USO08039817B2

(12) United States Patent (10) Patent No.: US 8,039,817 B2

Feng et al. (45) Date of Patent: Oct. 18, 2011
(54) COMPENSATOR FOR MULTIPLE SURFACE 5,744,305 A 4/1998 Fodor et al.
MAGING 5,750,341 A 5/1998 Macevicz
5,795,716 A 8, 1998 Chee
5,831,070 A 11/1998 Pease et al.
(75) Inventors: Wenyi Feng, San Diego, CA (US); 5,856,101 A 1/1999 Hubbell et al.
Jason Bryant, Essex (GB); Dale 5,858,659 A 1/1999 Sapolsky et al.
Buermann, Los Altos, CA (US) (Continued)
(73) Assignee: Illumina, Inc., San Diego, CA (US) FOREIGN PATENT DOCUMENTS
(*) Notice: Subject to any disclaimer, the term of this DE 100 14 204 A1 10, 2001
patent is extended or adjusted under 35 (Continued)
U.S.C. 154(b) by 372 days.
OTHER PUBLICATIONS
(21) Appl. No.: 12/434,495 Grover et al., “Monolithic membrane valves and diaphragm pumps
(22) Filed: May 1, 2009 for practical large-scale integration into glass microfluid devices.”
Sensors and Actuators B 89:315-323 (2003).
(65) Prior Publication Data
(Continued)
US 2009/0272914 A1 Nov. 5, 2009
Primary Examiner — Kiho Kim
Related U.S. Application Data (74) Attorney, Agent, or Firm — Fletcher Yoder, PC
(60) Provisional application No. 61/050,522, filed on May (57) ABSTRACT
5, 2008, provisional application No. 61/138,444, filed
on Dec. 17, 2008. A system and method for imaging biological samples on
multiple surfaces of a Support structure are disclosed. The
(51) Int. Cl. Support structure may be a flow cell through which a reagent
GOIN 2L/64 (2006.01) fluid is allowed to flow and interact with the biological
(52) U.S. Cl. .................................................... 250/459.1 samples. Excitation radiation from at least one radiation
(58) Field of Classification Search ............... 250/259.1, Source may be used to excite the biological samples on mul
250/458. 1,576 tiple Surfaces. In this manner, fluorescent emission radiation
See application file for complete search history. may be generated from the biological samples and Subse
quently captured and detected by detection optics and at least
(56) References Cited one detector. The detected fluorescent emission radiation may
then be used to generate image data. This imaging of multiple
U.S. PATENT DOCUMENTS Surfaces may be accomplished either sequentially or simul
5,324,633. A 6, 1994 Fodor et al. taneously. In addition, the techniques of the present invention
5,451,683 A 9, 1995 Barrett et al. may be used with any type of imaging system. For instance,
5,482,867 A 1/1996 Barrett et al.
5,491,074 A 2f1996 Aldwin et al. both epifluorescent and total internal reflection methods may
5,599,675 A 2f1997 Brenner benefit from the techniques of the present invention.
5,624,711 A 4/1997 Sundberg et al.
5,646,411 A 7, 1997 Kain et al. 27 Claims, 18 Drawing Sheets

al
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U.S. PATENT DOCUMENTS 2004/0248287 A1 12, 2004 Hu et al.

2005/0057798 A1 3, 2005 Osborne et al.
5,874,219 A 2, 1999 Rava et al. 2005, 0100900 A1 5/2005 Kawashima et al.
3.
-
A 1992 Fleet
e
al. 2005/0181394 A1
2005, 0191698 A1
8/2005 Steemers et al.
9, 2005 Chee et al.
5,981,185. A 1 1/1999 Matson et al. 2006/0188901 A1 8/2006 Barnes et al.
E. A 3. Se: 11 et all 2006/0240439 A1 10, 2006 Smith et al.
6,025.601 A 2, 2000 Malay 2006/0253035 A1 11, 2006 Stern
W4 - 2006/0281109 A1 12/2006 Barr Ost et al.
89A 3.399 thatal 2007/0114362 A1 5/2007 Feng et al.
6,090,555 A T/2000 Eyal 2007, 0166705 A1 7, 2007 Milton et al.
W-1 W 2008, O262747 A1 10, 2008 Kain et al.
