U.S. patent number RE48,561 [Application Number 16/000,720] was granted by the patent office on 2021-05-18 for compensator for multiple surface imaging.
This patent grant is currently assigned to ILLUMINA, INC.. The grantee listed for this patent is ILLUMINA, INC.. Invention is credited to Maria Candelaria Rogert Bacigalupo, Steven Barnard, Jason Bryant, Wenyi Feng.
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United States Patent |
RE48,561 |
Feng , et al. |
May 18, 2021 |
Compensator for multiple surface imaging
Abstract
A system and method for imaging biological samples on multiple
surfaces of a support structure are disclosed. The support
structure may be a flow cell through which a reagent fluid is
allowed to flow and interact with the biological samples.
Excitation radiation from at least one radiation source may be used
to excite the biological samples on multiple surfaces. In this
manner, fluorescent emission radiation may be generated from the
biological samples and subsequently captured and detected by
detection optics and at least one detector. The detected
fluorescent emission radiation may then be used to generate image
data. This imaging of multiple surfaces may be accomplished either
sequentially or simultaneously. In addition, the techniques of the
present invention may be used with any type of imaging system. For
instance, both epifluorescent and total internal reflection methods
may benefit from the techniques of the present invention.
Inventors: |
Feng; Wenyi (San Diego, CA),
Bryant; Jason (Essex, GB), Barnard; Steven (Del
Mar, CA), Bacigalupo; Maria Candelaria Rogert (Encinitas,
CA) |
Applicant: |
Name |
City |
State |
Country |
Type |
ILLUMINA, INC. |
San Diego |
CA |
US |
|
|
Assignee: |
ILLUMINA, INC. (San Diego,
CA)
|
Family
ID: |
41256502 |
Appl.
No.: |
16/000,720 |
Filed: |
June 5, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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15926322 |
Sep 22, 2020 |
RE48219 |
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14229455 |
Jun 30, 2015 |
9068220 |
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14056590 |
Oct 17, 2013 |
8698102 |
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13974976 |
Aug 23, 2013 |
8586947 |
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13629949 |
Sep 28, 2012 |
8546772 |
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13544716 |
Jul 9, 2012 |
8278630 |
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13399820 |
Feb 17, 2012 |
8242463 |
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13281237 |
Oct 25, 2011 |
8143599 |
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13209306 |
Aug 12, 2011 |
8071962 |
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12434495 |
May 1, 2009 |
8039817 |
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61050522 |
May 5, 2008 |
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61138444 |
Dec 17, 2008 |
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Reissue of: |
14721870 |
May 26, 2015 |
9365898 |
Jun 14, 2016 |
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Reissue of: |
14721870 |
May 26, 2015 |
9365898 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N
21/05 (20130101); G01N 21/6458 (20130101); C12Q
1/6825 (20130101); C12Q 1/6874 (20130101); C12Q
1/6869 (20130101); G02B 21/0024 (20130101); G01N
2021/0346 (20130101); G02B 2207/113 (20130101); G01N
2021/6421 (20130101); G01N 2021/6419 (20130101) |
Current International
Class: |
G01N
21/05 (20060101); C12Q 1/6825 (20180101); G01N
21/64 (20060101); C12Q 1/6874 (20180101); G01N
21/03 (20060101); G02B 21/00 (20060101); C12Q
1/6869 (20180101) |
References Cited
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WO |
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2010/099350 |
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Sep 2010 |
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WO |
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|
Primary Examiner: Gagliardi; Albert J
Attorney, Agent or Firm: Foley & Lardner LLP
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
.Iadd.More than one reissue application of U.S. Pat. No. 9,365,898
has been filed. A continuation reissue of the instant application,
application Ser. No. 17/214,093, has been filed on Mar. 26, 2021.
.Iaddend..[.This.]. .Iadd.The instant .Iaddend.application is
.Iadd.a continuation reissue of U.S. patent application Ser. No.
15/926,322, filed Mar. 20, 2018, which issued as U.S. Pat. No.
RE48,219 on Sep. 22, 2020, which is herein incorporated in its
entirety by reference, which is an application for reissue of U.S.
Pat. No. 9,365,898, which issued on Jun. 14, 2016 from U.S. patent
application Ser. No. 14/721,870, which is herein incorporated in
its entirety by reference, which .Iaddend.is a continuation of U.S.
patent application Ser. No. 14/229,455, entitled "Compensator for
Multiple Surface Imaging," filed Mar. 28, 2014, .Iadd.and issued as
U.S. Pat. No. 9,068,220 on Jun. 30, 2015, .Iaddend.which is herein
incorporated in its entirety by reference, and which is a
continuation of U.S. patent application Ser. No. 14/056,590,
entitled "Compensator for Multiple Surface Imaging," filed Oct. 17,
2013, and issued as U.S. Pat. No. 8,698,102 on Apr. 15, 2014, which
is herein incorporated in its entirety by reference, and which is a
continuation of U.S. patent application Ser. No. 13/974,976,
entitled "Compensator for Multiple Surface Imaging," filed Aug. 23,
2013, and issued as U.S. Pat. No. 8,586,947 on Nov. 19, 2013, which
is herein incorporated in its entirety by reference, and which is a
continuation of U.S. patent application Ser. No. 13/629,949,
entitled "Compensator for Multiple Surface Imaging," filed Sep. 28,
2012, and issued as U.S. Pat. No. 8,546,772 on Oct. 1, 2013, which
is herein incorporated in its entirety by reference, and which is a
continuation of U.S. patent application Ser. No. 13/544,716,
entitled "Compensator for Multiple Surface Imaging," filed Jul. 9,
2012, and issued as U.S. Pat. No. 8,278,630 on Oct. 2, 2012, which
is herein incorporated in its entirety by reference, and which is a
continuation of U.S. patent application Ser. No. 13/399,820,
entitled "Compensator for Multiple Surface Imaging," filed Feb. 17,
2012, and issued as U.S. Pat. No. 8,242,463 on Aug. 14, 2012, which
is herein incorporated in its entirety by reference, and which is a
continuation of U.S. patent application Ser. No. 13/281,237,
entitled "Compensator for Multiple Surface Imaging," filed Oct. 25,
2011, and issued as U.S. Pat. No. 8,143,599 on Mar. 27, 2012, which
is herein incorporated in its entirety by reference, and which is a
continuation of U.S. patent application Ser. No. 13/209,306,
entitled "Compensator for Multiple Surface Imaging," filed Aug. 12,
2011, and issued as U.S. Pat. No. 8,071,962 on Dec. 6, 2011, which
is herein incorporated in its entirety by reference, and which is a
continuation of U.S. patent application Ser. No. 12/434,495,
entitled "Compensator for Multiple Surface Imaging," filed May 1,
2009, and issued as U.S. Pat. No. 8,039,817 on Oct. 18, 2011, which
is herein incorporated in its entirety by reference, and which
claims priority of U.S. Provisional patent application Ser. No.
61/050,522, entitled "Multi-Surface Biological Sample Imaging
System and Method," filed May 5, 2008, which is herein incorporated
in its entirety by reference, and of U.S. Provisional patent
application Ser. No. 61/138,444, entitled "Compensator for Multiple
Surface Imaging," filed Dec. 17, 2008, which is herein incorporated
in its entirety by reference.
Claims
The invention claimed is:
.[.1. A system, comprising: a flow cell comprising first and second
surfaces separated by a fluid passage through which a fluorescent
reagent flows to add fluorescent tags to nucleic acid sites
distributed on the first and second surfaces; and a detection
system configured to detect the fluorescent tags to distinguish the
nucleic acid sites on the first surface, and to detect the
fluorescent tags to distinguish the nucleic acid sites on the
second surface..].
.[.2. The system of claim 1, wherein the nucleic acid sites are
distributed in a spatially ordered pattern on the first surface and
on the second surface..].
.[.3. The system of claim 1, wherein the nucleic acid sites are
distributed in a random spatial distribution on the first surface
and on the second surface..].
.[.4. The system of claim 1, wherein the nucleic acid sites are
separated with spaces between each other..].
.[.5. The system of claim 1, wherein the nucleic acid sites are
present at a density of at least 1,000 sites per square
millimeter..].
.[.6. The system of claim 1, wherein the detection system is
configured to detect the fluorescent tags at an optical resolution
between 0.1 and 50 microns..].
.[.7. The system of claim 1, wherein the flow cell is configured to
be translated, and the detection system if configured to detect the
fluorescent tags at successive regions of the first surface and at
successive regions of the second surface, respectively..].
.[.8. The system of claim 7, wherein the detection system is
configured to perform wide angle area detection of each successive
region..].
.[.9. The system of claim 1, wherein the first surface and the
second surface are detected from the same side of the flow
cell..].
.[.10. The system of claim 9, wherein the first surface is disposed
between an excitation source and the second surface during
detection by the detection system..].
.[.11. The system of claim 10, wherein the first surface and the
second surface are excited by total internal reflection..].
.[.12. The system of claim 9, wherein the first surface is disposed
between a detector and the second surface during detection by the
detection system..].
.[.13. The system of claim 12, comprising corrective optics
configured to be inserted and removed between the detector and the
flow cell after detection of the fluorescent tags on the first
surface by the detection system..].
.[.14. The system of claim 13, wherein the corrective optics
comprise a lens, objective, or cover slip..].
.[.15. The system of claim 12, wherein the first surface is
detected through a first objective and the second surface is
detected through a second objective..].
.[.16. The system of claim 1, wherein the first surface and the
second surface are detected from opposite sides of the flow
cell..].
.[.17. The system of claim 1, wherein the detection system is
configured to produce one or more images of the first surface and
the second surface..].
.[.18. The system of claim 17, wherein the one or more images have
a resolution of 10 microns or less..].
.[.19. The system of claim 1, comprising a radiation source
configured to direct excitation radiation toward the first and
second surfaces at several different wavelengths..].
.[.20. The system of claim 19, wherein the detection system is
configured to capture and detect emitted radiation returned in
response to each wavelength..].
.[.21. The system of claim 1, wherein the detection system is
configured to perform confocal detection of fluorescence emitted
from the nucleic acid sites..].
.[.22. The system of claim 1, wherein the detection system is
configured to repeat detection of the fluorescent tags in a process
of sequencing the nucleic acids at the nucleic acid sites..].
.[.23. The system of claim 22, wherein the sequencing comprises
sequencing by synthesis..].
.[.24. The system of claim 22, wherein the sequencing comprises
sequencing by ligation..].
