U.S. patent application number 10/832635 was filed with the patent office on 2004-10-07 for two-dimensional spectral imaging system.
This patent application is currently assigned to Quantum Dot Corporation. Invention is credited to Empedocles, Stephen A., Watson, Andrew R..
Application Number | 20040197816 10/832635 |
Document ID | / |
Family ID | 22721719 |
Filed Date | 2004-10-07 |
United States Patent
Application |
20040197816 |
Kind Code |
A1 |
Empedocles, Stephen A. ; et
al. |
October 7, 2004 |
Two-dimensional spectral imaging system
Abstract
Improved devices, systems, and methods for sensing and/or
identifying signals from within a signal detection region are
well-suited for identification of spectral codes. Large numbers of
independently identifiable spectral codes can be generated by quite
small bodies, and a plurality of such bodies or probes may be
present within a detection region. Simultaneously imaging of
identifiable spectra from throughout the detection region allows
the probes to be identified. As the identifiable spectra can be
treated as being generated from a point source within a much larger
detection field, a prism, diffractive grading, holographic
transmissive grading, or the like can spectrally disperse the
images of the labels across a sensor surface. A CCD can identify
the relative wavelengths of signals making up the spectra. Absolute
signal wavelengths may be identified by determining positions of
the labels, by an internal wavelength reference within the spectra,
or the like.
Inventors: |
Empedocles, Stephen A.;
(Mountain View, CA) ; Watson, Andrew R.; (Belmont,
CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
Quantum Dot Corporation
Hayward
CA
|
Family ID: |
22721719 |
Appl. No.: |
10/832635 |
Filed: |
April 26, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10832635 |
Apr 26, 2004 |
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09827076 |
Apr 5, 2001 |
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6759235 |
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60195520 |
Apr 6, 2000 |
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Current U.S.
Class: |
435/6.13 ;
435/7.1 |
Current CPC
Class: |
B01J 2219/00722
20130101; B01L 2400/0454 20130101; B01J 2219/00432 20130101; B01L
3/502715 20130101; G01N 21/274 20130101; G01N 2035/00742 20130101;
B01J 2219/00576 20130101; G01N 21/253 20130101; B01J 2219/00707
20130101; B01J 2219/00596 20130101; G01N 21/272 20130101; G01N
2201/12 20130101; B01L 2400/0457 20130101; G01N 2015/0092 20130101;
G01N 21/6428 20130101; B01J 2219/005 20130101; G01N 21/278
20130101; G01N 33/54373 20130101; B01J 2219/00585 20130101; G01N
21/25 20130101; B01J 2219/00657 20130101; B01L 3/545 20130101; B01L
2300/021 20130101; G01N 2201/06113 20130101; B01J 2219/00677
20130101; B82Y 30/00 20130101; G01N 21/6452 20130101; B01L 3/502761
20130101; B01J 2219/00574 20130101; G01N 21/6486 20130101; G01N
2015/1497 20130101; B01J 2219/00743 20130101; G01N 2035/00752
20130101; B01J 2219/00317 20130101; B01J 2219/00648 20130101; B01L
3/5085 20130101; G01N 21/6489 20130101; G01N 2201/04 20130101; B01J
2219/00659 20130101; G01N 2015/1472 20130101; G01N 2021/6441
20130101; G01N 2035/00158 20130101; G01N 21/6456 20130101; B01L
2200/0668 20130101; B01L 2300/0819 20130101; B01L 2300/0829
20130101; B01J 2219/00578 20130101; G01N 2021/6482 20130101; C40B
40/06 20130101; C40B 60/14 20130101; B01L 3/5025 20130101 |
Class at
Publication: |
435/006 ;
435/007.1 |
International
Class: |
C12Q 001/68; G01N
033/53 |
Claims
What is claimed is:
1. A method for identifying signals of differing strengths, the
method comprising: generating a plurality of signals in response to
excitation energy, the signals comprising higher intensity signals
and lower intensity signals; sensing the lower intensity signals by
simultaneously imaging the signals on a sensor; and sequentially
sensing at least some of the higher intensity signals.
2. The method of claim 1, wherein at least one of the signals is
generated by a semiconductor nanocrystal.
3. The method of claim 1, wherein sensing the lower intensity
signals comprises imaging for a first integration time, and wherein
sequentially sensing the higher intensity signals comprises
sequentially imaging for a second integration time shorter than the
first integration time.
4. The method of claim 1, further comprising filtering the higher
intensity signals from the simultaneously imaged signals.
5. The method of claim 4, wherein the higher intensity signals have
wavelengths that are different than wavelengths of the lower
intensity signals, and wherein the filtering step comprises
wavelength filtering the higher intensity signals.
6. The method of claim 1, wherein the higher intensity signals are
sequentially sensed by scanning markers generating the signals, and
wherein the markers generating the higher intensity signals are
spatially intermingled with the markers generating the lower
intensity signals.
7. The method of claim 6, wherein the scanning step comprises
scanning an aperture relative to the markers.
8. The method of claim 7, wherein the scanning step comprises
scanning a slit relative to the markers.
9. The method of claim 1, wherein the excitation energy comprises a
first energy, the first energy exciting high-energy markers to
generate the high energy signals, and a second energy, the second
energy exciting low-energy markers to generate the lower energy
signals.
10. The method of claim 9, wherein the second energy is less than
the first energy, and wherein the second energy selectively excites
the low energy markers.
11. The method of claim 1, wherein the high intensity signals are
generated by label markers and define an identifiable spectral
code, and wherein the low intensity signals are generated by assay
markers and indicate results of a plurality of assays, each assay
having an associated spectral code.
12. The method of claim 11, wherein the markers are supported by
probe bodies to define probes, each probe comprising a label with
at least one label marker to generate the spectral code, wherein at
least one assay marker is associated with the probe to indicate
results of an associated assay, and further comprising determining
each assay result by identifying each label and correlating the
label with the associated marker signal.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a divisional of and claims the benefit
of priority from U.S. patent application Ser. No. 09/827,076,
entitled "Two-Dimensional Spectral Imaging System," (Atty. Docket
No. 019916-004300US) filed on Apr. 5, 2001, which claims the
benefit under 35 USC 119(e) of U.S. Provisional Patent Application
No. 60/195,520, entitled "Method for Encoding Materials with
Semiconductor Nanocrystals, Compositions Made Thereby, and Devices
for Detection and Decoding Thereof," (Atty. Docket No.
019916-004700US) filed on Apr. 6, 2000, the full disclosures of
which are incorporated herein by reference.
[0002] The subject matter of the present application is also
related to the following co-pending patent applications, the
disclosures of which are also incorporated herein by reference:
U.S. patent application Ser. No. 09/160,458 filed on Sep. 24, 1998
(Atty. Docket No. 19916-000300US), entitled "Inventory Control";
U.S. patent application Ser. No. 09/397,432 filed on Sep. 17, 1999
(Atty. Docket No. 19916-002500US), entitled "Inventory Control";
PCT Patent Publication No. WO 99/50916 published on Apr. 1, 1999,
entitled "Quantum Dot White and Colored Light Emitting Diodes"; and
U.S. patent application Ser. No. 09/259,982 filed Mar. 1, 1999,
entitled "Semiconductor Nanocrystal Probes for Biological
Applications and Process for Making and Using Such Probes" (Atty.
Docket No. 19916-001600US).
BACKGROUND OF THE INVENTION
[0003] The present invention generally provides devices,
compositions of matter, kits, systems and methods for detecting and
identifying a plurality of signals from within a signal area. In a
particular embodiment, the invention provides systems and methods
for detecting and identifying a plurality of spectral barcodes from
throughout a sensing area, especially for identifying and/or
tracking inventories of elements, for high-throughput assay
systems, and the like. The invention will often use labels which
emit identifiable spectra that include a number of discreet signals
having measurable wavelengths and/or intensities.
[0004] Tracking the locations and/or identities of a large number
of items can be challenging in many settings. Barcode technology in
general, and the Universal Product Code in particular, has provided
huge benefits for tracking a variety of objects. Barcode
technologies often use a linear array of elements printed either
directly on an object or on labels which may be affixed to the
object. These barcode elements often comprise bars and spaces, with
the bars having varying widths to represent strings of binary ones,
and the spaces between the bars having varying widths to represent
strings of binary zeros.
[0005] Barcodes can be detected optically using devices such as
scanning laser beams or handheld wands. Similar barcode schemes can
be implemented in magnetic media. The scanning systems often
electro-optically decode the label to determine multiple
alphanumerical characters that are intended to be descriptive of
(or otherwise identify) the article or its character. These
barcodes are often presented in digital form as an input to a data
processing system, for example, for use in point-of-sale
processing, inventory control, and the like.
[0006] Barcode techniques such as the Universal Product Code have
gained wide acceptance, and a variety of higher density
alternatives have been proposed. Unfortunately, these standard
barcodes are often unsuitable for labeling many "libraries" or
groupings of elements. For example, small items such as jewelry or
minute electrical components may lack sufficient surface area for
convenient attachment of the barcode. Similarly, emerging
technologies such as combinatorial chemistry, genomics research,
microfluidics, micromachines, and other nanoscale technologies do
not appear well-suited for supporting known, relatively large-scale
barcode labels. In these and other developing fields, it is often
desirable to make use of large numbers of fluids, and identifying
and tracking the movements of such fluids using existing barcodes
is particularly problematic. While a few chemical encoding systems
for chemicals and fluids have been proposed, reliable and accurate
labeling of large numbers of small and/or fluid elements remains a
challenge.
[0007] Small scale and fluid labeling capabilities have recently
advanced radically with the suggested application of semiconductor
nanocrystals (also known as Quantum Dot.TM. particles), as detailed
in U.S. patent application Ser. No. 09/397,432, the full disclosure
of which is incorporated herein by reference. Semiconductor
nanocrystals are microscopic particles having size-dependent
optical and/or electrical properties. As the band gap energy of
such semiconductor nanocrystals vary with a size, coating and/or
material of the crystal, populations of these crystals can be
produced having a variety of spectral emission characteristics.
Furthermore, the intensity of the emission of a particular
wavelength can be varied, thereby enabling the use of a variety of
encoding schemes. A spectral label defined by a combination of
semiconductor nanocrystals having differing emission signals can be
identified from the characteristics of the spectrum emitted by the
label when the semiconductor nanocrystals are energized.
[0008] While semiconductor nanocrystal-based spectral labeling
schemes represent a significant advancement for tracking and
identifying many elements of interest, still further improvements
would be desirable. In general, it would be beneficial to provide
improved techniques for sensing or reading these new spectral
labels. It would be particularly beneficial to provide improved
techniques for applying these labeling and tracking technologies to
high-throughput assay systems now being developed.
[0009] Multiplexed assay formats would be highly desirable for
improved throughput capability, and to match the demands that
combinatorial chemistry is putting on established discovery and
validation systems for pharmaceuticals. For example, simultaneous
elucidation of complex protein patterns may allow detection of rare
events or conditions, such as cancer. In addition, the
ever-expanding repertoire of genomic information would benefit from
very efficient, parallel and inexpensive assay formats. Desirable
multiplexed assay characteristics include ease of use, reliability
of results, a high-throughput format, and extremely fast and
inexpensive assay development and execution.
[0010] A number of known assay formats may be employed for
high-throughput testing. Each of these formats has limitations,
however. By far the most dominant high-throughput technique is
based on the separation of different assays into different regions
of space. The 96-well plate format is the workhorse in this
arena.
[0011] In 96-well plate assays, the individual wells (which are
isolated from each other by walls) are often charged with different
components, and the assay is performed and then the assay result in
each well measured. The information about which assay is being run
is carried with the well number, or the position on the plate, and
the result at the given position determines which assays are
positive. These assays can be based on chemiluminescence,
scintillation, fluorescence, scattering, or absorbance/colorimetric
measurements, and the details of the detection scheme depend on the
reaction being assayed.
[0012] Multi-well assays have been reduced in size to enhance
throughput, for example, to accommodate 384 or 1536 wells per
plate. Unfortunately, the fluid delivery and evaporation of the
assay solution at this scale are significantly more confounding to
the assays. High-throughput formats based on multi-well arraying
often rely on complex robotics and fluid dispensing systems to
function optimally. The dispensing of the appropriate solutions to
the appropriate bins on the plate poses a challenge from both an
efficiency and a contamination standpoint, and pains must be taken
to optimize the fluidics for both properties. Furthermore, the
throughput is ultimately limited by the number of wells that one
can put adjacent on a plate, and the volume of each well.