6,136.269 A 10/2000 Winkler et al. 2008/0297911 A1* 12/2008 Christenson et al. ......... 359,666
3. R 858: SIEa. 2009/0309049 A1* 12/2009 Van Dijk et al. ... 25Of 578.1
6,327,410 B1 12/2001 Waltet al. FOREIGN PATENT DOCUMENTS
6,355.431 B1 3/2002 Chee et al.
6,416,949 B1 7/2002 Dower et al. WO 98.44151 A1 10, 1998
6.428,752 B1 8/2002 Montagu WO OOf 18957 A1 4, 2000
6,482,591 B2 11/2002 Lockhart et al. WO OO63437 A2 10, 2000
6,770,441 B2 8/2004 Dickinson et al. WO 2005/065814 A1 T 2005
7.057,026 B2 6/2006 Barnes et al. WO 2006/064199 A1 6, 2006
7,115,400 B1 10/2006 Adessi et al. WO 2007/O10251 A2 1/2007
7.244.559 B2 7/2007 Rothberg et al. WO 2007/123744 A2 11/2007
7,329,860 B2 2, 2008 Feng et al. OTHER PUBLICATIONS
2002/0102578 A1 8/2002 Dickinson et al.
2003. O108900 A1 6/2003 Oliphant et al. Shendure et al.; "Accurate Multiplex Polony Sequencing of an
58.3-6. A. '3. custon
USS
et al. Evolved Bacteria Genome.” Science 309: 1728-1732 (2005).
2004/O185483 A1 9/2004 Stuelpnagel et al. * cited by examiner
U.S. Patent Oct. 18, 2011 Sheet 1 of 18 US 8,039,817 B2
U.S. Patent Oct. 18, 2011 Sheet 2 of 18 US 8,039,817 B2
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U.S. Patent Oct. 18, 2011 Sheet 4 of 18 US 8,039,817 B2
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U.S. Patent Oct. 18, 2011 Sheet 13 of 18 US 8,039,817 B2
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COMPENSATOR FOR MULTIPLE SURFACE imaging and evaluation Subsystems to multiple Surfaces that
MAGING Support samples. The Support structure may, for instance, be
CROSS REFERENCE TO RELATED
a flow cell through which a reagent fluid is allowed to flow and
APPLICATIONS
interact with biological samples. Excitation radiation from at
least one radiation Source may be used to excite the biological
This application claims priority of U.S. Provisional Patent samples on multiple Surfaces. In this manner, fluorescent
Application No. 61/050,522, entitled “Multi-Surface Bio radiation may be emitted from the biological samples and
logical Sample Imaging System and Method, filed May 5, Subsequently captured and detected by detection optics and at
2008, which is herein incorporated in its entirety by refer 10
least one detector. The returned radiation may then be used to
ence, and of U.S. Provisional Patent Application No. 61/138, generate image data. This imaging of multiple Surfaces may
444, entitled “Compensator for Multiple Surface Imaging.” be accomplished either sequentially or simultaneously. In
filed Dec. 17, 2008, which is herein incorporated in its addition, the techniques of the present invention may be used
entirety by reference. with any of a variety of types of imaging systems. For
BACKGROUND 15 instance, both epifluorescent and total internal reflection
(TIR) methods may benefit from the techniques of the present
The present invention relates generally to the field of imag invention. In addition, the biological samples imaged may be
ing and evaluating analytical samples. More particularly, the present on the Surfaces of the Support structure in random
invention relates to a technique for imaging and evaluating locations or in patterns.
analytical samples on multiple Surfaces of a Support structure Accordingly, the invention provides a method for imaging
using a compensator. a biological sample. The method includes detecting radiation
There are an increasing number of applications for imaging emitted from a first emissive component of a biological
of analytical samples on a Support structure. These Support sample disposed on a first Surface of a flow cell using a
structures may include plates upon which biological samples detector. The flow cell is mounted on an imaging station. The
are present. For instance, these plates may include deoxyri 25 method also includes inserting corrective optics between the
bonucleic acid (DNA) and ribonucleic acid (RNA) probes detector and the flow cell. The method further includes detect
that are specific for nucleotide sequences present in genes in ing radiation emitted from a second emissive component of a
humans and other organisms. Individual DNA or RNA probes biological sample disposed on a second Surface of the flow
can be attached at specific locations in a small geometric grid
or array on the Support structure. Depending upon the tech cell using the detector and the corrective optics. The first and
nology employed, the samples may attach at random, semi 30 second Surfaces are in an arrangement whereby one of the
random or predetermined locations on the Support structure. surfaces is disposed between the detector and the other sur
A test sample, such as from a known person or organism, can face. In addition, the corrective optics reduce aberration of
be exposed to the array or grid. Such that complementary detection at one of the Surfaces due to the arrangement. The
genes or fragments hybridize to probes at the individual sites steps of the method are repeated while maintaining the flow
on a Surface of a plate. In certain applications, such as 35 cell on the imaging station.