.[.25. The system of claim 1, wherein the fluorescent reagent
comprises fluorescently labeled nucleic acids..].
.[.26. The system of claim 1, wherein the fluorescent reagent
comprises fluorescently labeled nucleotides..].
.[.27. The system of claim 1, wherein each of the nucleic acid
sites constitutes a population of nucleic acids having identical
sequences..].
.Iadd.28. A method of manufacturing a biological evaluation support
structure, the method comprising: providing a first substrate
having a first surface on which a first plurality of biological
sample sites are disposed; providing a second substrate having a
second surface on which a second plurality of biological sample
sites are disposed; forming a patterned adhesive on at least one of
the first surface of the first substrate or the second surface of
the second substrate; and bonding the first substrate to the second
substrate via the patterned adhesive, such that the second surface
of the second substrate, with the second plurality of biological
sample sites thereon, faces the first surface of the first
substrate, with the first plurality of biological sample sites
thereon, so as to form a biological evaluation support structure.
.Iaddend.
.Iadd.29. The method of claim 28, wherein the first plurality of
biological sample sites are disposed in a predetermined pattern on
the first surface of the first substrate, and the second plurality
of biological sample sites are disposed in a predetermined pattern
on the second surface of the second substrate. .Iaddend.
.Iadd.30. The method of claim 29, wherein each of the biological
sample sites of the first plurality of biological sample sites
comprises a well in the first surface of the first substrate, and
each of the biological sample sites of the second plurality of
biological sample sites comprises a well in the second surface of
the second substrate. .Iaddend.
.Iadd.31. The method of claim 28, wherein the adhesive is patterned
so as to form a plurality of flow passages through which reagent
fluids are flowable. .Iaddend.
.Iadd.32. The method of claim 28, wherein the adhesive comprises an
epoxy resin. .Iaddend.
.Iadd.33. The method of claim 28, wherein the patterned adhesive is
printed on the at least one of the first substrate or the second
substrate. .Iaddend.
.Iadd.34. The method of claim 28, where the adhesive is a
heat-curable adhesive, and the method further comprises curing the
adhesive by heating the adhesive. .Iaddend.
.Iadd.35. The method of claim 28, wherein the biological evaluation
support structure is a flow cell. .Iaddend.
.Iadd.36. The method of claim 28, wherein, in bonding the first
substrate to the second substrate via the patterned adhesive, the
first substrate is bonded to the second substrate via both the
patterned adhesive and an intermediate layer. .Iaddend.
.Iadd.37. The method of claim 36, wherein the intermediate layer is
made of a polymer material. .Iaddend.
.Iadd.38. The method of claim 36, wherein forming the patterned
adhesive comprises forming a first patterned adhesive on the first
substrate and forming a second patterned adhesive on the second
substrate. .Iaddend.
.Iadd.39. The method of claim 38, wherein forming the first
patterned adhesive and forming the second patterned adhesive
comprise printing the first patterned adhesive onto the first
substrate and printing the second patterned adhesive onto the
second substrate. .Iaddend.
.Iadd.40. The method of claim 28, wherein the second substrate has
a plurality of ports extending therethrough. .Iaddend.
.Iadd.41. The method of claim 28, wherein each of the biological
sample sites of the first and second plurality of biological sample
cites is a nucleic acid site. .Iaddend.
.Iadd.42. The method of claim 28, wherein: the patterned adhesive
defines a fluid passage through which a fluorescent reagent is
flowable to add fluorescent tags to the biological sample sites;
and the method further comprises using a detection system to detect
the fluorescent tags to distinguish the biological sample sites on
the first surface and to detect the fluorescent tags to distinguish
the biological sample sites on the second surface. .Iaddend.
.Iadd.43. A method of manufacturing a flow cell, the method
comprising: providing a first substrate having a first surface on
which a first plurality of biological sample sites are disposed;
providing a second substrate having a second surface on which a
second plurality of biological sample sites are disposed; providing
an intermediate layer having a predetermined pattern; and bonding
the first substrate to the second substrate via the intermediate
layer, such that the second surface of the second substrate, with
the second plurality of biological sample sites thereon, faces the
first surface of the first substrate, with the first plurality of
biological sample sites thereon, so as to form a flow cell.
.Iaddend.
.Iadd.44. The method of claim 43, wherein, in bonding the first
substrate to the second substrate via the intermediate layer, the
intermediate layer is heat bonded to the first and second
substrate. .Iaddend.
.Iadd.45. The method of claim 43, wherein the first plurality of
biological sample sites are disposed in a predetermined pattern on
the first surface of the first substrate, and the second plurality
of biological sample sites are disposed in a predetermined pattern
on the second surface of the second substrate. .Iaddend.
.Iadd.46. The method of claim 43, wherein each of the biological
sample sites of the first plurality of biological sample sites
comprises a well in the first surface of the first substrate, and
each of the biological sample sites of the second plurality of
biological sample sites comprises a well in the second surface of
the second substrate. .Iaddend.
.Iadd.47. The method of claim 43, wherein the intermediate layer is
patterned so as to form a plurality of flow passages through which
reagent fluids are flowable. .Iaddend.
.Iadd.48. The method of claim 43, wherein each of the first and
second plurality of biological sample sites has a density of at
least 1,000 sites per square millimeter. .Iaddend.
.Iadd.49. The method of claim 43, wherein the second substrate has
a plurality of ports extending therethrough. .Iaddend.
.Iadd.50. The method of claim 43, wherein each of the biological
sample sites of the first and second plurality of biological sample
cites is a nucleic acid site. .Iaddend.
.Iadd.51. The method of claim 43, wherein: the predetermined
pattern defines a fluid passage through which a fluorescent reagent
is flowable to add fluorescent tags to the biological sample sites;
and the method further comprises using a detection system to detect
the fluorescent tags to distinguish the biological sample sites on
the first surface and to detect the fluorescent tags to distinguish
the biological sample sites on the second surface. .Iaddend.
.Iadd.52. A support structure for use in evaluating biological
samples, the support structure comprising: a first substrate having
a first surface on which a first plurality of biological sample
sites are disposed; a second substrate having a second surface on
which a second plurality of biological sample sites are disposed;
and an intermediate layer having a predetermined pattern, wherein
the first substrate is bonded to the second substrate via the
intermediate layer, such that the second surface of the second
substrate, with the second plurality of biological sample sites
thereon, faces the first surface of the first substrate, with the
first plurality of biological sample sites thereon. .Iaddend.
.Iadd.53. A system comprising: the support structure of claim 52,
wherein the predetermined pattern defines a fluid passage through
which a fluorescent reagent is flowable to add fluorescent tags to
the biological sample sites; and a detection system configured to
detect the fluorescent tags to distinguish the biological sample
sites on the first surface and to detect the fluorescent tags to
distinguish the biological sample sites on the second surface.
.Iaddend.
Description
BACKGROUND
The present invention relates generally to the field of imaging and
evaluating analytical samples. More particularly, the invention
relates to a technique for imaging and evaluating analytical
samples on multiple surfaces of a support structure using a
compensator.
There are an increasing number of applications for imaging of
analytical samples on a support structure. These support structures
may include plates upon which biological samples are present. For
instance, these plates may include deoxyribonucleic acid (DNA) and
ribonucleic acid (RNA) probes that are specific for nucleotide
sequences present in genes in humans and other organisms.
Individual DNA or RNA probes can be attached at specific locations
in a small geometric grid or array on the support structure.
Depending upon the technology employed, the samples may attach at
random, semi-random or predetermined locations on the support
structure. A test sample, such as from a known person or organism,
can be exposed to the array or grid, such that complementary genes
or fragments hybridize to probes at the individual sites on a
surface of a plate. In certain applications, such as sequencing,
templates or fragments of genetic material may be located at the
sites, and nucleotides or other molecules may be caused to
hybridize to the templates to determine the nature or sequence of
the templates. The sites can then be examined by scanning specific
frequencies of light over the sites to identify which genes or
fragments in the sample were present, by fluorescence of the sites
at which genes or fragments hybridized.
These plates are sometimes referred to as microarrays, gene or
genome chips, DNA chips, gene arrays, and so forth, and may be used
for expression profiling, monitoring expression levels, genotyping,
sequencing, and so forth. For example, diagnostic uses may include
evaluation of a particular patient's genetic makeup to determine
whether a disease state is present or whether pre-disposition for a
particular condition exists. The reading and evaluation of such
plates are an important aspect of their utility. Although
microarrays allow separate biological components to be presented
for bulk processing and individual detection, the number of
components that can be detected in a single experiment is limited
by the resolution of the system. Furthermore, the bulk reagents
used in some methods can be expensive such that reduced volumes are
desired. The present invention provides methods and compositions
that increase the efficiency of array based detection to counteract
these limitations. Other advantages are provided as well and will
be apparent from the description below.
BRIEF DESCRIPTION
The present invention provides a novel approach to analytical
sample imaging and evaluation that expands the use of imaging and
evaluation subsystems to multiple surfaces that support samples.
The support structure may, for instance, be a flow cell through
which a reagent fluid is allowed to flow and interact with
biological samples. Excitation radiation from at least one
radiation source may be used to excite the biological samples on
multiple surfaces. In this manner, fluorescent radiation may be
emitted from the biological samples and subsequently captured and
detected by detection optics and at least one detector. The
returned radiation may then be used to generate image data. This
imaging of multiple surfaces may be accomplished either
sequentially or simultaneously. In addition, the techniques of the
present invention may be used with any of a variety of types of
imaging systems. For instance, both epifluorescent and total
internal reflection (TIR) methods may benefit from the techniques
of the present invention. In addition, the biological samples
imaged may be present on the surfaces of the support structure in
random locations or in patterns.
Accordingly, the invention provides a method for imaging a
biological sample. The method includes detecting radiation emitted
from a first emissive component of a biological sample disposed on
a first surface of a flow cell using a detector. The flow cell is
mounted on an imaging station. The method also includes inserting
corrective optics between the detector and the flow cell. The
method further includes detecting radiation emitted from a second
emissive component of a biological sample disposed on a second
surface of the flow cell using the detector and the corrective
optics. The first and second surfaces are in an arrangement whereby
one of the surfaces is disposed between the detector and the other
surface. In addition, the corrective optics reduce aberration of
detection at one of the surfaces due to the arrangement. The steps
of the method are repeated while maintaining the flow cell on the
imaging station.