Arbitrarily small wells have arbitrarily small volumes, resulting
in a signal that scales with the volume, shrinking proportionally
with the cube of the radius. The spatial isolation of each well,
and thereby each assay, has been much more common than running
multiple assays in a single well. Such single-well multiplexing
techniques are not widely used, due in large part to the difficulty
in "demultiplexing" or resolving the results of the different
assays in a single well.
[0013] For even higher throughput genomic and genetic analysis
techniques, positional array technology has been shrunk to
microscopic scales, often using high-density oligonucleotide
arrays. Over a 1-cm square of glass, tens to hundreds of thousands
of different nucleotides can be written in, for example, 25-.mu.m
spots, which are well resolved from each other. On this planar test
structure or "chip," which is emblazoned with an alignment grid, a
particular spot's x,y position determines which oligonucleotide is
present at that spot. Typically fluorescently-labeled amplified DNA
is added to the array, hybridized and is then detected using
fluorescence-based techniques. Although this is a very powerful
technique for assaying a large number of genetic markers
simultaneously, the cost is still very high, and the flexibility of
this assay is extremely limited.
[0014] Once a chip is made with particular DNA sequences at
particular locations, they are fixed and the addition thereto of
new markers comes at a very high price. The extremely small feature
size, and the highly parallel assay format, comes at the cost of
the flexibility inherent in a common platform system, such as the
96-well plates. In addition, this assay is ultimately performed at
the surface of the chip, and the results depend on the kinetics of
the hybridization to the surface, a process that is negatively
influenced by steric issues, mixing issues, and diffusion issues.
In fact, small microarray chips are not particularly suited to the
detection of rare events, as the diffusion of the solution over the
chip may not be sufficiently thorough. In order to perform the
hybridizations to the microarray chips more efficiently, a
dedicated fluidics workstation can be used to pump the solution
over the surface of the chip repeatedly; such instruments add cost
and time to execution of the assay.
[0015] The use of spectral barcodes holds great promise for
enhancing the throughput of assays, as described in an application
entitled "Semiconductor Nanocrystal Probes for Biological
Applications and Process for Making and Using such Probes," U.S.
application Ser. No. 09/259,982 filed Mar. 1, 1999, the full
disclosure of which is incorporated herein by reference.
Multiplexed assays may be performed using a number of probes which
include both a spectral label (often in the form of several
semiconductor nanocrystals) and one or more moieties. The moieties
may be capable of selectively bonding to one or more detectable
substances within a sample fluid, while the spectral labels can be
used to identify the probe within the fluid (and hence the
associated moiety). As the individual probes can be quite small,
and as the number of barcodes which can be independently identified
can be quite large, large numbers of individual assays might be
performed within a single fluid sample by including a large number
of differing probes. These probes may take the form of quite small
beads, with each bead optionally including a spectral label, a
moiety, and a bead body or matrix, often in the form of a
polymer.
[0016] Together with the substantial advantages provided by highly
multiplexed, spectrally-encoded assay bead systems, there will be
significant challenges in implementing these techniques. In
particular, determining multiplexed assay results might be quite
challenging. While the reaction times and accuracy of the spectral
labels can be quite advantageous, it can be challenging to
accurately read each spectral barcode and/or assay result from the
hundreds, and in many cases thousands, of beads within a highly
multiplexed bead assay system. Similarly, while spectral coding in
general allows labeling and/or identification of a large number of
elements, interpreting the spectral codes can be quite challenging
when the individual label structures are small, and when many
labels are located near each other.
[0017] In light of the above, it would generally be desirable to
provide improved systems and methods for detecting and identifying
signals. It would be particularly beneficial if these improved
techniques facilitated the identification of each spectral code
from among a plurality of spectral barcodes in a given region. To
take advantage of the potential capabilities of spectral coding of
minute probes and other structures, it would be highly desirable if
these enhanced techniques allowed detection and/or identification
of large numbers of spectral codes or other signals (such as assay
marker signals) in a highly time efficient manner.
SUMMARY OF THE INVENTION
[0018] The present invention generally provides improved devices,
systems, and methods for sensing and/or identifying signals. The
techniques of the present invention are particularly well-suited
for identification of labels which generate spectral codes. Large
numbers of independently identifiable spectral codes can be
generated by quite small bodies having such labels, and a plurality
of such bodies or probes may be present within a detection region.
In some embodiments, the invention allows simultaneously imaging of
identifiable spectra from throughout the detection region. This
simultaneous imaging allows the labels (and hence, the associated
probes, assay results, and the like) to be identified. A wavelength
dispersive element (for example, a prism, diffractive grating,
holographic transmissive grating, or the like) can simultaneously
spectrally disperse the images of the labels across a sensor
surface. A two-dimensional areal light sensor (such as a
Charge-Coupled Device or "CCD") can substantially simultaneously
sense the relative wavelengths of signals making up the spectra.
Taking advantage of a very small label size, the identifiable
spectra can be treated as being generated from point-sources within
a large detection field, thereby acting as their own "slit" in this
spectroscopic instrument. Absolute signal wavelengths may be
identified by determining positions of the labels, using an
internal wavelength reference within the spectra, and/or the
like.
[0019] Spectral labels may be used with other markers generating
signals that differ significantly from the identifiable spectra
from the labels. For example, spectrally encoded beads may be used
within parallel assay systems by generating assay signals in
addition to the label spectra. These assay signals may accurately
and reliably indicate the results of the assay, but these signals
may be significantly lower in intensity than the spectral label.
Hence, the present invention also provides techniques for
identifying signals of widely varying strengths. These techniques
often involve simultaneously sensing lower intensity signals using
a relatively long integration time with areal imaging. Higher
intensity signals can be sequentially sensed, often using a
scanning system. This dual sensing system enhances the overall
efficiency of signal detection and interpretation by allowing a
relatively long signal integration time for the lower intensity
signals, while the higher intensity signals are quickly scanned
with a shorter integration time. In some embodiments, a plurality
of excitation energies may be directed toward the signal
generators, with at least one of the excitation energies
selectively producing the lower energy signals. Such techniques are
particularly well-suited to take advantage of the capabilities of
semiconductor nanocrystals, which can accurately generate
detectable signals from minute bodies, and which can be selectively
energized by appropriate excitation sources.
[0020] In a first aspect, the invention provides a system
comprising a plurality of labels generating identifiable spectra in
response to excitation energy. A detector simultaneously images at
least some of the spectra for identification of the labels.
[0021] In many embodiments, at least some of the spectra will
comprise a plurality of detectable signals defining a plurality of
wavelengths. Label markers may generate these different label
signals, so that the labels can comprise a plurality of label
markers. The wavelengths from the spectra can be intermingled.
Preferably, the labels will comprise at least one semiconductor
nanocrystal. More typically, each label will comprise at least one
population of semiconductor nanocrystals, with each semiconductor
nanocrystal of each population generating a signal having an
associated population wavelength in response to the excitation
energy. In many embodiments, the labels will comprise a plurality
of populations supported by a matrix.
[0022] In some embodiments, at least one probe body will include a
label and an associated assay indicator marker. The indicator
markers generate indicator signals in response to an interaction
between the probe body and an associated test substance, thereby
indicating results of an assay.
[0023] The labels may be distributed across a two-dimensional
sensing field. The detector will often include a wavelength
dispersive element and a sensor, and each label will preferably be
sufficiently smaller than the surrounding sensing field to allow
the spectra to be wavelength-dispersed by the wavelength dispersive
element without excessive overlap of the dispersed spectra upon the
sensor. The dispersed spectra can often be analyzed as being
generated from discrete point-light sources. By using discrete
point source spectral labels, the system avoids any need for slit
apertures or the like, as generally found on linear spectrometers
and other spectral dispersion systems. In other words, the small
labels can act as their own slits. This also allows the detector to
admit signals from throughout a two-dimensional sensing field.
[0024] The wavelength dispersive element is usually disposed
between the sensing field and the light sensor. The sensor
simultaneously senses the spectra from the plurality of labels. An
open optical path often extends from the sensing field to the
wavelength dispersive element, and from the wavelength dispersive
element to the sensor, with optics typically imaging the sensing
field on the sensor. The sensor will typically comprise an areal
sensor (such as CCD), and the open optical path will have an open
cross-section with significant first and second open orthogonal
dimensions, in contrast to the slit or point apertures often used
in dispersive systems. The wavelength dispersive element may
comprise a prism, a dispersive reflective grating, a holographic
transmission grating, or the like.
[0025] In many embodiments, a spatial positioner provides label
positions within the sensor field. The detector will often sense
relative spectral data, while an analyzer coupled to the label
positioner and the detector can derive absolute wavelengths of the
spectra in response to both the relative spectral data and the
indicated label positions. In some embodiments, a beam splitter may
optically couple the label positioner with the sensing field along
a positioning optical path, and may also couple the detector with
the sensor field along a spectral optical path, so that at least a
portion of the positioning and spectral optical paths make use of
common optical elements. The beam splitter may direct most of the
energy from the sensing field toward the detector for relative
spectral information, and a minority of the energy from the sensing
field toward a positioning image. In some embodiments, a beam
splitter may direct a portion of an image from the sensing field to
a first dispersion member so as to distribute the spectra along a
first axis relative to the sensing field, and a second portion of
the image to a second dispersion member so as to distribute the
spectra along a second axis, the second axis being at an angle to
the first axis relative to the sensing field for resolving spectral
ambiguities from any overlapping wavelengths along the first axis.
Similar ambiguity resolution techniques may sequentially disperse
the spectra along differing axes.
[0026] At least some of the spectra will often comprise a plurality
of signals. The detector may include means for distributing these
signals across a sensor in response to wavelengths of the signals,
and in response to positions of the labels in the sensor fields.
The distributing means may be disposed between the sensing field
and the sensor. The system may also include means for determining
positions of the labels within the sensing field, with a spectral
analyzer coupled to the positioning means and the sensor so that
the analyzer can determine the spectra. The positioning means may
optionally comprise an areal sensor and a beam splitter, a
calibration reference signal within some or all of the spectra, or
the like.
[0027] In another aspect, the invention provides a system
comprising a plurality of labels distributed across a
two-dimensional sensing field. The labels generate spectra in
response to excitation energy. A wavelength dispersive element is
disposed in an open optical path of the spectra from the
two-dimensional sensing field. A sensor is disposed in the path
from the wavelength dispersive element. A label positioning system
is coupled to the labels and an analyzer is coupled to the sensor
for identifying the labels in response to the sensed spectral
information.
[0028] In another aspect, the invention provides a method
comprising generating spectra from a plurality of labels. The
spectra are sensed with a sensor by simultaneously imaging the
labels on the sensor, and the labels are identified in response to
the sensed spectra.
[0029] In many embodiments, the labels will be movably disposed
within a two-dimensional sensing field while the spectra are
sensed. The positions of the labels may be determined when the
spectra are sensed by the sensor, and the labels may be identified
in response to the label positions (as well as using the data from
the sensor). The spectra from the labels will often be dispersed.
In some embodiments, the spectra will be dispersed along a second
dispersion axis at an angle to a first dispersion axis so as to
resolve ambiguity from spectral overlap.
[0030] In another aspect, the invention provides a method for
identifying signals of differing strengths. The method comprises
generating a plurality of signals in response to excitation energy.
The signals include higher intensity signals and lower intensity
signals. The lower intensity signals are sensed by simultaneously
imaging the signals. At least some of the higher intensity signals
are sequentially sensed.
[0031] In many embodiments, the lower intensity signals will be
sensed by imaging a sensing field for a first integration time. The
higher intensity signals may be sequentially sensed by imaging a
portion of the sensing field for a second integration time, the
second integration time being shorter than the first integration
time. Optionally, the higher intensity signals may be filtered from
the simultaneous image. This is facilitated where the higher
intensity signals have wavelengths that are different than
wavelengths of the lower intensity signals, as wavelength filtering
may be employed to avoid saturation of the image.
[0032] The higher intensity signals may be sequentially sensed by
scanning labels which generate the signals. The labels generating
the higher intensity signals may be spatially intermingled with
markers generating the lower intensity signals. Scanning may
comprise scanning an aperture relative to the labels, such as a
slit, a pinhole aperture, or the like. In some embodiments,
scanning may be performed by scanning an excitation energy over a
portion of the sensing field.
[0033] In some embodiments, the excitation energy may comprise a
first energy for exciting the higher energy markers of the labels
to generate the high energy signals, and a second energy for
generating the lower energy signals. The second energy may
selectively excite the low energy markers.