sequencing, templates or fragments of genetic material may The invention further provides an imaging system for
be located at the sites, and nucleotides or other molecules may detecting radiation on a multi-surface flow cell. The imaging
be caused to hybridize to the templates to determine the system includes a multi-surface flow cell having first and
nature or sequence of the templates. The sites can then be second emissive components of a biological sample disposed
examined by Scanning specific frequencies of light over the 40 on respective first and second surfaces of the flow cell. The
sites to identify which genes or fragments in the sample were imaging system also includes an optical train including an
present, by fluorescence of the sites at which genes or frag objective, imaging optics configured to focus the optical train
ments hybridized. on the first emissive component via the objective, and correc
These plates are sometimes referred to as microarrays, tive optics configured to focus the optical train on the second
gene or genome chips, DNA chips, gene arrays, and so forth, 45 emissive component and configured to reduce aberration of
and may be used for expression profiling, monitoring expres detection at the first or second emissive component. The
sion levels, genotyping, sequencing, and so forth. For imaging system further includes a radiation source config
example, diagnostic uses may include evaluation of a particu ured to direct excitation radiation towards the first and second
lar patient’s genetic makeup to determine whether a disease emissive components. In addition, the imaging system
state is present or whether pre-disposition for a particular 50 includes detection optics configured to capture emitted radia
condition exists. The reading and evaluation of such plates are tion returned from the first and second emissive components
an important aspect of their utility. Although microarrays via the optical train. Further, the imaging system includes a
allow separate biological components to be presented for bulk detector for detecting the captured radiation.
processing and individual detection, the number of compo
nents that can be detected in a single experiment is limited by 55 DRAWINGS
the resolution of the system. Furthermore, the bulk reagents
used in Some methods can be expensive such that reduced FIG. 1 is a diagrammatical overview for a biological
volumes are desired. The present invention provides methods sample imaging system in accordance with the present inven
and compositions that increase the efficiency of array based tion;
detection to counteract these limitations. Other advantages 60 FIG. 2 is a diagrammatical perspective view of an exem
are provided as well and will be apparent from the description plary radiation line directed toward a Surface of a Support
below. structure to semi-confocally irradiate biological sites, and to
semi-confocally return radiation to a detector in accordance
BRIEF DESCRIPTION with the present invention;
65 FIG.3 is a sectional view of an exemplary support structure
The present invention provides a novel approach to ana with excitation radiation directed at two surfaces of the Sup
lytical sample imaging and evaluation that expands the use of port structure in accordance with the present invention;
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3 4
FIG. 4 is a diagrammatical perspective view of an exem the Support structure using focusing lenses along the excita
plary Support structure having an array of biological compo tion path in accordance with the present invention;
nent sites in a spatially ordered pattern in accordance with the FIG. 21 is a diagrammatical view of a biological sample
present invention; imaging system with dual radiation sources and dual detec
FIG. 5 is a diagrammatical perspective view of an exem tors configured to simultaneously scan multiple Surfaces of
plary Support structure having biological component sites in a the Support structure using focusing lenses along the excita
random spatial distribution in accordance with the present tion and emission paths in accordance with the present inven
invention; tion;
FIG. 6 is a sectional view of an exemplary support structure FIG. 22 is a diagrammatical view of a biological sample
with excitation radiation directed at multiple surfaces of the 10 imaging system with multiple radiation sources and multiple
Support structure in accordance with the present invention; detectors configured to simultaneously scan multiple Surfaces
FIG. 7 illustrates exemplary dimensions between the of the Support structure using focusing lenses along the exci
objective and the Support structure in accordance with the tation and emission paths in accordance with the present
present invention; invention;
FIG. 8 is an exemplary chart of spherical aberration vs. 15 FIG. 23 is a diagrammatical overview for a TIR biological
thickness of the upper plate of the support structure of FIG. 7 sample imaging system in accordance with the present inven
in accordance with the present invention; tion;
FIG.9A illustrates exemplary images expected for first and FIG. 24 is a sectional view of an exemplary Support struc
second Surfaces of a Support structure when obtained through ture, prism, and lens objective for use with TIR imaging of a
an upper surface thickness of 300 microns (plus 100 microns bottom surface of a flow lane in accordance with the present
of fluid) without corrective optics, where the imaging system invention;
is optimized for the second Surface; FIG.25 is a sectional view of an exemplary support struc
FIG.9B illustrates exemplary images expected for first and ture, prism, and lens objective for use with TIR imaging of a
second Surfaces of a Support structure when obtained through top Surface of a flow lane in accordance with the present
an upper surface thickness of 340 microns (plus 100 microns 25 invention;
of fluid) without corrective optics; FIG. 26 is a sectional view of another exemplary support
FIG. 10A illustrates an exemplary objective imaging the structure, prism, and lens objective for use with TIR imaging
second Surface without the assistance of a compensator in of a top Surface of a flow lane in accordance with the present
accordance with the present invention; invention; and
FIG. 10B illustrates an exemplary objective imaging the 30 FIG. 27 is a sectional view of an exemplary support struc
first Surface with the assistance of a compensator in accor ture being heated on both top and bottom surfaces in accor
dance with the present invention: dance with the present invention.