The invention further provides an imaging system for detecting
radiation on a multi-surface flow cell. The imaging system includes
a multi-surface flow cell having first and second emissive
components of a biological sample disposed on respective first and
second surfaces of the flow cell. The imaging system also includes
an optical train including an objective, imaging optics configured
to focus the optical train on the first emissive component via the
objective, and corrective optics configured to focus the optical
train on the second emissive component and configured to reduce
aberration of detection at the first or second emissive component.
The imaging system further includes a radiation source configured
to direct excitation radiation towards the first and second
emissive components. In addition, the imaging system includes
detection optics configured to capture emitted radiation returned
from the first and second emissive components via the optical
train. Further, the imaging system includes a detector for
detecting the captured radiation.
DRAWINGS
FIG. 1 is a diagrammatical overview for a biological sample imaging
system in accordance with the present invention;
FIG. 2 is a diagrammatical perspective view of an exemplary
radiation line directed toward a surface of a support structure to
semi-confocally irradiate biological sites, and to semi-confocally
return radiation to a detector in accordance with the present
invention;
FIG. 3 is a sectional view of an exemplary support structure with
excitation radiation directed at two surfaces of the support
structure in accordance with the present invention;
FIG. 4 is a diagrammatical perspective view of an exemplary support
structure having an array of biological component sites in a
spatially ordered pattern in accordance with the present
invention;
FIG. 5 is a diagrammatical perspective view of an exemplary support
structure having biological component sites in a random spatial
distribution in accordance with the present invention;
FIG. 6 is a sectional view of an exemplary support structure with
excitation radiation directed at multiple surfaces of the support
structure in accordance with the present invention;
FIG. 7 illustrates exemplary dimensions between the objective and
the support structure in accordance with the present invention;
FIG. 8 is an exemplary chart of spherical aberration vs. thickness
of the upper plate of the support structure of FIG. 7 in accordance
with the present invention;
FIG. 9A illustrates exemplary images expected for first and second
surfaces of a support structure when obtained through an upper
surface thickness of 300 microns (plus 100 microns of fluid)
without corrective optics, where the imaging system is optimized
for the second surface;
FIG. 9B illustrates exemplary images expected for first and second
surfaces of a support structure when obtained through an upper
surface thickness of 340 microns (plus 100 microns of fluid)
without corrective optics;
FIG. 10A illustrates an exemplary objective imaging the second
surface without the assistance of a compensator in accordance with
the present invention;
FIG. 10B illustrates an exemplary objective imaging the first
surface with the assistance of a compensator in accordance with the
present invention;
FIG. 11 is an exemplary compensator design, incorporating a first
objective and a second objective which may replace the first
objective in the optical train in accordance with the present
invention;
FIG. 12 is another exemplary compensator design, incorporating a
corrective device which may be inserted between the objective and
the support structure in accordance with the present invention;
FIG. 13 is another exemplary compensator design, incorporating a
correction collar in accordance with the present invention;
FIG. 14 is another exemplary compensator design, incorporating an
infinite space compensator in accordance with the present
invention;
FIG. 15 is a perspective view of an exemplary flow cell assembly
using patterned adhesives to form channel characteristics in
accordance with the present invention;
FIG. 16 is a perspective view of another exemplary flow cell
assembly using patterned adhesives to form channel characteristics
in accordance with the present invention;
FIG. 17 is a process flow diagram of an exemplary method of
assembling flow cells using patterned adhesives to form channel
characteristics in accordance with the present invention;
FIG. 18 is a diagrammatical view of a biological sample imaging
system with one radiation source and dual detectors configured to
sequentially scan multiple surfaces of the support structure in
accordance with the present invention;
FIG. 19 is a diagrammatical view of a biological sample imaging
system with dual radiation sources and dual detectors configured to
sequentially scan multiple surfaces of the support structure in
accordance with the present invention;
FIG. 20 is a diagrammatical view of a biological sample imaging
system with dual radiation sources and dual detectors configured to
simultaneously scan multiple surfaces of the support structure
using focusing lenses along the excitation path in accordance with
the present invention;
FIG. 21 is a diagrammatical view of a biological sample imaging
system with dual radiation sources and dual detectors configured to
simultaneously scan multiple surfaces of the support structure
using focusing lenses along the excitation and emission paths in
accordance with the present invention;
FIG. 22 is a diagrammatical view of a biological sample imaging
system with multiple radiation sources and multiple detectors
configured to simultaneously scan multiple surfaces of the support
structure using focusing lenses along the excitation and emission
paths in accordance with the present invention;
FIG. 23 is a diagrammatical overview for a TIR biological sample
imaging system in accordance with the present invention;
FIG. 24 is a sectional view of an exemplary support structure,
prism, and lens objective for use with TIR imaging of a bottom
surface of a flow lane in accordance with the present
invention;
FIG. 25 is a sectional view of an exemplary support structure,
prism, and lens objective for use with TIR imaging of a top surface
of a flow lane in accordance with the present invention;
FIG. 26 is a sectional view of another exemplary support structure,
prism, and lens objective for use with TIR imaging of a top surface
of a flow lane in accordance with the present invention; and
FIG. 27 is a sectional view of an exemplary support structure being
heated on both top and bottom surfaces in accordance with the
present invention.
DETAILED DESCRIPTION
Turning now to the drawings, and referring first to FIG. 1, a
biological sample imaging system 10 is illustrated
diagrammatically. The biological sample imaging system 10 is
capable of imaging multiple biological components 12, 14 within a
support structure 16. For instance, in the illustrated embodiment,
a first biological component 12 may be present on a first surface
18 of the support structure 16 while a second biological component
14 may be present on a second surface 20 of the support structure.
The support structure 16 may, for instance, be a flow cell with an
array of biological components 12, 14 on the interior surfaces 18,
20 which generally mutually face each other and through which
reagents, flushes, and other fluids may be introduced, such as for
binding nucleotides or other molecules to the sites of biological
components 12, 14. The support structure 16 may be manufactured in
conjunction with the present techniques or the support structure 16
may be purchased or otherwise obtained from a separate entity.
Fluorescent tags on the molecules that bind to the components may,
for instance, include dyes that fluoresce when excited by
appropriate excitation radiation. Assay methods that include the
use of fluorescent tags and that can be used in an apparatus or
method set forth herein include those set forth elsewhere herein
such as genotyping assays, gene expression analysis, methylation
analysis, or nucleic acid sequencing analysis.
Those skilled in the art will recognize that a flow cell or other
support structure may be used with any of a variety of arrays known
in the art to achieve similar results. Furthermore, known methods
for making arrays can be used, and if appropriate, modified in
accordance with the teaching set forth herein in order to create a
flow cell or other support structure having multiple surfaces
useful in the detection methods set forth herein. Such arrays may
be formed by disposing the biological components of samples
randomly or in predefined patterns on the surfaces of the support
by any known technique. In a particular embodiment, clustered
arrays of nucleic acid colonies can be prepared as described in
U.S. Pat. No. 7,115,400; U.S. Patent Application Publication No.
2005/0100900; PCT Publication No. WO 00/18957; or PCT Publication
No. WO 98/44151, each of which is hereby incorporated by reference.
Such methods are known as bridge amplification or solid-phase
amplification and are particularly useful for sequencing
applications.
Other exemplary random arrays, and methods for their construction,
that can be used in the invention include, without limitation,
those in which beads are associated with a solid support, examples
of which are described in U.S. Pat. Nos. 6,355,431; 6,327,410; and
U.S. Pat. No. 6,770,441; U.S. Patent Application Publication Nos.
2004/0185483 and US 2002/0102578; and PCT Publication No. WO
00/63437, each of which is hereby incorporated by reference. Beads
can be located at discrete locations, such as wells, on a
solid-phase support, whereby each location accommodates a single
bead.
Any of a variety of other arrays known in the art or methods for
fabricating such arrays can be used in the present invention.
Commercially available microarrays that can be used include, for
example, an Affymetrix.RTM. GeneChip.RTM. microarray or other
microarray synthesized in accordance with techniques sometimes
referred to as VLSIPS.TM. (Very Large Scale Immobilized Polymer
Synthesis) technologies as described, for example, in U.S. Pat.
Nos. 5,324,633; 5,744,305; 5,451,683; 5,482,867; 5,491,074;
5,624,711; 5,795,716; 5,831,070; 5,856,101; 5,858,659; 5,874,219;
5,968,740; 5,974,164; 5,981,185; 5,981,956; 6,025,601; 6,033,860;
6,090,555; 6,136,269; 6,022,963; 6,083,697; 6,291,183; 6,309,831;
6,416,949; 6,428,752; and 6,482,591, each of which is hereby
incorporated by reference. A spotted microarray can also be used in
a method of the invention. An exemplary spotted microarray is a
CodeLink.TM. Array available from Amersham Biosciences. Another
microarray that is useful in the invention is one that is
manufactured using inkjet printing methods such as SurePrint.TM.
Technology available from Agilent Technologies.
Sites or features of an array are typically discrete, being
separated with spaces between each other. The size of the sites
and/or spacing between the sites can vary such that arrays can be
high density, medium density, or lower density. High density arrays
are characterized as having sites separated by less than about 15
.mu.m. Medium density arrays have sites separated by about 15 to 30
.mu.m, while low density arrays have sites separated by greater
than 30 .mu.m. An array useful in the invention can have sites that
are separated by less than 100 .mu.m, 50 .mu.m, 10 .mu.m, 5 .mu.m,
1 .mu.m or 0.5 .mu.m. An apparatus or method of the invention can
be used to image an array at a resolution sufficient to distinguish
sites at the above densities or density ranges.
As exemplified herein, a surface used in an apparatus or method of
the invention is typically a manufactured surface. It is also
possible to use a natural surface or a surface of a natural support
structure; however the invention can be carried out in embodiments
where the surface is not a natural material or a surface of a
natural support structure. Accordingly, components of biological
samples can be removed from their native environment and attached
to a manufactured surface.
Any of a variety of biological components can be present on a
surface for use in the invention. Exemplary components include,
without limitation, nucleic acids such as DNA or RNA, proteins such
as enzymes or receptors, polypeptides, nucleotides, amino acids,
saccharides, cofactors, metabolites or derivatives of these natural
components. Although the apparatus and methods of the invention are
exemplified herein with respect to components of biological
samples, it will be understood that other samples or components can
be used as well. For example, synthetic samples can be used such as
combinatorial libraries, or libraries of compounds having species
known or suspected of having a desired structure or function. Thus,
the apparatus or methods can be used to synthesize a collection of
compounds and/or screen a collection of compounds for a desired
structure or function.