[0034] The higher intensity signals of the labels may be generated
by label markers and can define an identifiable spectral code. The
low intensity signals may be generated by assay markers and can
indicate results of a plurality of assays, with each assay having
an associated spectral code. The markers may be supported by probe
bodies to define probes. Each probe can include a plurality of
label markers, which together define a label (to generate the
spectral code), and at least one associated assay marker (to
indicate results of an associated assay). The results of each assay
may be determined by identifying each label, and by correlating the
label with an associated assay marker signal.
[0035] In another aspect, the invention provides a method for
acquiring signals. The method comprises generating a first
plurality of signals from a first plurality of markers in response
to a first excitation energy. A second plurality of signals are
generated from a second plurality of markers in response to a
second excitation energy. The first and second markers are
intermingled. Intensities of the first signals are tuned relative
to intensities of the second signals by selecting a characteristic
of at least one of the first and second excitation energies. The
tuned first and second signals are simultaneously imaged on a
sensor.
[0036] Typically, at least one of the markers will comprise a
semiconductor nanocrystal. Preferably, the first energy will
selectively energize the first plurality of markers. The
intensities will be tuned so that the signals are within an
acceptable intensity range of the sensor during a common
integration time by varying an intensity of at least one of the
first and second excitation energies.
[0037] In yet another aspect, the invention provides a
high-throughput assay method comprising performing a plurality of
assays, and generating assay signals with assay markers to indicate
the results of the assays. The assay markers are simultaneously
area imaged, and spectral codes associated with each assay marker
are generated. The assay results are interpreted by identifying the
spectral code and assay markers, and by correlating each spectral
code with an associated assay marker signal.
[0038] In another aspect, the invention provides a system for
detecting spectral information. Spectral information includes
higher intensity signals and lower intensity signals. The signals
are generated within a two-dimensional field. The systems comprises
a detector optically couplable with the two-dimensional field for
simultaneous imaging of the low intensity signals. A scanner has an
aperture movable relative to the two-dimensional field for
sequential imaging of the higher intensity signals.
[0039] In yet another aspect, the invention provides a system
comprising a plurality of labels generating identifiable spectra in
response to excitation energy. Other markers are intermingled with
the labels. The other markers generate other signals, with the
other signals being weaker than the spectra. A scanner has an
aperture movable relative to the labels for identifying the
spectra. A detector is optically coupled to the plurality of other
markers for simultaneously imaging the other signals.
[0040] Typically, groups of the markers will be held together by a
probe matrix so as to define a plurality of probes, with each probe
including at least one label and at least one associated other
marker. This allows each probe to indicate results of an associated
assay via the identifiable spectra of the label. A processor
coupled to the scanner and to the detector can determine the
results of the assay in response to the spectra as sensed by the
scanner, and in response to the associated assay markers as sensed
by the detector. An integration time of the detector can be longer
than an integration time of the scanner for the spectra without
overly delaying the identification time, as the other markers (or
assay markers) are simultaneously imaged throughout the sensing
field.
[0041] In yet another aspect, the invention provides a
high-throughput assay system comprising a fluid with an excitation
energy source transmitting excitation energy toward the fluid. A
plurality of assay probes are disposed in the fluid. Each probe has
a spectral label. The spectral labels generate identifiable
spectral codes in response to the excitation energy. The probes
generate assay signals in response to assay results. A scanner
moves a sensing region relative to the fluid (and/or at least one
of the fluid and fluid holder relative to the sensing region) for
identification of the probes from the spectral codes. The
two-dimensional imaging system images the assay markers from the
probes throughout the two-dimensional sensing field
simultaneously.
[0042] In yet another aspect, the invention provides a
high-throughput assay system comprising a fluid and a first
excitation energy source transmitting a first excitation energy
toward the fluid. The second excitation energy source transmits a
second excitation energy toward the fluid. A plurality of assay
probes are disposed in the fluid. Each probe has a spectral label,
and assay markers in the fluid are associated with the probes. The
assay markers transmit an assay signal in response to assay
results, and in response to the second excitation energy. A first
excitation energy selectively energizes the spectral labels so that
the spectral labels transmit identifiable spectral codes. A sensing
system senses the assay signals and the spectral codes. The sensing
system has an intensity range. Intensities of the first and second
excitation sources are selected so that the assay signals and the
spectral codes are within the intensity range, often at the same
integration time.
[0043] In yet another aspect, the invention provides a fluid-flow
assay system comprising a fluid and a probe movably disposed within
the fluid. The probe has a label to generate an identifiable
spectra and an assay marker to generate an assay signal in response
to interaction between the probe and a detectable substance. A
probe reader senses the spectra and signal when the probe and fluid
flow through a sensing region to determine an assay result.
[0044] Typically, a plurality of differing probes will flow through
the sensing region. The probe reader will determine results of a
plurality of different assays by identifying the probes from their
associated spectra, and by correlating the assay signals from the
probes with the associated assays of the identified probes. In the
exemplary embodiment, the fluid (and the probes) flow across a slit
aperture within a thin, flat channel so that the distance between
the probes and reader is substantially uniform. This facilitates
imaging of the probes within the sensing region.
[0045] In yet another aspect, the invention provides a fluid-flow
assay method comprising moving a probe by flowing a fluid. A
spectra from the moving probe is sensed while the probe acts as its
own aperture by dispersing the image, and results of an assay are
determined by identifying the probe from the spectra. Once again,
such methods are particularly useful for multiplexed assays, as a
plurality of differing probes can be identified and their assay
results correlated.
BRIEF DESCRIPTION OF THE DRAWINGS
[0046] FIG. 1 schematically illustrates an imaging system and
high-throughput assay method according the principles of the
present invention.
[0047] FIG. 1A schematically illustrates an exemplary processor for
the system of claim 1.
[0048] FIG. 2 schematically illustrates probes having spectral
labels and assay markers, in which the probes comprise bead
structures disposed within a test fluid.
[0049] FIGS. 2A-2E schematically illustrate spectral codes or
labels having a plurality of signals.
[0050] FIG. 3 schematically illustrates a system and method for
determining a spectrum from a relatively large object by use of an
aperture.
[0051] FIG. 4 schematically illustrates a method and structure for
determining a spectrum from a small object, such as an assay probe
having semiconductor nanocrystal markers, without using an
aperture.
[0052] FIGS. 5A and 5B schematically illustrate a system and method
for determining absolute spectra from a plurality of semiconductor
nanocrystals by limiting the viewing field with an aperture and by
spectrally dispersing the apertured image.
[0053] FIG. 6 schematically illustrates a system and method for
determining absolute spectra of a plurality of spectrally encoded
beads by simultaneously imaging the relative spectra of the beads,
and by deriving the absolute spectra from the bead positions.
[0054] FIG. 6A schematically illustrates a method for correlating
the bead positions and relative spectra sensed using the system of
FIG. 6 to derive the absolute spectra.
[0055] FIGS. 6B and 6C schematically illustrate the use of a beam
splitter and calibration signals within the spectral codes to
determine the absolute wavelengths of a spectrum.
[0056] FIGS. 7A-7C schematically illustrate a system and method for
resolving ambiguities among overlapping dispersed spectra.
[0057] FIGS. 8 and 8A-8C graphically illustrates a wide variation
in signal intensities between a spectral label and an assay marker
for the exemplary probes illustrated in FIG. 2, and a method for
identifying such signals.
[0058] FIG. 9 schematically illustrates a system and method for
simultaneously imaging a plurality of assay markers, and for
sequentially scanning associated spectral labels for a plurality of
spectrally encoded assay probes, and also illustrates the use of
differing excitation energy sources for selectively energizing the
assay markers.
[0059] FIG. 9A schematically illustrates a fluid flow assay
scanning system and method.
[0060] FIGS. 10A-10C schematically illustrate a plate for
positioning semiconductor nanocrystal assay probes, together with a
method for the use of positioned probes in multiplexed assays.
[0061] FIG. 11 schematically illustrates a method for reading the
spectral labels and/or identifying assay results using the probe
positioning plate of FIG. 10C.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
[0062] The present invention generally provides improved devices,
systems, methods, compositions of matter, kits, and the like for
sensing and interpreting spectral information. The invention is
particularly well-suited to take advantage of new compositions of
matter which can generate signals at specific wavelengths in
response to excitation energy. A particularly advantageous signal
generation structure for use of the present invention is the
semiconductor nanocrystal. Other useful signaling structures may
also take advantage of the improvements provided by the present
invention, including conventional fluorescent dyes, radiated
elements and compounds, and the like.
[0063] The invention can allow efficient sensing and/or
identification of a large number of spectral codes, particularly
when each code includes multiple signals. The invention may also
enhance the reliability and accuracy with which such codes are
read, and may thereby enable the use of large numbers of spectral
codes within a relatively small region. Hence, the techniques of
the present invention will find advantageous applications within
highly multiplexed assays, inventory control in which a large
number of small and/or fluid elements are intermingled, and the
like.
[0064] Spectral Labeling
[0065] Referring now to FIG. 1, an inventory system 10 includes a
library of labeled elements 12a, 12b, . . . (collectively referred
to as elements 12) and an analyzer 14. Analyzer 14 generally
includes a processor 16 coupled to a detector 18. An energy source
20 transmits an excitation energy 22 to a sensing field within a
first labeled element 12a of library 8. In response to excitation
energy 22, first labeled element 12a emits radiant energy 24
defining a spectral code. Spectral code of radiant energy 24 is
sensed by detector 18 and the spectral code is interpreted by
processor 16 so as to identify labeled element 12a.
[0066] Library 8 may optionally comprise a wide variety of
elements. In many embodiments, labeled elements 12 may be
separated. However, in the exemplary embodiment, the various
labeled elements 12a, 12b, 12c, . . . are intermingled within a
test fluid 34. Imaging is facilitated by maintaining the labeled
elements on or near a surface. As used therein, "areal imaging"
means imaging of a two-dimensional area. Hence, fluid 34 may be
contained in a thin, flat region between planar surfaces.
[0067] Preferably, detector 18 simultaneously images at least some
of the signals generated by elements 12 from within a
two-dimensional sensing field. In some embodiments, at least some
of the spectral signals from within the sensing field are
sequentially sensed using a scanning system. Regardless,
maintaining each label as a spatially integral unit will often
facilitate identification of the label. This discrete spatial
integrity of each label is encompassed within the term "spatially
resolved labels." Preferably, the spatial integrity of the beads
and the space between beads will be sufficient to allow at least
some of the beads to be individually resolved over all other beads,
preferably allowing most of the beads to be individually resolved,
and in many embodiments, allowing substantially all of the beads to
be individually resolved.
[0068] The spectral coding of the present invention is particularly
well-suited for identification of small or fluid elements which may
be difficult to label using known techniques. Elements 12 may
generally comprise a composition of matter, a biological structure,
a fluid, a particle, an article of manufacture, a consumer product,
a component for an assembly, or the like. All of these are
encompassed within the term "identifiable substance."
[0069] The labels included with labeled elements 12 may be adhered
to, applied to a surface of, and/or incorporated within the items
of interest, optionally using techniques analogous to those of
standard bar coding technologies. For example, spectral labeling
compositions of matter (which emit the desired spectra) may be
deposited on adhesive labels and applied to articles of
manufacture. Alternatively, an adhesive polymer material
incorporating the label might be applied to a surface of a small
article, such as a jewel or a component of an electronic assembly.
As the information in the spectral code does not depend upon the
aerial surface of the label, such labels can be quite small.
[0070] In other embodiments, the library will comprise fluids (such
as biological samples), powders, cells, and the like. While
labeling of such samples using standard bar coding techniques can
be quite problematic, particularly when a large number of samples
are to be accurately identified, the spectral codes of the present
invention can allow robust identification of a particular element
from among ten or more library elements, a hundred or more library
elements, a thousand or more library elements, and even ten
thousand or more library elements.
[0071] The labels of the labeled elements 12 will often include
compositions of matter which emit energy with a controllable
wavelength/intensity spectrum. To facilitate identification of
specific elements from among library 8, the labels of the elements
may include combinations of differing compositions of matter to
emit differing portions of the overall spectral code. In other
embodiments, the signals may be defined by absorption (rather than
emission) of energy, by Raman scattering, or the like. As used
herein, the term "markers" encompasses compositions of matter which
produce the different signals making up the overall spectra. A
plurality of markers can be combined to form a label, with the
signals from the markers together defining the spectra for the
label.
[0072] The present invention generally utilizes a spectral code
comprising one or more signals from one or more markers. The
markers may comprise semiconductor nanocrystals, with the different
markers often taking the form of different particle size
distributions of semiconductor nanocrystals having different signal
generation characteristics. The combined markers define labels
which can generate spectral codes, which are sometimes referred to
as "spectral barcodes." These spectral codes can be used to track
the location of a particular item of interest or to identify a
particular item of interest. The semiconductor nanocrystals used in
the spectral coding scheme can be tuned to a desired wavelength to
produce a characteristic spectral emission or signal by changing
the composition and/or size of the semiconductor nanocrystal.