FIG. 11 is an exemplary compensator design, incorporat
ing a first objective and a second objective which may replace DETAILED DESCRIPTION
the first objective in the optical train in accordance with the 35
present invention; Turning now to the drawings, and referring first to FIG. 1,
FIG. 12 is another exemplary compensator design, incor a biological sample imaging system 10 is illustrated diagram
porating a corrective device which may be inserted between matically. The biological sample imaging system 10 is
the objective and the support structure in accordance with the capable of imaging multiple biological components 12, 14
present invention; 40 within a support structure 16. For instance, in the illustrated
FIG. 13 is another exemplary compensator design, incor embodiment, a first biological component 12 may be present
porating a correction collar in accordance with the present on a first surface 18 of the support structure 16 while a second
invention; biological component 14 may be present on a second Surface
FIG. 14 is another exemplary compensator design, incor 20 of the support structure. The support structure 16 may, for
porating an infinite space compensator in accordance with the 45 instance, be a flow cell with an array of biological compo
present invention; nents 12, 14 on the interior surfaces 18, 20 which generally
FIG. 15 is a perspective view of an exemplary flow cell mutually face each other and through which reagents, flushes,
assembly using patterned adhesives to form channel charac and other fluids may be introduced, such as for binding nucle
teristics in accordance with the present invention; otides or other molecules to the sites of biological compo
FIG. 16 is a perspective view of another exemplary flow 50 nents 12, 14. The support structure 16 may be manufactured
cell assembly using patterned adhesives to form channel char in conjunction with the present techniques or the Support
acteristics in accordance with the present invention; structure 16 may be purchased or otherwise obtained from a
FIG. 17 is a process flow diagram of an exemplary method separate entity. Fluorescent tags on the molecules that bind to
of assembling flow cells using patterned adhesives to form the components may, for instance, include dyes that fluoresce
channel characteristics in accordance with the present inven 55 when excited by appropriate excitation radiation. Assay
tion; methods that include the use of fluorescent tags and that can
FIG. 18 is a diagrammatical view of a biological sample be used in an apparatus or method set forth herein include
imaging system with one radiation source and dual detectors those set forth elsewhere herein such as genotyping assays,
configured to sequentially scan multiple surfaces of the Sup gene expression analysis, methylation analysis, or nucleic
port structure in accordance with the present invention; 60 acid sequencing analysis.