Returning to the exemplary system of FIG. 1, the biological sample
imaging system 10 may include at least a first radiation source 22
but may also include a second radiation source 24 (or additional
sources). The radiation sources 22, 24 may be lasers operating at
different wavelengths. The selection of the wavelengths for the
lasers will typically depend upon the fluorescence properties of
the dyes used to image the component sites. Multiple different
wavelengths of the lasers used may permit differentiation of the
dyes at the various sites within the support structure 16, and
imaging may proceed by successive acquisition of a series of images
to enable identification of the molecules at the component sites in
accordance with image processing and reading logic generally known
in the art. Other radiation sources known in the art can be used
including, for example, an arc lamp or quartz halogen lamp.
Particularly useful radiation sources are those that produce
electromagnetic radiation in the ultraviolet (UV) range (about 200
to 390 nm), visible (VIS) range (about 390 to 770 nm), infrared
(IR) range (about 0.77 to 25 microns), or other range of the
electromagnetic spectrum.
For ease of description, embodiments utilizing fluorescence-based
detection are used as examples. However, it will be understood that
other detection methods can be used in connection with the
apparatus and methods set forth herein. For example, a variety of
different emission types can be detected such as fluorescence,
luminescence, or chemiluminescence. Accordingly, components to be
detected can be labeled with compounds or moieties that are
fluorescent, luminescent, or chemiluminescent. Signals other than
optical signals can also be detected from multiple surfaces using
apparatus and methods that are analogous to those exemplified
herein with the exception of being modified to accommodate the
particular physical properties of the signal to be detected.
Output from the radiation sources 22, 24 may be directed through
conditioning optics 26, 28 for filtering and shaping of the beams.
For example, in a presently contemplated embodiment, the
conditioning optics 26, 28 may generate a generally linear beam of
radiation, and combine beams from multiple lasers, for example, as
described in U.S. Pat. No. 7,329,860. The laser modules can
additionally include a measuring component that records the power
of each laser. The measurement of power may be used as a feedback
mechanism to control the length of time an image is recorded in
order to obtain uniform exposure, and therefore more readily
comparable signals.
After passing through the conditioning optics 26, 28, the beams may
be directed toward directing optics 30 which redirect the beams
from the radiation sources 22, 24 toward focusing optics 32. The
directing optics 30 may include a dichroic mirror configured to
redirect the beams toward the focusing optics 32 while also
allowing certain wavelengths of a retrobeam to pass therethrough.
The focusing optics 32 may confocally direct radiation to one or
more surfaces 18, 20 of the support structure 16 upon which
individual biological components 12, 14 are located. For instance,
the focusing optics 32 may include a microscope objective that
confocally directs and concentrates the radiation sources 22, 24
along a line to a surface 18, 20 of the support structure 16.
Biological component sites on the support structure 16 may
fluoresce at particular wavelengths in response to an excitation
beam and thereby return radiation for imaging. For instance, the
fluorescent components may be generated by fluorescently tagged
nucleic acids that hybridize to complementary molecules of the
components or to fluorescently tagged nucleotides that are
incorporated into an oligonucleotide using a polymerase. As noted
above, the fluorescent properties of these components may be
changed through the introduction of reagents into the support
structure 16 (e.g., by cleaving the dye from the molecule, blocking
attachment of additional molecules, adding a quenching reagent,
adding an acceptor of energy transfer, and so forth). As will be
appreciated by those skilled in the art, the wavelength at which
the dyes of the sample are excited and the wavelength at which they
fluoresce will depend upon the absorption and emission spectra of
the specific dyes. Such returned radiation may propagate back
through the directing optics 30. This retrobeam may generally be
directed toward detection optics 34 which may filter the beam such
as to separate different wavelengths within the retrobeam, and
direct the retrobeam toward at least one detector 36.
The detector 36 may be based upon any suitable technology, and may
be, for example, a charged coupled device (CCD) sensor that
generates pixilated image data based upon photons impacting
locations in the device. However, it will be understood that any of
a variety of other detectors may also be used including, but not
limited to, a detector array configured for time delay integration
(TDI) operation, a complementary metal oxide semiconductor (CMOS)
detector, an avalanche photodiode (APD) detector, a Geiger-mode
photon counter, or any other suitable detector. TDI mode detection
can be coupled with line scanning as described in U.S. Pat. No.
7,329,860.
The detector 36 may generate image data, for example, at a
resolution between 0.1 and 50 microns, which is then forwarded to a
control/processing system 38. In general, the control/processing
system 38 may perform various operations, such as analog-to-digital
conversion, scaling, filtering, and association of the data in
multiple frames to appropriately and accurately image multiple
sites at specific locations on a sample. The control/processing
system 38 may store the image data and may ultimately forward the
image data to a post-processing system (not shown) where the data
are analyzed. Depending upon the types of sample, the reagents
used, and the processing performed, a number of different uses may
be made of the image data. For example, nucleotide sequence data
can be derived from the image data, or the data may be employed to
determine the presence of a particular gene, characterize one or
more molecules at the component sites, and so forth. The operation
of the various components illustrated in FIG. 1 may also be
coordinated with the control/processing system 38. In a practical
application, the control/processing system 38 may include hardware,
firmware, and software designed to control operation of the
radiation sources 22, 24, movement and focusing of the focusing
optics 32, a translation system 40, and the detection optics 34,
and acquisition and processing of signals from the detector 36. The
control/processing system 38 may thus store processed data and
further process the data for generating a reconstructed image of
irradiated sites that fluoresce within the support structure 16.
The image data may be analyzed by the system itself, or may be
stored for analysis by other systems and at different times
subsequent to imaging.
The support structure 16 may be supported on a translation system
40 which allows for focusing and movement of the support structure
16 before and during imaging. The stage may be configured to move
the support structure 16, thereby changing the relative positions
of the radiation sources 22, 24 and detector 36 with respect to the
surface bound biological components for progressive scanning
Movement of the translation system 40 can be in one or more
dimensions including, for example, one or both of the dimensions
that are orthogonal to the direction of propagation for the
excitation radiation line, typically denoted as the X and Y
dimensions. In particular embodiments, the translation system 40
may be configured to move in a direction perpendicular to the scan
axis for a detector array. A translation system 40 useful in the
present invention may be further configured for movement in the
dimension along which the excitation radiation line propagates,
typically denoted as the Z dimension. Movement in the Z dimension
can also be useful for focusing.
FIG. 2 is a diagrammatical representation of an exemplary
semi-confocal line scanning approach to imaging the support
structure 16. In the illustrated embodiment, the support structure
16 includes an upper plate 42 and a lower plate 44 with an internal
volume 46 between the upper and lower plates 42, 44. The upper and
lower plates 42, 44 may be made of any of a variety of materials
but may preferably be made of a substrate material that is
substantially transparent at the wavelengths of the excitation
radiation and the fluoresced retrobeam, allowing for the passage of
excitation radiation and returned fluorescent emissions without
significant loss of signal quality. Moreover, when used in
epifluorescent imaging arrangements as shown, one of the surfaces
through which the radiation traverses may be substantially
transparent at the relevant wavelengths, while the other (which is
not traversed by radiation) may be less transparent, translucent,
or even opaque or reflective. The upper and lower plates 42, 44 may
both contain biological components 12, 14 on their respective,
inwardly facing surfaces 18, 20. As discussed above, the internal
volume 46 may, for instance, include one or more internal passages
of a flow cell though which reagent fluids may flow.
The support structure 16 may be irradiated by excitation radiation
48 along a radiation line 50. The radiation line 50 may be formed
by the excitation radiation 48 from the radiation sources 22, 24,
directed by the directing optics 30 through the focusing optics 32.
The radiation sources 22, 24 may generate beams that are processed
and shaped to provide a linear cross section or radiation line
including a plurality of wavelengths of radiation used to cause
fluorescence at correspondingly different wavelengths from the
biological components 12, 14, depending upon the particular dyes
used. The focusing optics 32 may then semi-confocally direct the
excitation radiation 48 toward the first surface 18 of the support
structure 16 to irradiate sites of biological component 12 along
the radiation line 50. In addition, the support structure 16, the
directing optics 30, the focusing optics 32, or some combination
thereof, may be slowly translated such that the resulting radiation
line 50 progressively irradiates the component as indicated by the
arrow 52. This translation results in successive scanning of
regions 54 which allow for the gradual irradiation of the entire
first surface 18 of the support structure 16. As will be discussed
in more detail below, the same process may also be used to
gradually irradiate the second surface 20 of the support structure
16. Indeed, the process may be used for multiple surfaces within
the support structure 16.
Exemplary methods and apparatus for line scanning are described in
U.S. Pat. No. 7,329,860, which is incorporated herein by reference,
and which describes a line scanning apparatus having a detector
array configured to achieve confocality in the scanning axis by
restricting the scan-axis dimension of the detector array. More
specifically, the scanning device can be configured such that the
detector array has rectangular dimensions such that the shorter
dimension of the detector is in the scan-axis dimension and imaging
optics are placed to direct a rectangular image of a sample region
to the detector array such that the shorter dimension of the image
is also in the scan-axis dimension. In this way, semi-confocality
can be achieved since confocality occurs in a single axis (i.e. the
scan axis). Thus, detection is specific for features on the surface
of a substrate, thereby rejecting signals that may arise from the
solution around the feature. The apparatus and methods described in
U.S. Pat. No.7,329,860 can be modified such that two or more
surfaces of a support are scanned in accordance with the
description herein.
Detection apparatus and methods other than line scanning can also
be used. For example, point scanning can be used as described below
or in U.S. Pat. No. 5,646,411, which is incorporated herein by
reference. Wide angle area detection can be used with or without
scanning motion. As set forth in further detail elsewhere herein,
TIR methods can also be used.
As illustrated generally in FIG. 2, the radiation line 50 used to
image the sites of biological components 12, 14, in accordance with
the present invention, may be a continuous or discontinuous line.
As such, some embodiments of the present invention may include a
discontinuous line made up of a plurality of confocally or
semi-confocally directed beams of radiation which nevertheless
irradiate a plurality of points along the radiation line 50. These
discontinuous beams may be created by one or more sources that are
positioned or scanned to provide the excitation radiation 48. These
beams, as before, may be confocally or semi-confocally directed
toward the first or second surfaces 18, 20 of the support structure
16 to irradiate sites of biological component 12, 14. As with the
continuous semi-confocal line scanning described above, the support
structure 16, the directing optics 30, the focusing optics 32, or
some combination thereof, may be advanced slowly as indicated by
arrow 52 to irradiate successive scanned regions 54 along the first
or second surfaces 18, 20 of the support structure 16, and thereby
successive regions of the sites of biological components 12,
14.
