described herein. The claims are also not limited to
implementations that address any identified deficiencies of any
prior schemes.
[0311] Various examples use optical readouts to detect biological
or other nanoparticles or other targets. Various examples use
BLU-RAY readout optics or discs to increase resolution and detect
targets much smaller than some prior schemes can detect. Various
examples permit detecting small biological particles without
pre-treatment that is likely to kill cells, such as drying or
exposure to high vacuum. Various examples use TPP signals to detect
particles, unlike optical-storage configurations that use HF
signals to detect pits and lands. Various examples include LOC
structures that move fluid samples via capillary action, permitting
effectively measuring samples without requiring vacuum pumps.
Various examples permit measuring multiple samples on a single
LOC.
[0312] Although the techniques have been described in language
specific to structural features and/or methodological acts, it is
to be understood that the appended claims are not necessarily
limited to the features and/or acts described. Rather, the features
and acts are described as example implementations of such
techniques. For example, network 1850, processor 1886, and other
structures described herein for which multiple types of
implementing devices or structures are listed can include any of
the listed types, and/or multiples and/or combinations thereof
[0313] Throughout this description, some aspects are described in
terms that would ordinarily be implemented as software programs.
Those skilled in the art will readily recognize that the equivalent
of such software can also be constructed in hardware, firmware, or
micro-code. Because data-manipulation algorithms and systems are
well known, the present description is directed in particular to
algorithms and systems forming part of, or cooperating more
directly with, systems and methods described herein. Other aspects
of such algorithms and systems, and hardware or software for
producing and otherwise processing signals or data involved
therewith, not specifically shown or described herein, are selected
from such systems, algorithms, components, and elements known in
the art. Given the systems and methods as described herein,
software not specifically shown, suggested, or described herein
that is useful for implementation of any aspect is conventional and
within the ordinary skill in such arts.
[0314] The operations of the example processes are illustrated in
individual blocks and summarized with reference to those blocks.
The processes are illustrated as logical flows of blocks, each
block of which can represent one or more operations that can be
implemented in hardware, software, and/or a combination thereof. In
the context of software, the operations represent
computer-executable instructions stored on one or more
computer-readable media that, when executed by one or more
processors, enable the one or more processors to perform the
recited operations. Generally, computer-executable instructions
include routines, programs, objects, modules, components, data
structures, and the like that perform particular functions and/or
implement particular abstract data types. The order in which the
operations are described is not intended to be construed as a
limitation, and any number of the described operations can be
executed in any order, combined in any order, subdivided into
multiple sub-operations, and/or executed in parallel to implement
the described processes. The described processes can be performed
by resources associated with one or more data-processing systems
1801 or 1802 or other computing device(s), or components thereof,
such as one or more internal and/or external CPUs and/or GPUs,
and/or one or more pieces of hardware logic such as FPGAs, DSPs,
and/or other types described above.
[0315] All of the methods and processes described above can be
embodied in, and fully automated via, software code modules
executed by one or more general purpose computers and/or
processors. The code modules can be stored in any type of
computer-readable storage medium and/or other computer storage
device. Some and/or all of the methods can be embodied in
specialized computer hardware.
[0316] Conditional language such as, among others, "can," "could,"
"might" and/or "may," unless specifically stated otherwise, are
understood within the context to present that certain examples
include, while other examples do not include, certain features,
elements and/or steps. Thus, such conditional language is not
generally intended to imply that certain features, elements and/or
steps are in any way required for one or more examples and/or that
one or more examples necessarily include logic for deciding, with
and/or without user input and/or prompting, whether certain
features, elements and/or steps are included and/or are to be
performed in any particular example. The word "or" and the phrase
"and/or" are used herein in an inclusive sense unless specifically
stated otherwise. Accordingly, conjunctive language such as the
phrases "X, Y, or Z," "X, Y, and/or Z," or "at least one of X, Y or
Z," unless specifically stated otherwise, is to be understood as
signifying that an item, term, etc., can be either X, Y, or Z, or a
combination thereof.
[0317] Any routine descriptions, elements and/or blocks in the flow
diagrams described herein and/or depicted in the attached figures
should be understood as potentially representing modules, segments,
and/or portions of code that include one or more executable
instructions for implementing specific logical functions and/or
elements in the routine. Alternative implementations are included
within the scope of the examples described herein in which elements
and/or functions can be deleted and/or executed out of order from
any order shown or discussed, including substantially synchronously
and/or in reverse order, depending on the functionality involved as
would be understood by those skilled in the art. It should be
emphasized that many variations and modifications can be made to
the above-described examples, the elements of which are to be
understood as being among other acceptable examples. All such
modifications and variations are intended to be included herein
within the scope of this disclosure. Moreover, in the claims, any
reference to a group of items provided by a preceding claim clause
is a reference to at least some of the items in the group of items,
unless specifically stated otherwise.
[0318] The invention is inclusive of combinations of the aspects
described herein. References to "a particular aspect" (or
"embodiment" or "version") and the like refer to features that are
present in at least one aspect of the invention. Separate
references to "an aspect" (or "embodiment") or "particular aspects"
or the like do not necessarily refer to the same aspect or aspects;
however, such aspects are not mutually exclusive, unless so
indicated or as are readily apparent to one of skill in the art.
The use of singular or plural in referring to "method" or "methods"
and the like is not limiting.
* * * * *