FIG. 19 is a diagrammatical view of a biological sample Those skilled in the art will recognize that a flow cell or
imaging system with dual radiation sources and dual detec other support structure may be used with any of a variety of
tors configured to sequentially scan multiple Surfaces of the arrays known in the art to achieve similar results. Further
Support structure in accordance with the present invention; more, known methods for making arrays can be used, and if
FIG. 20 is a diagrammatical view of a biological sample 65 appropriate, modified in accordance with the teaching set
imaging system with dual radiation sources and dual detec forth herein in order to create a flow cell or other support
tors configured to simultaneously scan multiple Surfaces of structure having multiple surfaces useful in the detection
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5 6
methods set forth herein. Such arrays may be formed by or derivatives of these natural components. Although the
disposing the biological components of samples randomly or apparatus and methods of the invention are exemplified
in predefined patterns on the Surfaces of the Support by any herein with respect to components of biological samples, it
known technique. In a particular embodiment, clustered will be understood that other samples or components can be
arrays of nucleic acid colonies can be prepared as described in 5 used as well. For example, synthetic samples can be used Such
U.S. Pat. No. 7,115,400; U.S. Patent Application Publication as combinatorial libraries, or libraries of compounds having
No. 2005/0100900; PCT Publication No. WO 00/18957; or species known or Suspected of having a desired structure or
PCT Publication No. WO98/44151, each of which is hereby function. Thus, the apparatus or methods can be used to
incorporated by reference. Such methods are known as bridge synthesize a collection of compounds and/or screen a collec
amplification or solid-phase amplification and are particu 10
tion of compounds for a desired structure or function.
larly useful for sequencing applications. Returning to the exemplary system of FIG.1, the biological
Other exemplary random arrays, and methods for their sample imaging system 10 may include at least a first radia
construction, that can be used in the invention include, with
out limitation, those in which beads are associated with a tion source 22 but may also include a second radiation source
solid support, examples of which are described in U.S. Pat. 15 24 (or additional sources). The radiation Sources 22, 24 may
Nos. 6,355,431; 6,327,410; and 6,770,441; U.S. Patent be lasers operating at different wavelengths. The selection of
Application Publication Nos. 2004/0185483 and US 2002/ the wavelengths for the lasers will typically depend upon the
01.02578; and PCT Publication No. WO 00/63437, each of fluorescence properties of the dyes used to image the compo
which is hereby incorporated by reference. Beads can be nent sites. Multiple different wavelengths of the lasers used
located at discrete locations, such as wells, on a Solid-phase may permit differentiation of the dyes at the various sites
Support, whereby each location accommodates a single bead. within the Support structure 16, and imaging may proceed by
Any of a variety of other arrays known in the art or methods Successive acquisition of a series of images to enable identi
for fabricating Such arrays can be used in the present inven fication of the molecules at the component sites in accordance
tion. Commercially available microarrays that can be used with image processing and reading logic generally known in
include, for example, an Affymetrix R GeneChip(R) microar 25 the art. Other radiation sources known in the art can be used
ray or other microarray synthesized in accordance with tech including, for example, an arc lamp or quartz halogen lamp.
niques sometimes referred to as VLSIPSTM (Very Large Scale Particularly useful radiation sources are those that produce
Immobilized Polymer Synthesis) technologies as described, electromagnetic radiation in the ultraviolet (UV) range (about
for example, in U.S. Pat. Nos. 5,324,633; 5,744,305; 5.451, 200 to 390 nm), visible (VIS) range (about 390 to 770 nm),
683; 5,482,867; 5,491,0745,624,711; 5,795,716; 5,831,070; 30 infrared (IR) range (about 0.77 to 25 microns), or other range
5,856,101; 5,858,659; 5,874,219; 5,968,740; 5,974,164: of the electromagnetic spectrum.
5,981,185; 5,981,956; 6,025,601; 6,033,860; 6,090,555; For ease of description, embodiments utilizing fluores
6,136,269; 6,022,963; 6,083,697; 6,291, 183; 6,309,831; cence-based detection are used as examples. However, it will
6,416,949; 6,428,752; and 6,482.591, each of which is hereby be understood that other detection methods can be used in
incorporated by reference. A spotted microarray can also be 35 connection with the apparatus and methods set forth herein.
used in a method of the invention. An exemplary spotted For example, a variety of different emission types can be
microarray is a CodeLinkTM Array available from Amersham detected Such as fluorescence, luminescence, or chemilumi
Biosciences. Another microarray that is useful in the inven nescence. Accordingly, components to be detected can be
tion is one that is manufactured using inkjet printing methods labeled with compounds or moieties that are fluorescent,
such as SurePrintTM Technology available from Agilent Tech 40 luminescent, or chemiluminescent. Signals other than optical
nologies. signals can also be detected from multiple Surfaces using
Sites or features of an array are typically discrete, being apparatus and methods that are analogous to those exempli
separated with spaces between each other. The size of the sites fied herein with the exception of being modified to accom
and/or spacing between the sites can vary Such that arrays can modate the particular physical properties of the signal to be
be high density, medium density, or lower density. High den 45 detected.