It should be noted that the system will typically form and direct
excitation and returned radiation simultaneously for imaging. In
some embodiments, confocal point scanning may be used such that the
optical system directs an excitation point or spot across a
biological component by scanning the excitation beam through an
objective lens. The detection system images the emission from the
excited point on the detector without "descanning" the retrobeam.
This occurs since the retrobeam is collected by the objective lens
and is split off the excitation beam optical path before returning
back through the scan means. Therefore, the retrobeam will appear
on the detector 36 at different points depending on the field angle
of the original excitation spot in the objective lens. The image of
the excitation point, at the detector 36, will appear in the shape
of a line as the excitation point is scanned across the sample.
This architecture is useful, for example, if the scan means cannot
for some reason accept the retrobeam from the sample. Examples are
holographic and acoustic optic scan means that are able to scan a
beam at very high speeds but utilize diffraction to create the
scan. Therefore, the scan properties are a function of wavelength.
The retrobeam of emitted radiation is at a different wavelength
from the excitation beam. Alternatively or additionally, emission
signals may be collected sequentially following sequential
excitation at different wavelengths.
In particular embodiments, an apparatus or method of the invention
can detect features on a surface at a rate of at least about 0.01
mm.sup.2/sec. Depending upon the particular application of the
invention, faster rates can also be used including, for example, in
terms of the area scanned or otherwise detected, a rate of at least
about 0.02 mm.sup.2/sec, 0.05 mm.sup.2/sec, 0.1 mm.sup.2/sec, 1
mm.sup.2/sec, 1.5 mm.sup.2/sec, 5 mm.sup.2/sec, 10 mm.sup.2/sec, 50
mm.sup.2/sec, 100 mm.sup.2/sec, or faster. If desired, for example,
to reduce noise, the detection rate can have an upper limit of
about 0.05 mm.sup.2/sec, 0.1 mm.sup.2/sec, 1 mm.sup.2/sec, 1.5
mm.sup.2/sec, 5 mm.sup.2/sec, 10 mm.sup.2/sec, 50 mm.sup.2/sec, or
100 mm.sup.2/sec.
In some instances, the support structure 16 may be used in such a
way that biological components are expected to be present on only
one surface. However, in many instances, biological material is
present on multiple surfaces within the support structure 16. For
instance, FIG. 3 illustrates a typical support structure 16 where
biological material has attached to the first surface 18 as well as
to the second surface 20. In the illustrated embodiment, an
attachment layer 56 has formed on both the first surface 18 and the
second surface 20 of the support structure 16. A first excitation
radiation 58 source may be used to irradiate one of many sites of
biological component 12 on the first surface 18 of the support
structure 16 and return a first fluorescent emission 60 from the
irradiated biological component 12. Simultaneously or sequentially,
a second source of excitation radiation 62 may be used to irradiate
one of many sites of biological component 14 on the second surface
20 of the support structure 16, and return a second fluorescent
emission 64 from the irradiated biological component 14.
Although the embodiment exemplified in FIG. 3 shows excitation from
source 58 and source 62 coming from the same side of the support
structure 16, it will be understood that the optical system can be
configured to impinge on the surfaces from opposite sides of the
support structure 16. Taking FIG. 3 as an example, upper surface 18
can be irradiated from excitation source 58 as shown and the lower
surface 20 can be irradiated from below. Similarly, emission can be
detected from one or more sides of a support structure. In
particular embodiments, different sides of the support structure 16
can be excited from the same radiation source by first irradiating
one side and then flipping the support structure to bring another
side into position for excitation by the radiation source.
The distribution of biological components 12, 14 may follow many
different patterns. For instance, FIG. 4 illustrates a support
structure 16 where the biological components 12, 14 at sites or
features on the first and second surfaces 18, 20 are distributed
evenly in a spatially ordered pattern 66 of biological component
sites 68. For example, certain types of microarrays may be used
where the location of individual biological component sites 68 may
be in a regular spatial pattern. The pattern can include sites at
pre-defined locations. In contrast, in other types of biological
imaging arrays, biological components attach to surfaces at sites
that occur in random or statistically varying positions such that
imaging the microarray is used to determine the location of each of
the individual biological component features. Thus, the pattern of
features need not be pre-determined despite being the product of a
synthetic or manufacturing process.
For instance, FIG. 5 illustrates a support structure 16 where the
sites on the first and second surfaces 18, 20 are located in a
random spatial distribution 70 of biological component sites 72.
However, with both fixed arrays 66 and random distribution 70 of
biological sample sites, imaging of multiple surfaces 18, 20 of the
support structure 16 may be possible. In addition, it should be
noted that in both instances, the biological components at the
individual sites may constitute either a population of identical
molecules or a random mix of different molecules. Furthermore, the
density of biological samples may vary and may be at least 1,000
sites per square millimeter.
The present techniques accommodate such varied physical
arrangements of the multiple surfaces within the support structure
16, as well as the varied disposition of the sites within
components on the surfaces. As discussed above with reference to
FIGS. 2 and 3, in the embodiments with a support structure 16
having a first surface 18 and a second surface 20, a first source
of excitation radiation 58 may irradiate sites of biological
component 12 on the first surface 18, and return a first
fluorescent emission 60, while a second source of excitation
radiation 62 may irradiate sites of biological component 14 on the
second surface 20 and return a second fluorescent emission 64
source, as illustrated in FIG. 3. Thus, components of the volume of
sample between two surfaces need not be detected and can be
rejected. Selective detection of a surface of a support structure
provides preferential detection of the surface compared to the
volume of the support structure adjacent the surface and compared
to one or more other surfaces of the support structure.
In more complex configurations, it may be useful to irradiate more
than two surfaces. For instance, FIG. 6 illustrates a support
structure 16 having N number of plates including a first plate 42,
a second plate 44, . . . , an N-2 plate 74, an N-1 plate 76, and an
N plate 78. These plates define M number of surfaces including a
first surface 18, a second surface 20, . . . , an M-3 surface 80,
an M-2 surface 82, an M-1 surface 84, and an M surface 86. In the
illustrated embodiment, not only the first surface 18 and the
second surface 20 of the support structure 16 may be irradiated
but, rather, all M number of surfaces may be irradiated. For
instance, a source of excitation radiation 88 may be used to
irradiate biological component sites on the Mth surface 86 of the
support structure 16 and return a fluorescent emission 90 from the
irradiated biological component. For support structures having a
plurality of surfaces it may be desirable to excite upper layers
from the top and lower layers from the bottom to reduce
photobleaching. Thus, components on layers that are closer to a
first exterior side of a support structure can be irradiated from
the first side, whereas irradiation from the opposite exterior side
can be used to excite components present on layers that are closer
to the opposite exterior side.
FIG. 7 illustrates an objective 92 through which radiation from
emissive biological components 12, 14 on first and second surfaces
18, 20, respectively, of the support structure 16 may be detected.
The objective 92 may be one of the components of the focusing
optics 32 described above. Although not drawn to scale, FIG. 7
illustrates exemplary dimensions between the objective 92 and the
support structure 16. For instance, the objective 92 may typically
be spaced approximately 600 or more microns from the upper plate 42
of the support structure 16. The biological sample imaging system
10 may be configured to detect emitted radiation from biological
components 12 on the first surface 18 through 300 microns of the
upper plate 42 which may, for instance, be made of glass and may
have a refractive index N.sub.d of 1.472. In addition, the
biological sample imaging system 10 may also be configured to
detect emitted radiation from biological components 14 on the
second surface 20 through 300 microns of the upper plate 42 plus
100 microns of the fluid within the internal volume 46 of the
support structure 46.
In certain embodiments, the objective 92 may be designed for
diffraction-limited focusing and imaging on only one of the first
or second surfaces 18, 20 of the support structure 16. For example
throughout the present description of FIGS. 7 through 14, the
objective 92 may be designed for pre-compensation of the 300
microns of the upper plate 42 plus the 100 micron read buffer of
the fluid within the internal volume 46 of the support structure
16. In such a scenario, diffraction-limited performance may only be
possible on the second surface 20. Furthermore, the spherical
aberration introduced by the 100 micron read buffer may severely
impact the imaging quality when imaging from the first surface 18.
However, reducing the lane thickness of the internal volume 46 of
the support structure 16 might increase the amount of
surface-to-surface "crosstalk." Therefore, perhaps the most
appropriate solution is to correct the aberration. As such, it may
be necessary to use a compensator capable of achieving
diffraction-limited imaging performance on both the first and
second surfaces 18, 20 of the support structure 16.
It should be noted that the need for a compensator may be more
pronounced when using objectives 92 with high numerical aperture
(NA) values. Exemplary high NA ranges for which the invention is
particularly useful include NA values of at least about 0.6. For
example, the NA may be at least about 0.65, 0.7, 0.75, 0.8, 0.85,
0.9, 0.95, or higher. Those skilled in the art will appreciate that
NA, being dependent upon the index of refraction of the medium in
which the lens is working, may be higher including, for example, up
to 1.0 for air, 1.33 for pure water, or higher for other media such
as oils. The compensator may also find use in objectives having
lower NA values than the examples listed above. In general, the NA
value of an objective 92 is a measure of the breadth of angles for
which the objective 92 may receive light. The higher the NA value,
the more light that may be collected by the objective 92 for a
given fixed magnification. This is because the collection
efficiency and the resolution increase. As a result, multiple
objects may be distinguished more readily when using objectives 92
with higher NA values because a higher feature density may be
possible. Therefore, in general, a higher NA value for the
objective 92 may be beneficial for imaging. However, as the NA
value increases, its sensitivity to focusing and imaging-through
media thickness variation also increases. In other words, lower NA
objectives 92 have longer depth of field and are generally not as
sensitive to changes in imaging-through media thickness.
FIG. 8 is an exemplary chart 94 of spherical aberration (in waves)
vs. thickness (in microns) of the upper plate 42 of the support
structure 16 of FIG. 7 in accordance with the present invention.