Additionally, the intensity of the signal at a particular
characteristic wavelength can also be varied (optionally by, at
least in part, varying a number of semiconductor nanocrystals
emitting or absorbing at a particular wavelength), thus enabling
the use of binary or higher order encoding schemes. The information
encoded by the semiconductor nanocrystals can be spectroscopically
decoded from the characteristics of their signals, thus providing
the location and/or identity of the particular item or component of
interest. As used herein, wavelength and intensity are encompassed
within the term "signal characteristics."
[0073] While spectral codes will often be described herein with
reference to the signal characteristics of signals emitted with
discrete, narrow peaks, it should be understood that semiconductor
nanocrystals and other marker structures may generate signals
having quite different properties. For example, signals may be
generated by scattering, absorption, or the like, and alternative
signal characteristics such as wavelength range width, slope,
shift, or the like may be used in some spectral coding schemes.
[0074] Semiconductor Nanocrystals
[0075] Semiconductor nanocrystals are particularly well-suited for
use as markers in a spectral code system because of their unique
characteristics. Semiconductor nanocrystals have radii that are
smaller than the bulk exciton Bohr radius and constitute a class of
materials intermediate between molecular and bulk forms of matter.
Quantum confinement of both the electron and hole in all three
dimensions leads to an increase in the effective band gap of the
material with decreasing crystallite size. Consequently, both the
optical absorption and emission of semiconductor nanocrystals shift
to the blue (higher energies) with decreasing size. Upon exposure
to a primary light source, each semiconductor nanocrystal
distribution is capable of emitting energy in narrow spectral
linewidths, as narrow as 20-30 nm, and with a symmetric, nearly
Gaussian line shape, thus providing an easy way to identify a
particular semiconductor nanocrystal. The linewidths are dependent
on the size heterogeneity, i.e., monodispersity, of the
semiconductor nanocrystals in each preparation. Single
semiconductor nanocrystal complexes have been observed to have full
width at half max (FWHM) as narrow as 12-15 nm. In addition
semiconductor nanocrystal distributions with larger linewidths in
the range of 40-60 nm can be readily made and have the same
physical characteristics as semiconductor nanocrystals with
narrower linewidths.
[0076] Exemplary materials for use as semiconductor nanocrystals in
the present invention include, but are not limited to group II-VI,
III-V, and group IV semiconductors such as ZnS, ZnSe, ZnTe, CdS,
CdSe, CdTe, GaN, GaP., GaAs, GaSb, InP, InAs, InSb, AlS, AlP, AlSb,
PbS, PbSe, Ge, Si, and ternary and quaternary mixtures or alloys
thereof. The semiconductor nanocrystals are characterized by their
nanometer size. By "nanometer" size, it is meant less than about
150 Angstroms (A), and preferably in the range of 12-150 A.
[0077] The selection of the composition of the semiconductor
nanocrystal, as well as the size of the semiconductor nanocrystal,
affects the signal characteristics of the semiconductor
nanocrystal. Thus, a particular composition of a semiconductor
nanocrystal as listed above will be selected based upon the
spectral region being monitored. For example, semiconductor
nanocrystals that emit energy in the visible range include, but are
not limited to, CdS, CdSe, CdTe, and ZnTe. Semiconductor
nanocrystals that emit energy in the near IR range include, but are
not limited to, InP, InAs, InSb, PbS, and PbSe. Finally,
semiconductor nanocrystals that emit energy in the blue to
near-ultraviolet include, but are not limited to, ZnS and GaN. For
any particular composition selected for the semiconductor
nanocrystals to be used in the inventive system, it is possible to
tune the emission to a desired wavelength within a particular
spectral range by controlling the size of the particular
composition of the semiconductor nanocrystal.
[0078] In addition to the ability to tune the signal
characteristics by controlling the size of a particular
semiconductor nanocrystal, the intensities of that particular
emission observed at a specific wavelength are also capable of
being varied, thus increasing the potential information density
provided by the semiconductor nanocrystal coding system. In some
embodiments, 2-15 different intensities may be achieved for a
particular emission at a desired wavelength, however, more than
fifteen different intensities may be achieved, depending upon the
particular application of the inventive identification units. For
the purposes of the present invention, different intensities may be
achieved by varying the concentrations of the particular size
semiconductor nanocrystal attached to, embedded within or
associated with an item or component of interest, by varying a
Quantum yield of the nanocrystals, by varyingly quenching the
signals from the semiconductor nanocrystals, or the like.
Nonetheless, the spectral coding schemes may actually benefit from
a simple binary structure, in which a given wavelength is either
present our absent, as described below.
[0079] In a particularly preferred embodiment, the surface of the
semiconductor nanocrystal is also modified to enhance the
efficiency of the emissions, by adding an overcoating layer to the
semiconductor nanocrystal. The overcoating layer is particularly
preferred because at the surface of the semiconductor nanocrystal,
surface defects can result in traps for electron or holes that
degrade the electrical and optical properties of the semiconductor
nanocrystal. An insulting layer (having a bandpass layer typically
with a bandgap energy greater than the core and centered thereover)
at the surface of the semiconductor nanocrystal provides an
atomically abrupt jump in the chemical potential at the interface
that eliminates energy states that can serve as traps for the
electrons and holes. This results in higher efficiency in the
luminescent process.
[0080] Suitable materials for the overcoating layer include
semiconductors having a higher band gap energy than the
semiconductor nanocrystal. In addition to having a band gap energy
greater than the semiconductor nanocrystals, suitable materials for
the overcoating layer should have good conduction and valence band
offset with respect to the semiconductor nanocrystal. Thus, the
conduction band is desirably higher and the valence band is
desirably lower than those of the semiconductor nanocrystal. For
semiconductor nanocrystals that emit energy in the visible (e.g.,
CdS, CdSe, CdTe, ZnSe, ZnTe, GaP, GaAs) or near IR (e.g., InP,
InAs, InSb, PbS, PbSe), a material that has a band gap energy in
the ultraviolet regions may be used. Exemplary materials include
ZnS, GaN, and magnesium chalcogenides, (e.g., MgS, MgSe, and MgTe).
For semiconductor nanocrystals that emit in the near IR, materials
having a band gap energy in the visible, such as CdS, or CdSe, may
also be used. While the overcoating will often have a higher
bandgap than the emission energy, the energies can be, for example,
both within the visible range. The overcoating layer may include as
many as 8 monolayers of the semiconductor material. The preparation
of a coated semiconductor nanocrystal may be found in U.S. patent
application Ser. No. 08/969,302 filed Nov. 13, 1997, entitled
"Highly Luminescent Color-Selective Materials"; Dabbousi et al., J.
Phys. Chem B., Vol. 101, 1997, pp. 9463; and Kuno et al., J. Phys.
Chem., Vol. 106, 1997, pp. 9869. Fabrication and combination of the
differing populations of semiconductor nanocrystals may be further
understood with reference to U.S. patent application Ser. No.
09/397,432, previously incorporated herein by reference.
[0081] It is often advantageous to combine different markers of a
label into one or more labeled body. Such labeled bodies may help
spatially resolve different labels from intermingled items of
interest, which can be beneficial during identification. These
label bodies may comprise a composition of matter including a
polymeric matrix and a plurality of semiconductor nanocrystals,
which can be used to encode discrete and different absorption and
emission spectra. These spectra can be read using a light source to
cause the label bodies to absorb or emit light. By detecting the
light absorbed and/or emitted, a unique spectral code may be
identified for the labels. In some embodiments, the labeled bodies
may further include markers beyond the label bodies. These labeled
bodies will often be referred to as "beads" herein, and beads which
have assay capabilities may be called "probes." The structure and
use of such probes, including their assay capabilities, are more
fully described in U.S. patent application Ser. No. 09/566,014,
previously incorporated herein by reference.
[0082] Fabrication of Labeled Beads
[0083] Referring now to FIG. 2, first and second labeled elements
12a, 12b within test fluid 34 are formed as separate semiconductor
nanocrystal probes 34'. Each probe includes an associated label 36
formed from one or more populations of substantially mono-disperse
semiconductor nanocrystals 37. The individual populations of
semiconductor nanocrystals will often be mono-disperse so as to
provide a sufficient signal intensity at a uniform wavelength for
convenient sensing of the various signals within the code. The
exemplary probes further include one or more binding moieties 35',
together with a probe matrix or body material 39, which acts as a
binding agent to keep the various markers together in a structural
unit or bead. Binding moieties 35' help (indirectly) to generate
signals indicating results of an assay, each probe moiety having
selective affinity for an associated test substance 35 which may be
present within sample fluid 34. Probe moieties 35' may comprise an
antibody, DNA, or the like, and test substances 35 may carry
reporters or assay markers 38 for generating signals indicating
results of the assays. Alternatively, the assay markers may have
selective affinity for the combination of a particular test
substance and bound probe moiety, or the like. Preparation of the
spectrally encoded probes will now be described, followed by a
brief description of the use and structure of assay markers 38.
[0084] A process for encoding spectra into label body materials
using a feedback system can be based on the absorbance and
luminescence of the semiconductor nanocrystals in a solution that
can be used to dye the materials. More specifically, this solution
can be used for encoding of a plurality of semiconductor
nanocrystals into a material when that material is a polymeric
bead.
[0085] A variety of different materials can be used to prepare
these compositions. In particular, polymeric bead materials are an
appropriate format for efficient multiplexing and demultiplexing of
finite-sized materials. These label body beads can be prepared from
a variety of different polymers, including but not limited to
polystyrene, cross-linked polystyrene, polyacrylic, polysiloxanes,
polymeric silica, latexes, dextran polymers, epoxies, and the like.
The materials have a variety of different properties with regard to
swelling and porosity, which are well understood in the art.
Preferably, the beads are in the size range of approximately 10 nm
to 1 mm, more preferably in a size range of approximately 100 nm to
0.1 mm, often being in a range from 1000 nm to 10,000 nm, and can
be manipulated using normal solution techniques when suspended in a
solution.
[0086] Discrete emission spectra can be encoded into these
materials by varying the amounts and ratios of different
semiconductor nanocrystals, either the size distribution of
semiconductor nanocrystals, the composition of the semiconductor
nanocrystals, or other property of the semiconductor nanocrystals
that yields a distinguishable emission spectrum, which are embedded
into, attached to or otherwise associated with the material. The
semiconductor nanocrystals of the invention can be associated with
the material by adsorption, absorption, covalent attachment, by
co-polymerization or the like. The semiconductor nanocrystals have
absorption and emission spectra that depend on their size and
composition. These semiconductor nanocrystals can be prepared as
described in Murray et. al., (1993) J. Am. Chem. Soc.
115:8706-8715; Guzelian et. al., (1996) J. Phys. Chem.
100;7212-7219; or International Publication No. WO 99/26299
(inventors Bawendi et al.). The semiconductor nanocrystals can be
made further luminescent through overcoating procedures as
described in Danek et. al., (1966) Chem. Mat. 8(1):173-180; Hines
et. al., (1996) J. Phys. Chem. 100:468-471; Peng et. al., (1997) J.
Am. Chem. Soc. 119:7019-7029; or Daboussi et. al., (1997) J. Phys.
Chem.-B, 101:9463-9475.
[0087] The desired spectral emission properties may be obtained by
mixing semiconductor nanocrystals of different sizes and/or
compositions in a fixed amount and ratio to obtain the desired
spectrum. The spectral emission of this staining solution can be
determined prior to treatment of the material therewith. Subsequent
treatment of the material (through covalent attachment,
co-polymerization, passive absorption, swelling and contraction, or
the like) with the staining solution results in a material having
the designed spectral emission property. These spectra may be
different under different excitation sources. Accordingly, it is
preferred that the light source used for the encoding procedure be
as similar as possible (preferably of the same wavelength and/or
intensity) to the light source that will be used for the decoding.
The light source may be related in a quantitative manner, so that
the emission spectrum of the final material may be deduced from the
spectrum of the staining solution.
[0088] A number of semiconductor nanocrystal solutions can be
prepared, each having a distinct distribution of sizes and
compositions, and consequently a distinct emission spectrum, to
achieve a desired emission spectrum. These solutions may be mixed
in fixed proportions to arrive at a spectrum having the
predetermined ratios and intensities of emission from the distinct
semiconductor nanocrystals suspended in that solution. Upon
exposure of this solution to a light source, the emission spectrum
can be measured by techniques that are well established in the art.