sity arrays are characterized as having sites separated by less Output from the radiation sources 22, 24 may be directed
than about 15um. Medium density arrays have sites separated through conditioning optics 26, 28 for filtering and shaping of
by about 15 to 30 um, while low density arrays have sites the beams. For example, in a presently contemplated embodi
separated by greater than 30 Jum. An array useful in the inven ment, the conditioning optics 26, 28 may generate a generally
tion can have sites that are separated by less than 100 um, 50 50 linear beam of radiation, and combine beams from multiple
um, 10um, 5um, 1 um or 0.5um. An apparatus or method of lasers, for example, as described in U.S. Pat. No. 7,329,860.
the invention can be used to image an array at a resolution The laser modules can additionally include a measuring com
Sufficient to distinguish sites at the above densities or density ponent that records the power of each laser. The measurement
ranges. of power may be used as a feedback mechanism to control the
As exemplified herein, a Surface used in an apparatus or 55 length of time an image is recorded in order to obtain uniform
method of the invention is typically a manufactured Surface. It exposure, and therefore more readily comparable signals.
is also possible to use a natural Surface or a surface of a natural After passing through the conditioning optics 26, 28, the
Support structure; however the invention can be carried out in beams may be directed toward directing optics 30 which
embodiments where the Surface is not a natural material or a redirect the beams from the radiation sources 22, 24 toward
Surface of a natural Support structure. Accordingly, compo 60 focusing optics 32. The directing optics 30 may include a
nents of biological samples can be removed from their native dichroic mirror configured to redirect the beams toward the
environment and attached to a manufactured Surface. focusing optics 32 while also allowing certain wavelengths of
Any of a variety of biological components can be present a retrobeam to pass therethrough. The focusing optics 32 may
on a Surface for use in the invention. Exemplary components confocally direct radiation to one or more surfaces 18, 20 of
include, without limitation, nucleic acids such as DNA or 65 the support structure 16 upon which individual biological
RNA, proteins such as enzymes or receptors, polypeptides, components 12, 14 are located. For instance, the focusing
nucleotides, amino acids, Saccharides, cofactors, metabolites optics 32 may include a microscope objective that confocally
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7 8
directs and concentrates the radiation sources 22, 24 along a The Support structure 16 may be Supported on a translation
line to a surface 18, 20 of the support structure 16. system 40 which allows for focusing and movement of the
Biological component sites on the Support structure 16 Support structure 16 before and during imaging. The stage
may fluoresce at particular wavelengths in response to an may be configured to move the support structure 16, thereby
excitation beam and thereby return radiation for imaging. For 5 changing the relative positions of the radiation sources 22, 24
instance, the fluorescent components may be generated by and detector 36 with respect to the surface bound biological
fluorescently tagged nucleic acids that hybridize to comple components for progressive Scanning. Movement of the
mentary molecules of the components or to fluorescently translation system 40 can be in one or more dimensions
tagged nucleotides that are incorporated into an oligonucle including, for example, one or both of the dimensions that are
otide using a polymerase. As noted above, the fluorescent 10 orthogonal to the direction of propagation for the excitation
properties of these components may be changed through the radiation line, typically denoted as the X and Y dimensions. In
introduction of reagents into the Support structure 16 (e.g., by particular embodiments, the translation system 40 may be
cleaving the dye from the molecule, blocking attachment of configured to move in a direction perpendicular to the scan
additional molecules, adding a quenching reagent, adding an axis for a detector array. A translation system 40 useful in the
15 present invention may be further configured for movement in
acceptor of energy transfer, and so forth). As will be appre the dimension along which the excitation radiation line
ciated by those skilled in the art, the wavelength at which the propagates, typically denoted as the Z dimension. Movement
dyes of the sample are excited and the wavelength at which in the Z dimension can also be useful for focusing.
they fluoresce will depend upon the absorption and emission FIG. 2 is a diagrammatical representation of an exemplary
spectra of the specific dyes. Such returned radiation may semi-confocal line Scanning approach to imaging the Support
propagate back through the directing optics 30. This retro structure 16. In the illustrated embodiment, the support struc
beam may generally be directed toward detection optics 34 ture 16 includes an upper plate 42 and a lowerplate 44 with an
which may filter the beam such as to separate different wave internal volume 46 between the upper and lowerplates 42, 44.