Specifically, the upper line 96 of the graph depicts the amount of
spherical aberration of an image taken from biological components
12 on the first surface 18 of the support structure 16 while the
lower line 98 of the graph depicts the amount of spherical
aberration of an image taken from biological components 14 on the
second surface 20 of the support structure 16. In the illustrated
embodiment, the spherical aberration generated by the 100 micron
read buffer is around 4 waves, which is much higher than the
diffraction-limited performance requirement of less than 0.25
waves, for instance. As illustrated, at 300 microns (i.e. the
thickness of the upper plate 42), the spherical aberration for the
first surface 18 is around -13.2 waves (e.g., point 100) while the
spherical aberration for the second surface 20 is around -17.2
waves (e.g., point 102). FIG. 9A illustrates exemplary images
expected for the first and second surfaces 18, 20 of the support
structure 16 corresponding to the thickness of the upper plate 42
(i.e., 300 microns) in accordance with the present invention, where
the imaging system is optimized for the second surface 20
(pre-compensated for -17.2 waves). As shown, the imaging system is
capable of providing high image quality on the second surface 20
since, according to the present scenario, it was designed to do so.
However, the images taken for the first surface 18 contain
aberrations.
To balance out the spherical aberration, it is beneficial to
introduce an additional thickness (e.g., by introducing an
additional coverslip) between the objective 92 and the support
structure 16. For instance, returning now to FIG. 8, if an
additional thickness of approximately 40 microns were to be
introduced between the objective 92 and the first and second
surfaces 18, 20 of support structure 16, the difference between the
spherical aberrations at the design thickness (i.e., 300 micron
upper plate plus 100 microns of fluid) may be split such that the
image produced for both the first and second surfaces 18, 20 may
have similar quality. For instance, as illustrated, at 340 microns
(i.e. the thickness of the upper plate 42 plus an additional 40
micron thickness), the spherical aberration for the first surface
18 is around -15.2 waves (e.g., point 104) while the spherical
aberration for the second surface 20 is around -19.2 waves (e.g.,
point 106), splitting the difference of -17.2 waves (e.g., point
108) which may be characterized as the design point for the
objective 92. FIG. 9B illustrates exemplary images expected for the
first and second surfaces 18, 20 of the support structure 16
corresponding to the thickness of the upper plate 42 plus an
additional thickness (i.e., 300 microns plus 40 microns) in
accordance with the present invention, illustrating how the
additional thickness may allow for balance between images taken for
the first and second surfaces 18, 20 of the support structure
16.
However, merely introducing an additional thickness between the
objective 92 and the support structure 16 may not be desired for
all uses of the imaging system set forth herein. For instance, as
illustrated in FIGS. 9A and 9B, by simply introducing the
additional 40 micron thickness between the objective 92 and the
support structure 16, images from both the first and second
surfaces 18, 20 may still experience residual aberration from the
design point 108 of the objective 92. Therefore, a more precise
solution may be to only introduce the additional thickness when
detecting radiation from biological components 12 on the first
surface 18 of the support structure 16. In such a scenario, the
spherical aberration corresponding to the design point 108 of the
objective 92 may generally be achieved for both the first and
second surfaces 18, 20. It should be noted that the particular
dimensions and measurements (e.g., thicknesses, spherical
aberration values, and so forth) described with respect to FIGS. 9A
and 9B are merely intended to be exemplary of the manner in which
the present invention functions. As such, these dimensions and
measurements are not intended to be limiting. Indeed, the
particular geometries and resulting measurement values may vary
between implementations.
For example, FIG. 10A illustrates an exemplary objective 92 imaging
the second surface 20 of the support structure 16 without the
assistance of a compensator 110 in accordance with the present
invention. Without the compensator 110, the objective 92 may focus
and detect images from the second surface 20 of the support
structure 16 according to its design and experiencing the design
spherical aberration. However, FIG. 10B illustrates an exemplary
objective 92 imaging the first surface 20 of the support structure
16 with the assistance of a compensator 110 in accordance with the
present invention. By using the compensator 110 (e.g., similar to
the additional 40 micron thickness described above with respect to
FIGS. 8 and 9), the objective 92 may focus and detect images from
the first surface 18 of the support structure 16 under similar
conditions to that of its design point for the second surface 20 of
the support structure 16. Therefore, by detecting images for the
second surface 20 without the compensator 110 and detecting images
for the first surface 18 with the compensator 110, the objective 92
may be capable of detecting images on both surfaces with
diffraction-limited performance similar to the design of the
objective 92.
The chromatic shift curve may be limited to wavelength ranges of
between 530 nm to 780 nm. Chromatic shifts of different color
wavelength bands may be compensated for by focusing the focusing
optics 32 in each band. The compensator 110 should preferably be
"invisible" to the focusing optics 32. In other words, the
compensator 110 should correct the spherical aberration difference
of the read buffer but should maintain the chromatic shift curve in
the wavelength range of 530-780 nm. More specifically, the
chromatic shift relationships among the peak wavelengths of 560 nm,
610 nm, 687 nm, and 720 nm should be maintained. In addition, other
specifications, including NA, field curvature, field distortion,
detection magnification, and so forth, should also be maintained.
Furthermore, the compensator 110 package should be relatively small
(e.g., no more than 10 mm of total thickness). Moreover,
insensitivity to positioning error of the compensator 110 may be
preferred.
Several various designs may be implemented to introduce the
corrective optics of the compensator 110 into the optical train of
the imaging optics of the biological sample imaging system 10. For
example, FIG. 11 is an exemplary compensator 110 design,
incorporating a first objective 92 and a second objective 112 which
may replace the first objective 92 in the optical train in
accordance with the present invention. In the illustrated
embodiment, each respective objective 92, 112 may contain the
optics required to image respective surfaces, such as the first and
second surfaces 18, 20 of the support structure 16. For instance,
the first objective 92 may contain the imaging optics necessary to
focus on and image emissive biological components 14 on the second
surface 20 of the support structure 16 while the second objective
112 may contain the imaging optics plus the corrective optics
necessary to focus on and image emissive biological components 12
on the first surface 18 of the support structure 16. In operation,
the first objective 92 may detect images from the second surface 20
of the support structure 16. The first objective 92 may be replaced
by the second objective 112 in the optical train, at which point
the second objective 112 may detect images from the first surface
18 of the support structure 16. An advantage of the embodiment
illustrated in FIG. 11 is that the optics may be decoupled and may
operate independently. However, a disadvantage in some situations
is that having two entirely separate objectives 92, 112 may not be
cost-effective since certain components may be duplicated for each
objective 92, 112. Furthermore, in embodiments where multiple
images of an object are obtained, the use of two objectives may
increase the computational resources required for registration
between images. In particular embodiments, imaging of both surfaces
may occur through the same objective to provide particular
advantages as set forth below. In other words, the first objective
92 need not be removed or replaced with the second objective 112
for imaging of the different surfaces.
FIG. 12 is another exemplary compensator 110 design, incorporating
a corrective device 114 which may be inserted between the objective
92 and the support structure 16 in accordance with the present
invention. The corrective device 114 may, for instance, be a
coverslip or other thin layer of glass. As illustrated, the
corrective device 114 may simply be inserted into and removed from
the optical path between the objective 92 and the support structure
16 depending on the particular surface 16 being imaged. For
instance, the corrective device 114 may be removed from the optical
path when the objective 92 is used to focus on and image emissive
biological components 14 on the second surface 20 of the support
structure 16. Conversely, the corrective device 114 may be inserted
into the optical path when the objective 92 is used to focus on and
image emissive biological components 12 on the first surface 18 of
the support structure 16. An advantage of the embodiment
illustrated in FIG. 12 is that it is relatively straightforward.
The required additional compensator thickness may simply be
inserted into the optical path. Typically, the corrective device
114 may be placed such that it does not physically contact the
support structure 16 or the objective 92.
FIG. 13 is another exemplary compensator 110 design, incorporating
a correction collar 116 in accordance with the present invention.
In the illustrated embodiment, the correction collar 116 may be
adjusted between binary states. For instance, the first state 118
may correspond to the situation where the objective 92 is focused
on and detecting images from the second surface 20 of the support
structure 16 while the second state 120 may correspond to the
situation where the objective 92 is focused on and detecting images
from the first surface of the support structure 16. As such, when
the correction collar 116 is in the first state 118, the imaging
optics within the objective 92 may not include the corrective
optics within the optical path. Conversely, when the correction
collar 116 is in the second state 120, the imaging optics within
the objective 92 may include the corrective optics within the
optical path. Although illustrated as consisting of binary states
118, 120, the correction collar 116 may, in fact, include multiple
states. For instance, when more than two surfaces of the support
structure 16 are used for imaging, the correction collar 116 may be
configured to adjust between multiple states such that the imaging
and corrective optics vary for each respective surface of the
support structure 16. An advantage of the embodiment illustrated in
FIG. 13 is that it may be relatively easy to operate. For instance,
the correction collar 116 may simply be adjusted between states
whenever different surfaces of the support structure 16 are being
imaged.
FIG. 14 is another exemplary compensator 110 design, incorporating
an infinite space compensator 122 in accordance with the present
invention. This embodiment is somewhat similar to the corrective
device 114 embodiment of FIG. 12 in that the infinite space
compensator 122 may be inserted into and removed from the optical
path. However, a main difference between the embodiments is that,
in the embodiment of FIG. 14, there may be more space available
(e.g., up to 10 mm, as opposed to 600 microns in the embodiment of
FIG. 12) within which to insert the infinite space compensator 122
into the optical path. Therefore, the embodiment of FIG. 14 may
allow for greater flexibility than the corrective device 114
embodiment of FIG. 12.
In addition to the embodiments presented in FIGS. 11 through 14,
there may be other compensator 110 designs which may prove
beneficial. For instance, a fluidic corrector may be inserted
between the objective 92 and the support structure 16. In this
fluidic corrector design, the fluidic corrector may be filled with
a fluid, which may effectively act as the compensator 110. The
optics may be configured such that the fluid matches the upper
surface of the support structure 16 and, in the absence of fluid
air, matches the bottom surface of support structure 16. This
design may prove beneficial in that it may make automation easier
since the fluid would simply be inserted into and extracted from
the fluidic corrector depending on which surface is imaged.
Regardless of the particular embodiment selected, all of the
embodiments disclosed herein are characterized by repeatability and
the ability to automate the use of the embodiments. These are
important considerations in that the embodiments allow for the
detection of images from biological components 12, 14 on multiple
surfaces 18, 20 of the support structure 16 in an automated
fashion. This may allow not only for increased imaging production
but may also allow for greater flexibility in switching between the
multiple surfaces, depending on the particular imaging needs.