If the spectrum is not the desired spectrum, then more of a
selected semiconductor nanocrystal solution can be added to achieve
the desired spectrum and the solution titrated to have the correct
emission spectrum. These solutions may be colloidal solutions of
semiconductor nanocrystals dispersed in a solvent, or they may be
pre-polymeric colloidal solutions, which can be polymerized to form
a matrix with semiconductor nanocrystals contained within. While
ratios of the quantities of constituent solutions and the final
spectrum intensities need not be the same, it will often be
possible to derive the final spectra from the quantities (and/or
the quantities from the desired spectra.)
[0089] The solution luminescence will often be adjusted to have the
desired intensities and ratios under the exact excitation source
that will be used for the decoding. The spectrum may also be
prepared to have an intensity and ratio among the various
wavelengths that are known to produce materials having the desired
spectrum under a particular excitation source. A multichannel
auto-pipettor connected to a feedback circuit can be used to
prepare a semiconductor nanocrystal solution having the desired
spectral characteristics, as described above. If the several
channels of the titrator/pipettor are charged or loaded with
several unique solutions of semiconductor nanocrystals, each having
a unique excitation and emission spectrum, then these can be
combined stepwise through addition of the stock solutions. In
between additions, the spectrum may be obtained by exposing the
solution to a light source capable of causing the semiconductor
nanocrystals to emit, preferably the same light source that will be
used to decode the spectra of the encoded materials. The spectrum
obtained from such intermediate measurements may be judged by a
computer based on the desired spectrum. If the solution
luminescence is lacking in one particular semiconductor nanocrystal
emission spectrum, stock solution containing that semiconductor
nanocrystal may be added in sufficient amount to bring the emission
spectrum to the desired level. This procedure can be carried out
for all different semiconductor nanocrystals simultaneously, or it
may be carried out sequentially.
[0090] Once the staining solution has been prepared, it can be used
to incorporate a unique luminescence spectrum into the materials of
this invention. If the method of incorporation of the semiconductor
nanocrystals into the materials is absorption or adsorption, then
the solvent that is used for the staining solution may be one that
is suitable for swelling the materials. Such solvents are commonly
from the group of solvents including dichloromethane, chloroform,
dimethylformamide, tetrahydrofuran and the like. These can be mixed
with a more polar solvent, for example methanol or ethanol, to
control the degree and rate of incorporation of the staining
solution into the material. When the material is added to the
staining solution, the material will swell, thereby causing the
material to incorporate a plurality of semiconductor nanocrystals
in the relative proportions that are present in the staining
solution. In some embodiments, the semiconductor nanocrystals may
be incorporated in a different but predictable proportion. When a
more polar solvent is added, after removal of the staining solution
from the material, material shrinks, or unswells, thereby trapping
the semiconductor nanocrystals in the material. Alternatively,
semiconductor nanocrystals can be trapped by evaporation of the
swelling solvent from the material. After rinsing with a solvent in
which the semiconductor nanocrystals are soluble, yet that does not
swell the material, the semiconductor nanocrystals are trapped in
the material, and may not be rinsed out through the use of a
non-swelling, non-polar solvent. Such a non-swelling, non-polar
solvent is typically hexane or toluene. The materials can be
separated and then exposed to a variety of solvents without a
change in the emission spectrum under the light source. When the
material used is a polymer bead, the material can be separated from
the rinsing solvent by centrifugation or evaporation or both, and
can be redispersed into aqueous solvents and buffers through the
use of detergents in the suspending buffer, as is well known in the
art.
[0091] The above procedure can be carried out in sequential steps
as well. A first staining solution can be used to stain the
materials with one population of semiconductor nanocrystals. A
second population of semiconductor nanocrystals can be prepared in
a second staining solution, and the material exposed to this second
staining solution to associate the semiconductor nanocrystals of
the second population with the material. These steps can be
repeated until the desired spectral properties are obtained from
the material when excited by a light source, optionally using
feedback from measurements of the interim spectra generated by the
partially stained bead material to adjust the process.
[0092] The semiconductor nanocrystals can be attached to the
material by covalent attachment, and/or by entrapment in pores of
the swelled beads. For instance, semiconductor nanocrystals are
prepared by a number of techniques that result in reactive groups
on the surface of the semiconductor nanocrystal. See, e.g., Bruchez
et. al., (1998) Science 281:2013-2016; and Ghan et. al., (1998)
Science 281:2016-2018, Golvin et. al., (1992) J. Am. Chem. Soc.
114:5221-5230; Katari et. al. (1994) J. Phys. Chem. 98:4109-4117;
Steigerwald et. al. (1987) J. Am. Chem. Soc. 110:3046. The reactive
groups present on the surface of the semiconductor nanocrystals can
be coupled to reactive groups present on the surface of the
material. For instance, semiconductor nanocrystals which have
carboxylate groups present on their surface can be coupled to beads
with amine groups using a carbo-diimide activation step, or a
variety of other methods well known in the art of attaching
molecules and biological substances to bead surfaces. In this case,
the relative amounts of the different semiconductor nanocrystals
can be used to control the relative intensities, while the absolute
intensities can be controlled by adjusting the reaction time to
control the number of reacted sites in total. After the bead
materials are stained with the semiconductor nanocrystals, the
materials are optionally rinsed to wash away unreacted
semiconductor nanocrystals.
[0093] Referring once again to FIG. 2, labeled elements 12a, 12b
(here in the form of semiconductor nanocrystal probes) may be
useful in assays in a wide variety of forms. Utility of the probes
for assays benefits significantly from the use of moieties or
affinity molecules 35', as schematically illustrated in FIG. 2,
which may optionally be supported directly by a label marker 37 of
label 36, by the probe body matrix 39, or the like. Moieties 35'
can have selective affinity for an associated detectable substance
35, as schematically illustrated by correspondence symbol shapes in
FIG. 2. The probes may, in some embodiments, also include an
integrated assay marker 38 which is activated or enabled to
generate a signal by the binding of probe moiety 35' to test
substance 35. In many embodiments, the assay marker will instead be
coupled to the probes by coupling of detectable substance 35 to
moiety 35'. In other words, the assay marker 38 may (at least
initially) be coupled to the detectable substance 35, typically by
binding of a dye molecule, incorporation of a radioactive isotope,
or the like. The assay markers may thus be coupled to the probe by
the interaction between the moieties 35' and the test or detectable
substances 35. In other assays, the assay results may be determined
by the presence or absence of the probe or bead (for example, by
washing away probes having an unattached moiety) so that no
dedicated assay marker need be provided.
[0094] In alternative embodiments, the material used to make the
codes does not need to be semiconductor nanocrystals. For example,
any fluorescent material or combination of fluorescent materials
that can be finely tuned throughout a spectral range and can be
excited optically or by other means might be used. For organic
dyes, this may be possible using a number of different dyes that
are each spectrally distinct.
[0095] This bead preparation method can be used generically to
identify identifiable substances, including cells and other
biological matter, objects, and the like. Pre-made mixtures of
semiconductor nanocrystals, as described above, are attached to
objects to render them subsequently identifiable. Many identical or
similar objects can be coded simultaneously, for example, by
attaching the same semiconductor nanocrystal mixture to a batch of
microspheres using a variety of chemistries known in the art.
Alternatively, codes may be attached to objects individually,
depending on the objects being coded. In this case, the codes do
not have to be pre-mixed and may be mixed during application of the
code, for example using an inkjet printing system to deliver each
species of semiconductor nanocrystals to the object. The use of
semiconductor nanocrystal probes in chemical and/or biological
assays is more fully described in U.S. patent application Ser. No.
09/566,014, the full disclosure of which is incorporated herein by
reference.
[0096] The semiconductor nanocrystal probes of FIG. 2 may also be
utilized to detect the occurrence of an event. This event, for
example, may cause the source from which energy is transferred to
assay marker 38 to be located spatially proximal to the
semiconductor nanocrystal probe. Hence, the excitation energy from
energy source 20 may be transferred either directly to assay
markers 38, 38', or indirectly via excitation of one or more energy
sources adjacent the semiconductor nanocrystal probes due to
bonding of the test substances 35 to the moiety 35'. For example, a
laser beam may be used to excite a proximal source such as a
semiconductor nanocrystal probe 38' attached to one of the test
substances 35 (to which the affinity molecule selectively
attaches), and the energy emitted by this semiconductor nanocrystal
38' may then excite an assay marker 38 affixed to the probe matrix.
As mentioned above, still further assay marker structures and
methods are described in detail in co-pending U.S. patent
application Ser. No. 09/566,014.
[0097] Reading Beads
[0098] Referring once again to FIG. 1, energy source 20 generally
directs excitation energy 22 in such a form as to induce emission
of the spectral code from labeled element 12a. In one embodiment,
energy source 20 comprises a source of light, the light preferably
having a wavelength shorter than that of the spectral code. Energy
source 20 may comprise a source of blue or ultraviolet light,
optionally comprising a broad band ultraviolet light source such a
deuterium lamp, optionally with a filter. Alternatively, energy
source 20 may comprise an Xe or Hg UV lamp, or a white light source
such as a xenon lamp or a deuterium lamp, preferably with a short
pass or bandpass filter disposed along the excitation energy path
from the lamp to the labeled elements 12 so as to limit the
excitation energy to the desired wavelengths. Still further
alternative excitation energy sources include any of a number of
continuous wave (cw) gas lasers, including (but not limited to) any
of the argon ion laser lines (457 nm, 488 nm, 514 nm, etc.), a HeCd
laser, a solid-state diode laser (preferably having a blue or
ultraviolet output such as a GaN based laser, a GaAs based laser
with frequency doubling, a frequency doubled or tripled output of a
YAG or YLF based laser, or the like), any of the pulsed lasers with
an output in the blue or ultraviolet ranges, light emitting diodes,
or the like, or any other laser source (solid, liquid, or gas
based) with emissions to the blue of the code spectrum.
[0099] The excitation energy 22 from energy source 20 will induce
labeled element 12a to emit identifiable energy 24 having the
spectral code, with the spectral code preferably comprising signals
having relatively narrow peaks so as to define a series of
distinguishable peak wavelengths and associated intensities. The
peaks will typically have a half width of about 100 nm or less,
preferably of 70 nm or less, more preferably 50 nm or less, and
ideally 30 nm or less. In many embodiments, a plurality of separate
signals will be included in the spectral code as sensed by sensor
18. As semiconductor nanocrystals are particularly well-suited for
generating luminescent signals, identifiable energy 24 from label
12a will often comprise light energy. To help interpret the
spectral code from the identifiable energy 24, the light energy may
pass through one or more monochromator or other wavelength
dispersive element. A Charge-Coupled Device (CCD) camera or some
other two-dimensional detector of sensor 18 can sense and/or record
the images for later analysis. In other embodiments, a scanning
system maybe employed, in which the labeled element to be
identified is scanned with respect to a microscope objective, with
the luminescence put through a single monochromator or a grating or
prism to spectrally resolve the colors. The detector can be a diode
array that records the colors that are emitted at a particular
spatial position, a two-dimensional CCD, or the like.
[0100] Information regarding these spectra from the labeled
elements 12 will generally be transmitted from sensor 18 to
processor 16, the processor typically comprising a general purpose
computer. Processor 16 will typically include a central processing
unit, ideally having a processing capability at least equivalent to
a Pentium I.RTM. processor, although simpler systems might use
processing capabilities of a Palms.RTM. handheld processor or more.
Processor 16 will generally have input and output capabilities and
associated peripheral components, including an output device such
as a monitor, an input such as a keyboard, mouse, and/or the like,
and will often have a networking connection such as an Ethernet, an
Intranet, an Internet, and/or the like. An exemplary processing
block diagram is schematically illustrated in FIG. 1A.
[0101] Processor 16 will often make use of a tangible media 30
having a machine-readable code embodying method steps according to
one or more methods of the present invention. A database 32,
similarly embodied on a machine-readable code, will often include a
listing of the elements included in library 8, the spectral codes
of the labels associated with the elements, and a correlation
between specific library elements and their associated codes.
Processor 16 uses the information from database 32 together with
the spectrum characteristics sensed by sensor 18 to identify a
particular library element 12a. The machine-readable code of
program instructions 30 and database 32 may take a wide variety of
forms, including floppy disks, optical discs (such as CDs, DVDs,
rewritable CDs, and the like), alternative magnetic recording media
(such as tapes, hard drives, and the like), volatile and/or
non-volatile memories, software, hardware, firmware, or the
like.