lengths within the retrobeam, and direct the retrobeam toward The upper and lower plates 42, 44 may be made of any of a
at least one detector 36. 25 variety of materials but may preferably be made of a substrate
The detector 36 may be based upon any suitable technol material that is Substantially transparent at the wavelengths of
ogy, and may be, for example, a charged coupled device the excitation radiation and the fluoresced retrobeam, allow
(CCD) sensor that generates pixilated image databased upon ing for the passage of excitation radiation and returned fluo
photons impacting locations in the device. However, it will be rescent emissions without significant loss of signal quality.
understood that any of a variety of other detectors may also be 30 Moreover, when used in epifluorescent imaging arrange
used including, but not limited to, a detector array configured ments as shown, one of the Surfaces through which the radia
for time delay integration (TDI) operation, a complementary tion traverses may be substantially transparent at the relevant
metal oxide semiconductor (CMOS) detector, an avalanche wavelengths, while the other (which is not traversed by radia
photodiode (APD) detector, a Geiger-mode photon counter, tion) may be less transparent, translucent, or even opaque or
or any other suitable detector. TDI mode detection can be 35 reflective. The upper and lowerplates 42, 44 may both contain
coupled with line scanning as described in U.S. Pat. No. biological components 12, 14 on their respective, inwardly
7,329,860. facing surfaces 18, 20. As discussed above, the internal vol
The detector 36 may generate image data, for example, at a ume 46 may, for instance, include one or more internal pas
resolution between 0.1 and 50 microns, which is then for sages of a flow cell though which reagent fluids may flow.
warded to a control/processing system 38. In general, the 40 The support structure 16 may be irradiated by excitation
control/processing system 38 may perform various opera radiation 48 along a radiation line 50. The radiation line 50
tions, such as analog-to-digital conversion, Scaling, filtering, may be formed by the excitation radiation 48 from the radia
and association of the data in multiple frames to appropriately tion sources 22, 24, directed by the directing optics 30
and accurately image multiple sites at specific locations on a through the focusing optics 32. The radiation sources 22, 24
sample. The control/processing system 38 may store the 45 may generate beams that are processed and shaped to provide
image data and may ultimately forward the image data to a a linear cross section or radiation line including a plurality of
post-processing system (not shown) where the data are ana wavelengths of radiation used to cause fluorescence at corre
lyzed. Depending upon the types of sample, the reagents spondingly different wavelengths from the biological com
used, and the processing performed, a number of different ponents 12, 14, depending upon the particular dyes used. The
uses may be made of the image data. For example, nucleotide 50 focusing optics 32 may then semi-confocally direct the exci
sequence data can be derived from the image data, or the data tation radiation 48 toward the first surface 18 of the support
may be employed to determine the presence of a particular structure 16 to irradiate sites of biological component 12
gene, characterize one or more molecules at the component along the radiation line 50. In addition, the support structure
sites, and so forth. The operation of the various components 16, the directing optics 30, the focusing optics 32, or some
illustrated in FIG.1 may also be coordinated with the control/ 55 combination thereof, may be slowly translated such that the
processing system 38. In a practical application, the control/ resulting radiation line 50 progressively irradiates the com
processing system 38 may include hardware, firmware, and ponent as indicated by the arrow 52. This translation results in
Software designed to control operation of the radiation Successive scanning of regions 54 which allow for the gradual
Sources 22, 24, movement and focusing of the focusing optics irradiation of the entire first surface 18 of the support structure
32, a translation system 40, and the detection optics 34, and 60 16. As will be discussed in more detail below, the same
acquisition and processing of signals from the detector 36. process may also be used to gradually irradiate the second
The control/processing system 38 may thus store processed surface 20 of the support structure 16. Indeed, the process
data and further process the data for generating a recon may be used for multiple surfaces within the support structure
structed image of irradiated sites that fluoresce within the 16.