As described in greater detail above, a support structure 16 useful
in the apparatus or methods set forth herein can have two or more
surfaces upon which a biological component is attached. In
particular embodiments, the surface is a fabricated surface. Any of
a variety of surfaces known in the art can be used including, but
not limited to, those used for making arrays as set forth above.
Examples include, glass, silicon, polymeric structures, plastics,
and the like. Surfaces and flow cells that are particularly useful
are described in PCT Publication No. WO 2007/123744, which is
incorporated herein by reference. The surfaces of a support
structure can have the same or different properties. For example,
in the embodiment shown in FIG. 3, plate 42 can be transparent to
the excitation and emission wavelengths used in a detection method,
whereas plate 44 can optionally be transparent or opaque to the
excitation or emission wavelengths. Accordingly, the surfaces can
be made of the same material or the two or more surfaces can be
made of different materials.
A support structure having two or more surfaces can be formed by
adhering the surfaces to each other or to other supports. For
example, an adhesive material, such as epoxy resin, can be
dispensed in the form of a paste onto a planar substrate in a
pattern forming one or more channel characteristics of a flow cell.
An exemplary flow cell 124 is shown in FIG. 15. Utilizing a
programmable, automated adhesive dispenser, such as the
Millennium.RTM. M-2010 from Asymtek Corp., Carlsbad Calif., a
desired pattern of adhesive 126 can be designed and laid down onto
the surface of a planar lower substrate 128. The thickness of the
flow cell (and cross sectional height in the fluidic channels) can
be set by means of precision mechanical spacers 130 placed between
the lower substrate 128 and an upper substrate 132. Another
exemplary flow cell 134 is shown in FIG. 16. To create a
multi-layer cell, an interim transparent substrate layer 136,
shorter in length than the lower and upper substrate layers 128,
132 can be included. The shorter length allows fluidic access to
both/all layers from ports 138 passing through only one substrate.
This intermediate layer 136 bifurcates the flow cell cavity
horizontally and nearly doubles the available surface area for the
attachment of biologically interesting molecules.
An exemplary method 140 for fabricating such a flow cell is shown
in FIG. 17. A planar substrate acting as the structural base of the
cell is provided (block 142). Desired canalizing features of the
cell are designed, for example, using a computer assisted design
program (block 144). A pattern designed in this way can be exported
to a file compatible with driving an automatic adhesive dispensing
system (block 146). A program can be executed to dispense the
adhesive in the desired pattern onto the substrate (block 148).
Precision mechanical spacers can be placed onto the base substrate
before or after the adhesive is dispensed (block 150). A second
transparent substrate can then be placed onto the adhesive pattern,
pressing downward until the lower surface is in full contact with
the mechanical spacers (block 152). A weight or other force is
applied to the top substrate to hold it in full contact with the
adhesive. The spacers will typically have a height that is
equivalent or slightly less than the height of the adhesive layer
such that bonding can occur without causing undesirable aberrations
in the shape of the canalized features. The steps for adhering
substrates may be repeated for any number of layers desired.
Optionally, the assembly can be heat treated, for example, in an
oven or exposed to UV light, depending upon the cure requirements
of the adhesive (block 154).
Another exemplary method for fabricating a flow cell is to use an
intermediate layer that is cut to a desired pattern in place of an
adhesive layer. A particularly useful material for the intermediate
layer is silicone. The silicone layer can be heat bonded to the
lower substrate 128 and upper substrate 132. Exemplary methods
utilizing Bisco Silicone HT 6135 as an intermediate layer are
described, for example, in Grover et al., Sensors and Actuators B
89:315-323 (2003).
Still further, FIG. 18 illustrates an embodiment utilizing one
radiation source and dual detectors. Radiation from the radiation
source 22 is directed by the directing optics 30 toward the
focusing optics 32. From the focusing optics 32, the excitation
radiation 58 irradiates a biological component 12 on a first
surface 18 of the support structure 16. The biological component 12
emits a fluorescent emission 60 back through the focusing optics 32
toward the directing optics 30. This retrobeam is allowed to pass
through the directing optics 30 to the detection optics 34 which,
in this illustrated embodiment, may include a wavelength filter 156
or some other device for separating the retrobeam, and first and
second color filters 158, 160 for achieving multiple color
channels. The wavelength filter 156 may split the retrobeam into
two beams with one beam directed toward the first detector 36 via
the first color filter 158 and the other beam directed toward a
second detector 162 via the second color filter 160. In this
manner, the biological sample imaging system 10 may sequentially
scan the first and second surfaces 18, 20, first scanning the first
surface 18 of the support structure 16 using the first excitation
radiation 58 from the radiation source 22 and the returned first
fluorescent emission 60 (as depicted in the left portion of FIG.
18), and next scanning the second surface 20 of the support
structure 16 using the second excitation radiation 62 from the same
radiation source 22 and the returned second fluorescent emission 64
(as depicted in the right portion of FIG. 18).
Alternatively, FIG. 19 illustrates an embodiment utilizing dual
radiation sources and dual detectors. Again, the two surfaces 18,
20 of the support structure 16 may be scanned sequentially.
However, in this embodiment, the first surface 18 of the support
structure 16 is first scanned using the first radiation source 22
which generates the first excitation radiation 58 and the first
fluorescent emission 60 (as depicted in the left portion of FIG.
19) and, the second surface 20 of the support structure 16 is
scanned using the second radiation source 24 which generates the
second excitation radiation 62 and the second fluorescent emission
64 (as depicted in the right portion of FIG. 19). This embodiment
may also be extended to use any number of detectors in order to
reduce movement of the filters.
In the embodiments described above where scanning of the first and
second surfaces 18, 20 of the support structure 16 may be performed
sequentially, the individual steps of scanning the first and second
surfaces 18, 20 of the support structure 16 may be performed in a
number of ways. For instance, it may be possible to scan a single
line of the first surface 18, then scan a single line of the second
surface 20, then gradually move the first and second surfaces 18,
20 relative to the excitation radiation 58, 62 by translating the
support structure 16, the directing optics 30, the focusing optics
32, or some combination thereof, in order to repeat these steps of
scanning individual lines. Alternatively, entire regions of the
first surface 18 may be scanned before regions of the second
surface 20 are scanned. The individual processing steps taken may
depend upon several variables including the particular
configuration of the biological component sites 12, 14 on the
surfaces 18, 20 as well as other variables, including environmental
and operating conditions.
Particular embodiments may allow for simultaneous excitation of
multiple surfaces of the support structure 16. For instance, FIG.
20 illustrates an embodiment utilizing dual radiation sources and
dual detectors. However, in this embodiment, the first surface 18
and the second surface 20 of the support structure 16 may be
simultaneously scanned. This may be accomplished using focusing
lenses 164, 166, 168, 170 and a dichroic mirror 172 along the
excitation path in order to switch surfaces and filters 158, 160 to
achieve multiple color channels. Again, this illustrated embodiment
may also be extended to any number of detectors to improve
throughput, scanning efficiency, and to reduce movement of the
filters and other system components.
FIG. 21 illustrates another embodiment utilizing dual radiation
sources and dual detectors which allows for simultaneous scanning
of the first and second surfaces 18, 20 of the support structure
16. In this illustrated embodiment, however, not only are focusing
lenses 164, 166, 168, 170 and a dichroic mirror 172 used in the
excitation path but focusing lenses 174, 176 may be used just
upstream of the first and second detectors 36, 162 in conjunction
with the filters 158, 160 along the emission path in order to
switch surfaces and achieve multiple color channels. Once again,
this illustrated embodiment may also be extended to use any number
of detectors to increase throughput and scanning efficiency.
For instance, FIG. 22 illustrates an embodiment utilizing multiple
radiation sources and multiple detectors which are capable of
simultaneously outputting multiple channels with few moving parts.
In the illustrated embodiment, radiation sources 22 and 24 have
been replaced by radiation source groups 178 and 180 which are
capable of outputting multiple radiation sources and varying
wavelengths. In addition, detectors 36 and 38 have been replaced by
detector groups 182 and 184 in the illustrated embodiment. These
detector groups 182, 184 are similarly capable of detecting
multiple color channels. This embodiment therefore illustrates the
considerable adaptability of the present techniques to a range of
configurations capable of imaging components on multiple surfaces
of the support.
In the embodiments described above where scanning of the first and
second surfaces 18, 20 of the support structure 16 may be performed
simultaneously, focusing of the excitation radiation 58 source may
be accomplished in several various ways. For instance, it may be
possible to focus the excitation radiation 58 on one of the
surfaces preferentially over the other surface. In fact, due to the
nature of the configuration of the first surface 18 with respect to
the second surface 20, it may be necessary to do so. However,
alternate focusing techniques may be employed depending on the
specific configuration of the support structure 16. Moreover, it
may be advantageous in these various configurations to first image
the upper surface (i.e., the surface closer to the radiation
source) in order to reduce photobleaching of the components on that
surface that could result from first imaging the lower surface
(i.e., the surface farther from the radiation source). Such
selection of which surface to image may apply both when the
surfaces are imaged sequentially as well as when they are imaged
simultaneously.
In addition, the embodiments disclosed above have illustrated an
epifluorescent imaging scheme wherein the excitation radiation is
directed toward the surfaces of the support structure 16 from a top
side, and returned fluorescent radiation is received from the same
side. However, the techniques of the present invention may also be
extended to alternate arrangements. For instance, these techniques
may also be employed in conjunction with TIR imaging whereby the
surfaces of the support structure are irradiated from a lateral
side with radiation directed at an incident angle within a range of
critical angles so as to convey the excitation radiation within the
support or into the support from a prism positioned adjacent to it.
TIR techniques can be carried out as described, for example, in
U.S. Patent Application Publication No. 2005/0057798, which is
hereby incorporated by reference. Such techniques cause fluorescent
emissions from the components that are conveyed outwardly for
imaging, while the reflected excitation radiation exits via a side
opposite from that through which it entered. Here again, biological
components on the multiple surfaces may be imaged sequentially or
simultaneously.
For example, in FIG. 23, a TIR biological sample imaging system 186
is illustrated diagrammatically. A support structure 188 may be
used which includes multiple flow lanes 190 containing biological
components. For example, the support structure 188 may be a flow
cell through which reagents, flushes, and other fluids may be
introduced using the flow lanes 190 to contact emissive components
attached to the surface of the flow cell. The support structure 188
may be supported by a prism 192. In the TIR biological sample
imaging system 186, the radiation source 194 may output a radiation
beam 196 through the prism 192 from a lateral side of the support
structure 188. The radiation beam 196 may, for instance, be
directed toward a bottom surface of one of the flow lanes 190 of
the support structure 188, thereby exciting emissive components
attached to the surface.