[0102] As illustrated in FIG. 1, methods for detecting and
classifying spectral labels (such as encoded materials and beads)
may comprise exposing the labels to light of an excitation source
so that the semiconductor nanocrystals of the label are
sufficiently excited to emit light. This excitation source is
preferably of an energy capable of exciting the semiconductor
nanocrystals to emit light and may be of higher energy (and hence,
shorter wavelength) than the shortest emission wavelength of the
semiconductor nanocrystals in the label. Alternatively the
excitation source can emit light of longer wavelength if it is
capable of exciting some of the semiconductor nanocrystals disposed
in the matrix to emit light, such as using two-photon excitation.
This excitation source is preferably chosen to excite a sufficient
number of different populations of semiconductor nanocrystals to
allow unique identification of the encoded materials. For example,
using materials stained in a 1:2 ratio of red to blue and a 1:3
ratio of red to blue, it may not be sufficient to only excite the
red emitting semiconductor nanocrystals (e.g., by using green or
yellow light) of the sample in order to resolve these beads. It
would be desirable to use a light source with components that are
capable of exciting the blue emitting and the red emitting
semiconductor nanocrystals simultaneously, (e.g., violet or
ultraviolet). There may be one or more light sources used to excite
the populations of the different semiconductor nanocrystals
simultaneously or sequentially, but each light source may
selectively excite sub-populations of semiconductor nanocrystals
that emit at lower energy than the light source (to a greater
degree than higher energy emitting sub-populations), due to the
absorbance spectra of the semiconductor nanocrystals. Ideally, a
single excitation energy source will be sufficient to induce the
labels to emit identifiable spectra.
[0103] Spectral Codes
[0104] Referring now to FIGS. 2A-2E, the use of a plurality of
different signals within a single spectral label can be understood.
In this simple example, a coding system is shown having two
signals. A first signal has a wavelength peak 40a at a first
discreet wavelength, while a separate signal has a different
wavelength peak 40b. As shown in FIGS. 2A-2D, varying peak 40b
while the first peak 40a remains at a fixed location defines a
first family of spectral codes 1a through 4a. Moving the first peak
40a to a new location allows a second family of spectral codes to
be produced, as can be understood with reference to FIG. 2E.
[0105] The simple code system illustrated in FIGS. 2A-2E includes
only two signals, but still allows a large number of identifiable
spectra. More complex spectral codes having larger numbers of peaks
can significantly increase the number of codes. Additionally, the
intensities of one or more of the peaks may also be varied, thereby
providing still higher order codes having larger numbers of
separately identifiable members.
[0106] Spectral Code Reading Systems
[0107] In general, fluorescent labeling is a powerful technique for
tracking components in biological systems. For instance, labeling a
portion of a cell with a fluorescent marker can allow one to
monitor the movement of that component within the cell. Similarly,
labeling an analyte in a bioassay can allow one to determine its
presence or absence, even at vanishingly small concentrations. The
use of multiple fluorophores with different emission wavelengths
allows different components to be monitored simultaneously.
Applications such as spectral encoding can take full advantage of
multicolor fluorophores, potentially allowing the simultaneous
detection of millions of analytes.
[0108] When imaging samples labeled with multiple chromophores, it
is desirable to resolve spectrally the fluorescence from each
discrete region within the sample. As an example, an assay may be
prepared in which polymer beads have been labeled with two
different chromophores and the results of the assay may be
determined by the ratio of the two types of beads within the final
sample. One could imagine immobilizing the beads and counting each
of the colors. Electronic imaging requires a technique for
acquiring an image of the sample in which spectral information is
available at each discrete point. While the human eye is
exceptionally good at distinguishing colors, typical electronic
photodetectors are often effectively color-blind. As such,
additional optical components are often used in order to acquire
spectral information.
[0109] Many techniques might be applied to solve this problem.
Fourier transform spectral imaging (Malik et al. (1996) J. Microsc.
182:133; Brenan et al. (1994) Appl. Opt 33:7520) and Hadamard
transform spectral imaging (Treado et al. (1989) Anal. Chem
61:732A; Treado et al. (1990) Appl. Spectrosc. 44:1-4; Treado et
al. (1990) Appl. Spectrosc. 44:1270; Hammaker et al. (1995) J. Mol.
Struct. 348:135; Mei et al. (1996) J. Anal. Chem. 354:250; Flateley
et al. (1993) Appl. Spectrosc. 47:1464), imaging through variable
interference (Youvan (1994) Nature 369:79; Goldman et al. (1992)
Biotechnology 10:1557), acousto-optical (Mortensen et al. (1996)
IEEE Trans. Inst. Meas. 45:394; Turner et al (1996) Appl.
Spectrosc. 50:277) or liquid crystal filters (Morris et al. (1994)
Appl. Spectrosc. 48:857) or simply scanning a slit or point across
the sample surface (Colarusso et al. (1998) Appl. Spectrosc.
52:106A) are methods capable of generating spectral and spatial
information across a two-dimensional region of a sample. Most of
these techniques, however, benefit from the mechanical scanning of
one component of the system as well as the acquisition of multiple
data frames in order to generate a spectral image. For instance,
Fourier transform imaging scans an interferometer, acquiring a full
image at each mirror position. The spectral information is then
extracted from the complete set of spatial images. Similarly,
"point scanning" typically relies on a full spectrum from each
position within the image and scans all positions to generate the
full image. These techniques may allow precise spectral fitting and
analysis, but may be too cumbersome and slow for highly multiplexed
systems.
[0110] Referring now to FIG. 3, a system and method for reading
spectral information from an arbitrarily large object 50 generally
makes use of a detector 52 including a wavelength dispersive
element 54 and a sensor 56. Imaging optics 58 image object 50 onto
a surface of sensor 56. Wavelength dispersive element 54 spectrally
disperses the image across the surface of the sensor, distributing
the image based on the wavelengths of the image spectra.
[0111] As object 50 is relatively large when imaged upon sensor 56,
differentiation of the discreet wavelengths within a spectrum 60 is
facilitated by the use of an aperture 62. As aperture 62 allows
only a small region of the image through wavelength dispersive
element 54, the wavelength dispersive element separates the image
components based on wavelength alone (rather than on a combination
of wavelength and position along the surface of image 50). Spectra
60 may then be directly determined based on the position of the
diffracted image upon sensor 56, together with the intensity of
image wavelength components as measured by the sensor. As the
position of object 50 is scanned past aperture 62, the remainder of
the spectral image can be collected. Referring now to FIG. 4, a
spectra 60 of a spectrally labeled nanocrystal bead 64 may be
performed using a detector 66 without an aperture. As bead 64 has a
signal generating area (as imaged by imaging optics 58) which is
much smaller than a sensing surface of sensor 56, bead 64 can act
as a point-source of spectra 60. Optics 58 would typically, in the
absence of dispersive element 54, image bead 64 on detector 56 so
that the bead image has a size similar to or smaller than an
aperture of a monochrometer (the undispensed image size typically
being about 250 .mu.m or less, ideally about 120 .mu.m or less).
The various signals of the spectral code emanate from small surface
area of the bead, so that the signal distribution across the sensor
surface is dominated by the wavelength dispersion, and no limiting
of the image via an aperture is required. As used herein, a "true
point source" is a light source with a dimension which is at least
as small as a minimum, diffraction limited determinable dimension.
A light source which is larger than a true point source may be
"treated" or "analyzed" as a point source if it has a dimension or
size which is sufficiently small that its size acts like an
aperture.
[0112] As described above, it will often be advantageous to include
a plurality of different spectrally labeled beads within a fluid.
These labeled beads will often be supported by the surrounding
fluid, and/or will be movable with the fluid, particularly in
high-throughput multiplexed bead-based assays. Optionally, the
beads may have a size sufficient to define a suspension within the
surrounding test fluid. In some embodiments, the beads may comprise
a colloid within the test fluid. In some embodiments, beads 64 may
be movably supported by a surface of a vessel containing the test
fluid, for example, being disposed on the bottom surface of the
vessel (where probe 64 has a density greater than that of the test
fluid). In other embodiments, the beads may be affixed to a support
structure and/or to each other. Still further alternatives are
possible, such as for probe 64 to be floating on an upper surface
of the test fluid, for the bead or beads to be affixed to or
disposed between cooperating surfaces of the vessel to maintain the
positioning of the bead or beads, for the bead or beads to be
disposed at the interface between two fluids, and the like.
[0113] As was described above, it will often be advantageous to
include numerous beads 64 within a single test fluid so as to
perform a plurality of assays. Similarly, it will often be
advantageous to identify a large number of fluids or small discreet
elements within a single viewing area without separating out each
spectral label from the combined labeled elements. As illustrated
in FIG. 4, the dispersed spectral image 68 of bead 64 upon sensor
56 will depend on both the relative spectra generated by the bead,
and on the position of the bead. For example, bead 64' is imaged
onto a different portion 68' of sensor 56, which could lead to
misinterpretation of the wavelengths of the spectra if the location
of bead 64' is not known. So long as an individual bead 64 can be
accurately aligned with the imaging optics 58 and sensor system 66,
absolute spectral information can be obtained. However, as can be
understood with reference to FIG. 5A, a plurality of beads 64 will
often be distributed throughout an area 70.
[0114] To ensure that only beads 64 which are aligned along an
optical axis 72 are imaged onto sensor 56, aperture 62 restricts a
sensing field 74 of the sensing system. Where sensor 56 comprises
an areal sensor such a charge couple device (CCD), aperture 62 may
comprise a slit aperture so that spectral wavelengths .lambda. can
be determined from the position of the dispersed images 68 along a
dispersion axis of wavelength dispersive element 54 for multiple
beads 64 distributed along the slit viewing field 74 along a second
axis y, as can be understood with reference to FIG. 5B. Absolute
accuracy of the spectral readings will vary inversely with a width
of aperture slit 62, and the number of readings (and hence total
reading time) for reading all the beads in area 70 will be longer
as the slit gets narrower. Nonetheless, the beads 64 within the
two-dimensional area 70 may eventually be read by the system of
FIGS. 5A and 5B with a scanning system which moves the slit
relative to beads 64 (using any of a variety of scanning
mechanisms, such as movable mirrors, a movable aperture, a flow of
the beads passed a fixed aperture, a movement of the surface of the
vessel relative to the aperture, or the like).
[0115] While the techniques described above are capable of
producing spectral images, there are at least two distinct
disadvantages to most scanning systems. First, most scanning
systems are susceptible to mechanical or electronic failure that
would not exist in a static (non-scanning) system. Second, since
many data points are used to generate a single spectral image, a
limit is placed on the minimum time required in order to acquire a
full image. Depending on the signal levels, this time could be
several minutes or more. This generally precludes the use of
scanning techniques in any system in which the spatial position of
each point is not fixed. For instance, imaging the two-color beads
described above in an aqueous medium may be difficult with a
scanning system, since the beads can diffuse to different spatial
positions during the acquisition of a single spectral image.
[0116] Static spectral imaging systems, in which spectral
information is acquired without scanning, are very appealing since
data is acquired in a single step. An example of a static spectral
imaging system is one in which a spatial image is passed though
several beam-splitters, separating it into multiple images, each of
which is passed though a different band-pass filter. Each resulting
image provides information about a discrete region of the spectrum.
The images are then projected onto a detector and the signals are
recombined to produce an image that contains information about the
amount of light within each band-pass. Such systems are appealing
because all spectral information may be acquired simultaneously,
eliminating difficulties arising from non-stationary samples. The
disadvantage of a band-pass imaging system is that only a discrete
number of wavelengths can be monitored, precluding detailed
spectral analysis and fitting. At the same time, band-pass filters
and dichroic mirrors are not 100% efficient, reducing the potential
detection efficiency of multiple colors. For instance, a band-pass
system using ten 10% beamsplitters and 10 bandpass filters results
in a maximum of 10% detection efficiency along each channel. A
system of dichroic mirrors, each with 85% transmission efficiency
yields approximately 20% efficiency along the final channel.
[0117] Referring once again to FIGS. 5A and 5B, one-dimensional
spectral imaging can be achieved by projecting a fluorescent image
onto and/or through the entrance slit of a linear spectrometer, as
shown. In this configuration, spatial information is retained along
the y-axis, while spectral information (wavelengths .lambda.) is
dispersed along the x-axis, as described by Empedocles, et al. in
Phys. Rev. Lett., 77 (18); p. 3873 (1996). The entrance slit
restricts the spatial position of the light entering the
spectrometer, thereby (at least in part) defining the calibration
for each spectrum. The width of the entrance slit, in part, defines
the spectral resolution of the system.
[0118] Referring now to FIG. 6, a two-dimensional imaging system 80
allows simultaneous sensing of spectral information from bead 64
distributed throughout a two-dimensional sensing field 81. System
80 generally makes use of a detector 82 and a system for
restraining and/or identifying a position of beads 64 within
two-dimensional sensing field 81. In the exemplary embodiment, the
positioning system or means makes use of a bead position indicator
84. Positioning indicator 84 is optically coupled to sensing field
81. More specifically, a beam splitter 86 separates a portion of an
image generated by optical train 58, and directs the image portion
88 onto a position sensor. As described above, detector 82 makes
use of a wavelength dispersive element 54 and an areal sensor 56
aligned with optical train 58, hence, at least a portion of the
optical path between two-dimensional sensing field 81 and the
positioning system 84 is coaxial with the optical path between the
sensing field 81 and sensor 56 of the detector 82.
[0119] As beads 64 are distributed across two-dimensional sensing
field 81 and are not limited to a single lateral axis, wavelength
dispersive element 54 will distribute the spectra from beads 64
across the surface of sensor 56 based on both the wavelength of the
spectra from each bead and the associated position of the bead
within the sensing field. Where beads 64 are sufficiently small in
area so as to be treated as point-light sources, and where there is
significantly more area surrounding the beads than the total
surface area of the beads themselves so that the distributed
spectra from the labels on the beads do not overlap excessively,
sensor 56 can be used to determine relative spectra of the beads.
For example, analyzer 90, in response to signals from sensor 56,
may determine that a particular bead 64a has three equally-spaced
wavelength peaks of substantially even intensity, with a fourth
wavelength peak of twice the intensity of the other peaks separated
from the lowest of the peaks by three times the wavelength
differential between the other peaks. While such relative spectral
information is useful (and may be sufficient to identify codes in
some coding systems) it will often be advantageous to provide both
relative and absolute spectral information for each of beads
64.
[0120] Fortunately, positioning image 88 generated upon a sensor
surface 92 of position indicator 84 defines the position of beads
64 within sensing field 81. Signals transmitted from the sensor of
position indicator 84 to analyzer 90 can define positions for each
bead 64, and the analyzer can correlate each bead position with its
associated spectra (and hence the sensed relative spectra) to
determine the absolute spectrum from each bead. By taking advantage
of the point-light source qualities of the relatively small beads
within sensing field 81, no aperture need be included within
two-dimensional system 80. In some embodiments, the positioning
image and the spectrally disbursed image may be projected onto a
common sensor, either sequentially or on different positions of the
common sensor. Still further alternatives are possible, such as the
projection of a zero-order image on the CCD for spatial
information.
[0121] Stated differentially, two-dimensional images can be
obtained by eliminating the entrance slit from a linear
spectrometer and allowing the discrete images from individual
points to define the spatial position of the light entering the
spectrometer (FIG. 6). In this case, the spectral resolution of the
system is defined, in part, by the size of the discrete images.
Since the spatial position of the light from each point varies
across the x-axis, however, the calibration for each spectrum will
be different, resulting in an error in the absolute energy values.
Splitting the original image and passing one half through a
dispersive grating to create a separate image and spectra can
eliminate this calibration error. With appropriate alignment, a
correlation can be made between the spatial position and the
absolute spectral energy (FIG. 6A).
[0122] Correlation of the positioning image 88 with the spectrally
dispersed image 68 can be understood with FIGS. 6A through 6C.
Positioning image 88 generally indicates positions of beads 64
within sensing field 81, while spectrally dispersed image 68
reflects both the position and spectral wavelengths of each signal
within the spectra generated by beads 64. Using an accurately
calibrated system, analyzer 90 can determine the absolute
wavelengths of a particular dispersed image 96a by identifying the
associated bead position 64a, particularly where beads 64 do not
overlap along the y-axis. As can be understood with reference to
FIGS. 6B and 6C, correlation of beads' locations and spectrally
dispersed images may be facilitated by including a calibration
signal 40c within at least one of the spectra generated by a bead.
Such calibration signals will often be included in at least some of
the bead spectra, optionally being included in each bead spectrum.
Where the calibration signal wavelength is known, the location of
the associated bead along the x-axis can be determined from the
location of the calibration signal energy within the dispersed
image 68 from the diffracting characteristics of wavelength
dispersive element 54.
[0123] Referring now to FIGS. 7A-7C, ambiguity may arise when
images of beads 64 fall along and/or adjacent to a dispersion axis,
along a horizontal line in the example of FIG. 7A. To avoid such
ambiguity, an alternative two-dimensional spectral sensing system
80' includes an additional beam splitter 86' with a second
dispersive element or wavelength dispersive element 54', with the
second wavelength dispersive element having a diffraction axis
oriented at an angle to the dispersive axis of the first wavelength
dispersive element 54 relative to the image of the two-dimensional
sensing field 81. Typically, the second wavelength dispersive
element will have a dispersive axis oriented at 90.degree. to the
dispersive axis of the first wavelength dispersive element,
although any angle between 0.degree. and 180.degree. could be used.
This second wavelength dispersive element generates a dispersed
image 68' along the second dispersive axis (typically orthogonal to
the first dispersive axis), allowing analyzer 60 to unambiguously
distinguish the spectra from each discrete point within the image.
In related embodiments, two orthogonal (or otherwise angularly
offset) dispersive elements may be disposed along the same imaging
path, or possibly even jowled together to disperse a single image
spectrally along two offset dispersive axes. The tow offset spectra
may be imaged onto a single sensor. Positions of the beads may be
determined from the intersections of each spectral pair, so that a
processor derives the position form the combined images 68 and 68',
as can be understood with reference to FIG. 7C.
[0124] In the preferred embodiment, the original image is split
into 2 (or 3) images at ratios that provide more light to the
spectrally dispersed images, which have several sources of light
loss, than the direct image. In the preferred embodiment, the
spectral dispersion is performed using holographic transmission
gratings, however, similar results can be obtained using standard
reflection gratings.
[0125] This system will be useful for any spectral imaging
application where the image is made up of discrete points, such as
discrete labeled cellular material. It should also be useful for
high throughput screening of discrete spectral images such as
single molecules or ensembles of molecules immobilized on a
substrate such as a surface or bead. This technique can also be
used to perform highly parallel reading of spectrally encoded
beads.
[0126] Varying Signal Strengths
[0127] Referring now to FIGS. 2 and 8, test fluid 34 may generate
two very different types of signals for interpretation of parallel
assays: semiconductor nanocrystals 37 affixed to the bead bodies
generate a relatively robust, high intensity label spectra 100,
while assay markers 38 may generate a significantly lower intensity
assay signal 102. The significant difference in the strengths of
these two types of signals may complicate the interpretation of an
actual individual spectra from a highly multiplexed assay, such as
that illustrated schematically in FIG. 8A.
[0128] Since it is relatively trivial to detect arbitrarily large
signals, a large dynamic range generally requires detection of as
few markers as possible; ideally the detection limit will be a
single marker. Since it is possible to detect single molecules and
single semiconductor nanocrystals, it should in principle be
possible to reach this level of detection in an assay. One problem
arises in optimizing the detection of both the spectral code from a
bead, which is typically very bright, and the marker signal from
the bead, which is typically very dim.
[0129] One issue in the detection of very low signal levels is
integration time, i.e., how long must a signal be integrated to be
detected. In the case of single molecules, the answer is
approximately 0.1 to 1.0 second. If it were necessary to scan a
point or even a slit across a sample in order to get a
two-dimensional spectral image, this could take an extremely long
time. Two-dimensional spectral imaging allows one to take spectra
from an entire image in the same time that it would take to get a
single spectrum. However, to do two-dimensional spectral imaging,
the spacing between adjacent beads on the sample should be large
enough to limit the overlap of spectra from adjacent beads falling
on the CCD detector. Even with precise placement of beads, it is
still desirable to devote a large portion of the CCD (and therefore
the sample surface) for the spectra of each bead. This means that
the density of beads, or other materials, in a two-dimensional
spectral image should be fairly low. This reduces the number of
beads that can be read simultaneously. The same is true of using
multiple slits to scan multiple regions of a sample simultaneously.
The spacing between the slits, and therefore the number of regions
that can be scanned, is limited by the region of the CCD dedicated
to reading the spectra from each slit. Furthermore, in the case of
large signals, e.g., for instance the signal from a spectrally
encoded bead, the integration time for each image may be less than
the readout rate of the CCD. In that case, the advantage of
two-dimensional spectral imaging is lost, because the readout time
increases linearly with the number of pixels, and thus with the
number of beads being detected. It is only when the integration
time is long relative to the readout rate that this type of
parallel imaging becomes valuable.
[0130] An alternative form of spectral imaging is scanning a single
slit over the sample and creating a spectral image by plotting
spectra as a function of position. In this case, when the
integration time is less than the readout rate, the time required
to get a complete spectral image is the same as with
two-dimensional spectral imaging. When the integration time is
longer than the readout rate, however, this method is considerably
slower. While slit scanning can never be faster than
two-dimensional spectral imaging, it does have the added advantage
that high density samples can be used, since no portion of the CCD
and sample must be devoted to spectra.
[0131] As described above, there are different approaches for
spectral imaging. The appropriate choice depends on the integration
time required to collect signal from the bead. For very short
integration times needed for, e.g., spectral code reading, a
scanned slit is preferred. For long integration times, e.g., for
marker reading, two-dimensional spectral imaging is most
appropriate. Since the above described encoded beads include an
assay marker associated therewith, both long and short integration
time acquisitions would be beneficial. It would therefore be
desirable to develop a system that can maximize simultaneously the
detection speed of both short and long integration time
signals.
[0132] Referring to FIGS. 8 and 8B, a relatively short integration
time, such as that provided by a scanning system, might provide a
first dynamic range 104. Unfortunately, a scanning system having
dynamic range 104 may exhibit a background noise level 106 which
makes interpretation of assay signal 102 problematic.
Alternatively, as shown in FIGS. 8 and 8C, a reading system which
could efficiently gather information despite a relatively long
integration time, so as to provide a lower intensity dynamic range
108 appropriate for reading assay signal 102, may exhibit
saturation (schematically illustrated as the flat region of
long-integration measured signal 110) induced by the relatively
high-intensity label spectra. In many embodiments, overcoming these
potentially conflicting criteria is facilitated by maintaining the
label spectra within a first wavelength range 112a, and the assay
marker signals within a second wavelength range 112b which is
separate from the first wavelength range.
[0133] FIG. 9 schematically illustrates a technique designed to
maximize the rate of decoding and reading the markers from
spectrally encoded beads. It involves both slit scanning and
two-dimensional imaging. In this system, beads 64 are scanned
rapidly under a slit (by movement of the beads and/or scanning of
the slit). During this time, the spectral codes are read at a rate
that is fast relative to the read-out rate of the CCD detector.
After the beads pass the slit or are scanned, they may move into an
imaging area. Once the image area has been filled, the scanning
stops and a single image is taken of the beads. The image is passed
though a band-pass filter 128 that selects only the signal from the
marker. This image is acquired on a two-dimensional array. The
spectral codes from the scanned slit are then correlated with the
two-dimensional image to combine the code and marker data. Once
completed, a new sample of beads is scanned past the slit and into
the image area and the process is repeated. Alternatively,
two-dimensional imaging may occur before or during scanning and/or
the scanning and imaging may be performed in the same static
viewing area, as shown.
[0134] With this system, it is possible to maximize the acquisition
efficiency of both types of signals. As an example, a set of
brightly encoded beads may generate low intensity marker signals.
For this example, it is assumed that: (1) the spectral code can he
read with an integration time of 10 ms and the marker can be read
with an integration time of 1 second; and (2) that it takes 100
steps to scan a slit across the entire image and that the spacing
between multiple, adjacently scanned slits may be 20% of the image
size. To acquire a spectral image using slit scanning, the
integration time at each position might be 1 second to detect both
the code and the marker. Therefore, the total acquisition time for
a single image would be 100 seconds. To use two-dimensional
spectral imaging, the scanning rate is increased; however, the
density of the sample scanned is decreased. This might reduce the
number of beads per image by a factor of 20. While the
two-dimensional spectral image can then be acquired in 1 second, 20
such areas should be scanned to accumulate the same data as in the
single slit scanning example. Therefore, the data is acquired in 20
seconds. One final disadvantage of using the two-dimensional
spectral imaging system is that the signal from the spectral code
should not saturate in the time required to detect the marker.
[0135] By using the combination scanning/imaging system described
herein, the acquisition time is greatly reduced. The spectral codes
are read at 10 ms/step over 100 steps. The marker image is detected
with a single 1-second integration time. The total acquisition time
is then 2 seconds for the whole spectral image.