As discussed in further detail below, as long as the incident angle
of the radiation beam 196 is within the range of critical angles
(as described, for example, in US 2005/0057798), a portion of the
radiation beam 196 will be reflected off the bottom surface whereas
a separate fluorescent emission beam from surface-bound emissive
components will be directed toward focusing optics 198. Typically,
a well collimated radiation beam is used to prevent spread of
angles within the beam, thereby preventing unwanted hindrance of
total internal reflectance. The fluorescent emission beam may
propagate back through the focusing optics 198, directing optics
200, and detection optics 202 which may direct the beam toward a
detector 204. The focusing optics 198, directing optics 200,
detection optics 202, and detector 204 may operate in much the same
manner as with the epifluorescent techniques discussed above. In
the TIR biological sample imaging system 186, the focusing light
source 206 may be used as a separate light source from the
radiation source 194 to focus the optics on a particular surface to
be imaged. For instance, the focusing light source 206 may be
directed to the directing optics 200 where it is redirected toward
the focusing optics 198 which are used to focus the system on a
particular surface of the support structure 188.
The TIR biological sample imaging system 186 may also include a
translation system 208 for moving the support structure 188 and
prism 192 in one or more dimensions. The translation system 208 may
be used with focusing, redirecting the radiation source 194 to
different areas of the support structure 188, as well as for moving
the support structure 188 and prism 192 to a heating/cooling
station 210. The heating/cooling station 210 may be used to heat
and cool the support structure 188 before and after imaging. In
addition, a control/processing system 212 may be used to control
operation of the radiation source 194, the focusing light source
206, and the heating/cooling station 210, movement and focusing of
the focusing optics 198, the translation system 208, and the
detection optics 202, and acquisition and processing of signals
from the detector 204.
As discussed above, the TIR method of imaging may be used to direct
the radiation beam 196 from a lateral side of the support structure
188, as illustrated in FIG. 24. Each flow lane 190 of the support
structure 188 may include a bottom surface 214 and a top surface
216 and emissive components can optionally be attached to either or
both surface. In the illustrated embodiment, the radiation beam 196
is directed toward a bottom surface 214 of one of the flow lanes
190 of the support structure 188. Part of the radiation beam 196
may be reflected off the bottom surface 214 of the flow lane 190,
as depicted by reflected light beam 218. However, as long as the
incident angle of the radiation beam 196 is within the range of
critical angles, a separate fluorescent emission beam 220 may be
emitted from emissive components toward the focusing optics 198
which in the illustrated embodiment is a lens objective 222.
Indeed, directing the radiation beam 196 at a bottom surface 214 of
a flow lane 190 of the support structure 188 is a typical
implementation of the TIR imaging method. However, in doing so,
imaging data which may be collected from a top surface 216 of a
flow lane 190 of the support structure 188 may be overlooked.
Therefore, the orientation of the radiation source 194 and/or the
support structure 188 and prism 192 may be adjusted in order to
allow the radiation beam 196 to not be directed at a bottom surface
214 of a flow lane 190 of the support structure 188, as illustrated
in FIG. 25. In the illustrated embodiment, the radiation beam 196
is oriented so that the radiation beam 196 passes through the prism
192 and support structure 188 until contacting an air/glass
interface 224 of the support structure 188 at which point the
radiation beam 196 is redirected toward a top surface 216 of a flow
lane 190 of the support structure 188. At this point, part of the
radiation beam 196 may be reflected back toward another air/glass
interface 224 of the support structure 188. However, a separate
fluorescent emission beam 220 may be emitted from an emissive
component on the top surface 216 toward the lens objective 222.
Using this technique, top surfaces 216 of the flow lanes 190 of the
support structure 188 may be imaged using TIR imaging methods.
This, in effect, may allow for double the imaging data output for
cluster based sequencing applications while keeping other
variables, such as surface coating, cluster creation, and
sequencing, the same.
In order to accomplish this TIR imaging of top surfaces 216 of the
flow lanes 190 of the support structure 188, the radiation beam 196
reaches the air/glass interface 224 of the support structure 188
unperturbed. To do so, the radiation beam 196 does not first come
into contact with emissive components in adjacent flow lanes 190.
To do so, either the radiation beam 196 may be directed around the
adjacent flow lanes 190 or the adjacent flow lanes 190 may be index
matched with the support structure 188 material. In some
embodiments, the flow lanes 190 may be spaced within the support
structure 188, leaving sufficient room between the flow lanes 190
for the radiation beam 196 to pass. However, spacing the flow lanes
190 in this manner may ultimately reduce the amount of emissive
components which may be imaged. Therefore, in other embodiments, it
may be possible to accomplish the same effect by temporarily
filling alternate flow lanes 190 with index matching fluid. Doing
so may allow for easier direction of the radiation beam 196 toward
a top surface 216 of a flow lane 190 of the support structure
188.
It may also be possible to direct the radiation beam 196 in such a
way that it bounces off multiple top surfaces 216 of flow lanes 190
of the support structure 188, as illustrated in FIG. 26. In order
to accomplish this, the spacing of the flow lanes 190 can be
matched with the angle of radiation beam 196 such that the
radiation beam 196 is able to pass by the flow lanes 190, such that
it reaches the air/glass interface 224 of the support structure 188
unperturbed, while also being able to bounce back and forth between
top surfaces 216 of flow lanes 190 and the air/glass interface 224
of the support structure 188. As described above, in certain
embodiments, some of the flow lanes 190 may be filled with an index
matching fluid, such that these index-matched flow lanes 190
effectively become "invisible" to the radiation beam 196. In other
words, the radiation beam 196 may be allowed to pass through the
index-matched flow lanes 190. By allowing the radiation beam 196 to
pass through the index-matched flow lanes 190, the support
structure 188 may be used in multiple configurations without the
need of varying the spacing of the flow lanes 190.
In some embodiments, mirrors 226 or other suitable reflective
material may be used within certain flow lanes 190, facilitating
this multi-bounce technique. In any event, assuming N number of
flow lanes 190, it may only be possible to image N-2 number of top
surfaces 216 of the flow lanes 190 in this manner due to the fact
that the outer flow lanes 190 on either side of the support
structure 188 may not be accessible using these techniques.
However, modification of the prism 192 and/or support structure 188
may allow for imaging of the top surfaces 216 of these outermost
flow lanes 190. For instance, the support structure 188 may be
designed to fit within the prism 192, allowing the radiation beam
196 to propagate into a lateral side of the support structure
188.
In some embodiments, as discussed above briefly with respect to
FIG. 23, the support structure 188 may be moved to a
heating/cooling station 210, for example, by the action of the
translation system 208. The heating/cooling station 210 may be
configured to both heat and cool the support structure 188 before
and after imaging. The heating/cooling station 210 may, in fact, be
configured to heat and cool both a top surface 228 and a bottom
surface 230 of the support structure 188, as illustrated in FIG.
27. Indeed, all surfaces of the support structure 188 may be heated
or cooled at the heating/cooling station 210. In this manner, it
may further be possible to heat and cool both the top surfaces 216
and bottom surfaces 214 of the flow lanes 190 of the support
structure 188 by directly contacting one or more surfaces of the
flow cell with a heating or cooling device. This, of course, may
facilitate the development of biological components within the flow
lanes 190 of the support structure 188 and, therefore, facilitate
imaging. Although use of the heating/cooling station 210 has been
presented herein with respect to the TIR imaging methods, the
heating/cooling station 210 may also be used to heat and cool
multiple sides of a support structure used in conjunction with the
epifluorescent imaging methods discussed herein.
In particular embodiments, the current invention utilizes
sequencing-by-synthesis (SBS). In SBS, four fluorescently labeled
modified nucleotides are used to determine the sequence of
nucleotides for nucleic acids present on the surface of a support
structure such as a flow cell. Exemplary SBS systems and methods
which can be utilized with the apparatus and methods set forth
herein are described in U.S. Pat. No. 7,057,026; U.S. Patent
Application Publication Nos. 2005/0100900, 2006/0188901,
2006/0240439, 2006/0281109, and 2007/0166705; and PCT Publication
Nos. WO 05/065814, WO 06/064199, and WO 07/010251; each of which is
incorporated herein by reference.
In particular uses of the apparatus and methods herein, flow cells
containing arrayed nucleic acids are treated by several repeated
cycles of an overall sequencing process. The nucleic acids are
prepared such that they include an oligonucleotide primer adjacent
to an unknown target sequence. To initiate the first SBS sequencing
cycle, one or more differently labeled nucleotides and a DNA
polymerase are flowed into the flow cell. Either a single
nucleotide can be added at a time, or the nucleotides used in the
sequencing procedure can be specially designed to possess a
reversible termination property, thus allowing each cycle of the
sequencing reaction to occur simultaneously in the presence of all
four labeled nucleotides (A, C, T, G). Following nucleotide
addition, the features on the surface can be imaged to determine
the identity of the incorporated nucleotide (based on the labels on
the nucleotides). Then, reagents can be added to the flow cell to
remove the blocked 3' terminus (if appropriate) and to remove
labels from each incorporated base. Such cycles are then repeated
and the sequence of each cluster is read over the multiple
chemistry cycles.
Other sequencing methods that use cyclic reactions wherein each
cycle includes steps of delivering one or more reagents to nucleic
acids on a surface and imaging the surface bound nucleic acids can
also be used such as pyrosequencing and sequencing by ligation.
Useful pyrosequencing reactions are described, for example, in U.S.
Pat. No. 7,244,559 and U.S. Patent Application Publication No.
2005/0191698, each of which is incorporated herein by reference.
Sequencing by ligation reactions are described, for example, in
Shendure et al. Science 309:1728-1732 (2005); and U.S. Pat. Nos.
5,599,675 and 5,750,341, each of which is incorporated herein by
reference.
The methods and apparatus described herein are also useful for
detection of features occurring on surfaces used in genotyping
assays, expression analyses and other assays known in the art such
as those described in U.S. Patent Application Publication Nos.
2003/0108900, 2003/0215821, and US 2005/0181394, each of which is
incorporated herein by reference.
While only certain features of the invention have been illustrated
and described herein, many modifications and changes will occur to
those skilled in the art. It is, therefore, to be understood that
the appended claims are intended to cover all such modifications
and changes as fall within the true spirit of the invention.
* * * * *
References