[0136] Referring to FIGS. 8 and 9, a scanning/imaging system 120
generally comprises a detector which is optically coupled with
two-dimensional sensing field 81 by optics 58, and a scanner 124
having an aperture 62. Aperture 62 will generally be movable
relative to bead 64 of two-dimensional sensing field 81, either
through movement of the aperture (and associated apertured sensing
field 74), by software coupled to the CCD, or movement of the
beads.
[0137] To allow scanning/imaging system 120 to detect relatively
low-intensity signals within the two-dimensional sensing field 81,
optics 58 image the sensing field upon a surface of sensor 56. A
spectral filter 128 selectively transmits marker signals 102 to
sensor 56 of the detector, thereby avoiding saturation from the
relatively high-intensity spectral label signals. Using our simple
marker/label separation scheme illustrated in FIG. 8, filter 128
may comprise a dichroic filter which selectively transmits the
marker signals within second range 112b. Clearly, more complex
filtering and signal separation arrangements are possible.
Regardless, as numerous beads 64 within two-dimensional sensing
field 81 can have their assay markers detected simultaneously, a
relatively long integration time may be employed without adding
excessively to the overall sensing time.
[0138] In the schematic embodiment illustrated in FIG. 9, a beam
splitter 86 directs a separate signal portion to a sensor 56 of
scanner 124. Aperture 62 restricts an apertured sensing field of
the scanner 74 so that beads 64 are read sequentially in a line.
Each reading of the relatively bright spectral codes from the beads
can make use of a quite short integration time, optionally during
the long integration time employed by the two-dimensional marker
imaging system.
[0139] In an alternative embodiment, spectra and image/position
data may be sensed by the same sensor. Any of the scanning systems
described herein may be applied. After the spectra are scanned (or
before) a bandpass filter may remove the spectral information,
leaving assay signals and bead location information for each
associated signal in the 2-D image. Assay results may then be
determined from the locations of the signals and the dispersion of
the grating.
[0140] As mentioned above, sequential sensing of the spectra may be
performed by moving the aperture relative to the sensing field, by
software, by moving the beads (or other signal sources) relative to
the optical train or scanning system, or even by scanning one of an
excitation energy or the beads relative to the other. Aperture
scanning may be effected by a galvanometer, by a liquid crystal
display (LCD) selective transmission arrangement, by other digital
arrays, or by a digital micro-mirror array (DMD). Bead scanning
systems may use a fluid flow past a slit aperture, with the beads
flowing with the fluid. Such bead flow systems result in movement
of the aperture relative to the beads, even when the aperture
remains fixed, as movement may be determined relative to the bead's
frame of reference.
[0141] Referring now to FIG. 9A, a simple fluid-flow assay system
can make use of many of the structures and methods described herein
above. In the illustrated embodiment, a test fluid 34 flows through
a channel 131 so that beads 64 move across sensing field 74. Beads
64 within the slit-apertured sensing region are spectrally
dispersed and imaged as described above. As the location of the
slit-aperture is known, absolute spectral information regarding the
label spectra and assay signals may be determined from dispersed
image 68. When a plurality of beads are within sensing region 74
but separated along the x axis as shown, multiple beads may be read
simultaneously by a CCD, or the like. Flowing of the beads
sequentially through sensing region 74 may allow simultaneous assay
preparation and reading using flow injection analysis techniques,
or the like.
[0142] Imaging of sensing region 74 may be facilitated by providing
a thin, flat channel 131 so that beads 64 are near opposed major
surfaces of the channel, with at least one of the channel surfaces
being defined by a material which is transparent to the spectra and
marker signals. This fluid-flow system may be combined with many
aspects of the systems described hereinabove, for example, by
providing two different energy sources for the label spectra and
assay markers, by areal imaging of beads 64 distributed throughout
a two-dimensional sensing region adjacent to or overlapping with
slit-apertured sensing region 74, and the like.
[0143] A variety of modifications of the scanning/imaging system
120, and of the other imaging systems described herein above, are
encompassed within the present invention. For example, the optics
schematically illustrated in the figures may include optical
elements along the optical path before any apertures, after any
apertures, and/or on either side of any apertures. Similarly, at
least a portion of the optical train may be disposed after any beam
splitters. Rather than relying on separate sensors 56 for scanning,
position indication, two-dimensional imaging, and/or diffraction
image sensing, the optics may be arranged so as to direct these
differing images to a common sensor. Differing images may also be
acquired simultaneously or sequentially. Where areal sensing is not
required, it may be possible to make use of linear, point, or bulk
light sensors or photodetectors.
[0144] The systems of the present invention are particularly
well-suited for identification of label spectra that are spatially
intermingled with other markers, especially where at least one
label and/or at least one assay marker comprises a semiconductor
nanocrystal. As described above, an analyzer 90 will often
correlate the labels from each bead with an associated marker
signal (which may comprise an absence or absorbance of energy
having a characteristic wavelengths, scattering, a change in
signal/energy characteristics, or the like).
[0145] In one preferred embodiment, the two detection pathways
follow the same optical path and fall on the same detector. In this
embodiment, the excitation of the sample under the slit is
shuttered during image acquisition or is otherwise oriented such
that no spectra are obtained during the image acquisition. In
separate embodiments, the multiple detection pathways can be used
as well as multiple detectors.
[0146] The scanning/imaging system of FIG. 9 illustrates yet
another advantageous aspect of the present invention which may find
applications in other signal detection systems including those
described above. A simplified system for sensing both
high-intensity signals (such as spectral labels) and normally
low-intensity signals (such as assay markers) may include a first
excitation energy source 20a transmitting an excitation energy
toward fluid 34 (see FIG. 1) for generation of spectral codes from
the beads. First excitation energy source 20a may also, at least to
some extent, induce marker signals 102. However, a second
excitation energy source 20b also transmits an excitation energy
source toward the beads, with the excitation energy from this
second source selectively energizing the assay markers. This may be
accomplished, for example, by limiting the second excitation energy
source to a wavelength that is higher in energy than the
low-intensity marker signals, but which is lower in energy than the
high-intensity label signals. By selectively energizing the first
and/or second excitation energy sources, and/or by varying at least
one of the excitation energies relative to the other, the dynamic
range of the overall system can be effectively broadened to
accurately and reliably sense both the otherwise relatively weak
assay marker signals and the quite strong spectral labels. Either
or both of these excitation energy sources might be scanned
relative to the beads to effectively control the location and/or
size of the sensing field for the labels and/or assay markers.
[0147] In many preferred embodiments of signal detection systems,
two light sources are used. The first light source is an
inexpensive blue light source for exciting the spectral code and
the marker simultaneously. The blue source illuminates the slit
region of the sample, and the apertured region of the sample is
then dispersed and sensed as described above. Since it does not
require much light to detect the spectral code, this light source
can be very inexpensive. The two-dimensional image region of the
sample is then excited with a higher power red laser, which excites
only the marker semiconductor nanocrystals. This allows efficient
detection of the marker while eliminating the possibility of the
spectral codes saturating due to high excitation intensity.
[0148] In an alternative embodiment, the two light sources are used
to tune the relative intensities of the code and marker during
simultaneous detection. For example, if both marker and code are
detected using slit scanning or two-dimensional spectral imaging
alone, it is likely that the code would saturate in the time
required to detect the marker. This is avoided if the relative
excitation intensity for the code (blue light) is very weak
relative to the excitation intensity for the marker (red light).
The advantage of such a setup is that the relative intensity of the
code to marker signal can be tuned by adjusting the two light
sources. This reduces any concerns about dynamic range limitations
between the code and the marker. This two-light source system is
advantageous in any detection scheme that involves a wide dynamic
range that must be simultaneously detected (such as the
marker/bar-code system). It should therefore be useful in systems
other than that described in the current disclosure.
[0149] Fixed Position Beads
[0150] Techniques to analyze bead-based assays can be flow based
and/or imaging based. In the flow-based analysis, an instrument
such as sheath flow cytometer is used to read the fluorescence and
scatter information from each bead individually. Flow methods have
the disadvantage of requiring a relatively large volume of sample
to fill dead volume in the lines and do not allow averaging or
re-analysis of data points. Flow methods do allow a large number of
beads to be analyzed from a given sample. Imaging based systems,
such as the Biometric Image.TM. system, scan a surface to find
fluorescence signals. Advantages over the flow system include small
(<20 microlitre) sample volumes and the ability to average data
to improve signal to noise. The disadvantage is the need for a
large area in order to keep beads separated, and the dependence on
beads being an appropriate dilution to ensure that a sufficient
number can be analyzed without too many forming into doublets,
triplets, or the like.
[0151] Referring now to FIGS. 10A-10C, beads can be immobilized on
a planar surface such that they are regularly spaced in a chosen
geometry. The beads can be immobilized by micromachining wells into
the planar surface. For example, 7-micron wells that are 7 microns
deep, can be created by ablating a 7 micron layer of parylene on a
glass surface using a focused laser. Other methods can be used to
create microstructures on the glass surface that behave as wells.
The well dimensions are chosen such that only a single bead is
captured in the well and such that, when a lateral flow of fluid
passes the beads, the single beads remain trapped in the wells (see
FIG. 10C). The 7-micron well described may be suitable for analysis
of beads from around 4 microns to 6 microns, or "monodisperse" 5
micron beads. Other methods for capturing beads include selective
deposition of polymers using light-activated polymerization, where
the pattern of light is determined using a photoresist. The
polymers then bind non-specifically to single beads and other beads
can be washed away.
[0152] In use, the mixture of spectrally encoded beads that have
undergone an assay are deposited onto the capture surface and
allowed to settle into wells (by gravity) or to bind to the capture
surface. Excess beads are then washed away leaving single beads
filling up some portion, for example, >90% of the wells or
capture positions.
[0153] Still further structures might be used to immobilize and/or
position the beads, including superparamagnetic bead positioners
being developed by IMMUNICON CORPORATION of Pennsylvania, and by
ILLUMINA, INC. of San Diego, Calif.
[0154] The captured beads can then be analyzed using an imaging
system to capture fluorescence data at various emission wavelengths
for each bead. This method provides advantages over a simple scan
of randomly placed beads because (1) beads are known to be
separated so the spatial resolution required for detection can be
reduced as doublets do not have to be found and rejected--this
leads to greater analysis efficiency, (2) the packing of beads can
be considerably higher while still retaining spatially separated
singlet beads, (3) the beads do not move relative to the support
and so can be scanned multiple times without concerns about
movement, and (4) the concentration of beads in the sample that is
applied does not need to be precise (in the random scattering
approach too high a concentration leads to a high packing and
eventually a multi-layer structure whereas too low a concentration
leads to too few beads being analyzed).
[0155] In a system where spatial and spectral information are
combined by placing a coarse grating (reflection or transmission)
in the emission path, such that the emitted light from each bead is
spectrally dispersed in one dimension, the use of micromachined
wells is particularly useful. The wells are machined such that the
dispersed images of each bead cannot overlap. In addition,
knowledge of the bead positions means that absolute wavelength
determination can be carried out rather than relative
determinations or using a spectral calibrator (See FIG. 11).
[0156] Still further alternative bead positioning means are
possible. In one variation of the positioning wells illustrated in
FIGS. 10A-10C, a closely packed array of collimated holes may be
distributed across a surface. Where the holes extend through a
substrate defining the surface, a pressure system may be provided
along an opposed surface so as to actively pull beads 64 and test
fluid 32 into the array of holes. Such a system would allow a set
of beads to be pulled into positioning wells, to have the assay
results (optionally including bead labels and assay markers) read
from the entrained beads, and then optionally, to push the beads
out of the through holes. Such a positioning and reading cycle may
be repeated many times to read a large number of beads within a
test fluid. While there may be difficulty in transporting the beads
and test fluid to the positioning surface, such a system has
significant advantages.
[0157] Specific structures for containing test fluids with beads,
and/or for directing flows of such fluids and beads, may improve
spectral code reading performance. Codes may be read from above,
from below, or from an angle relative to vertical. Reading codes
from below, for example, may be enhanced by using a fluid
containing body with an opaque material over the fluid. The fluid
surrounding the beads may have an index of refraction which
substantially matches that of the material of the lower portion of
the fluid containing body. Such structures may be particularly
beneficial when reading dense bead codes.
[0158] While the exemplary embodiments of the present inventions
have been described in some detail for clarity of understanding, a
variety of modifications, adaptations, and changes will be obvious
to those of skill in the art. Hence, the scope of the present
invention is limited solely by the appended claims.
* * * * *