Support structure 16. The image data may be analyzed by the 65 Exemplary methods and apparatus for line Scanning are
system itself, or may be stored for analysis by other systems described in U.S. Pat. No. 7,329,860, which is incorporated
and at different times Subsequent to imaging. herein by reference, and which describes a line Scanning
 US 8,039,817 B2
10
apparatus having a detector array configured to achieve con beam. Alternatively or additionally, emission signals may be
focality in the scanning axis by restricting the scan-axis collected sequentially following sequential excitation at dif
dimension of the detector array. More specifically, the scan ferent wavelengths.
ning device can be configured Such that the detector array has In particular embodiments, an apparatus or method of the
rectangular dimensions such that the shorter dimension of the invention can detect features on a Surface at a rate of at least
detector is in the scan-axis dimension and imaging optics are about 0.01 mm/sec. Depending upon the particular applica
placed to direct a rectangular image of a sample region to the tion of the invention, faster rates can also be used including,
detector array Such that the shorter dimension of the image is for example, in terms of the area Scanned or otherwise
also in the scan-axis dimension. In this way, semi-confocality detected, a rate of at least about 0.02 mm/sec. 0.05mm/sec.
can be achieved since confocality occurs in a single axis (i.e. 10 0.1 mm/sec. 1 mm/sec. 1.5 mm/sec. 5 mm/sec. 10 mm/
the scan axis). Thus, detection is specific for features on the sec, 50 mm/sec. 100 mm/sec, or faster. If desired, for
Surface of a Substrate, thereby rejecting signals that may arise example, to reduce noise, the detection rate can have an upper
from the Solution around the feature. The apparatus and meth limit of about 0.05 mm/sec, 0.1 mm/sec. 1 mm/sec, 1.5
ods described in U.S. Pat. No. 7,329,860 can be modified such
mm/sec. 5 mm/sec. 10 mm/sec. 50 mm/sec, or 100 mm/
15 SCC.
that two or more surfaces of a Support are scanned in accor In some instances, the Support structure 16 may be used in
dance with the description herein. Such a way that biological components are expected to be
Detection apparatus and methods other than line Scanning present on only one surface. However, in many instances,
can also be used. For example, point Scanning can be used as biological material is present on multiple Surfaces within the
described below or in U.S. Pat. No. 5,646,411, which is support structure 16. For instance, FIG. 3 illustrates a typical
incorporated herein by reference. Wide angle area detection support structure 16 where biological material has attached to
can be used with or without Scanning motion. As set forth in the first surface 18 as well as to the second surface 20. In the
further detail elsewhere herein, TIR methods can also be illustrated embodiment, an attachment layer 56 has formed on
used. both the first surface 18 and the second surface 20 of the
As illustrated generally in FIG. 2, the radiation line 50 used 25 support structure 16. A first excitation radiation 58 source
to image the sites of biological components 12, 14, in accor may be used to irradiate one of many sites of biological
dance with the present invention, may be a continuous or component 12 on the first surface 18 of the support structure
discontinuous line. As such, Some embodiments of the 16 and return a first fluorescent emission 60 from the irradi
present invention may include a discontinuous line made up ated biological component 12. Simultaneously or sequen
of a plurality of confocally or semi-confocally directed beams 30 tially, a second source of excitation radiation 62 may be used
of radiation which nevertheless irradiate a plurality of points to irradiate one of many sites of biological component 14 on
along the radiation line 50. These discontinuous beams may the second surface 20 of the support structure 16, and return
be created by one or more sources that are positioned or a second fluorescent emission 64 from the irradiated biologi
cal component 14.
scanned to provide the excitation radiation 48. These beams, 35 Although the embodiment exemplified in FIG. 3 shows
as before, may be confocally or semi-confocally directed excitation from source 58 and source 62 coming from the
toward the first or second surfaces 18, 20 of the support same side of the support structure 16, it will be understood
structure 16 to irradiate sites of biological component 12, 14. that the optical system can be configured to impinge on the
As with the continuous semi-confocal line Scanning surfaces from opposite sides of the support structure 16. Tak
described above, the support structure 16, the directing optics 40 ing FIG.3 as an example, upper surface 18 can be irradiated
30, the focusing optics 32, or some combination thereof, may from excitation source 58 as shown and the lower surface 20
be advanced slowly as indicated by arrow 52 to irradiate can be irradiated from below. Similarly, emission can be
Successive scanned regions 54 along the first or second Sur detected from one or more sides of a Support structure. In
faces 18, 20 of the support structure 16, and thereby succes particular embodiments, different sides of the support struc
sive regions of the sites of biological components 12, 14. 45 ture 16 can be excited from the same radiation source by first
It should be noted that the system will typically form and irradiating one side and then flipping the Support structure to
direct excitation and returned radiation simultaneously for bring another side into position for excitation by the radiation
imaging. In some embodiments, confocal point Scanning may SOUC.
be used such that the optical system directs an excitation point The distribution of biological components 12, 14 may fol
or spot across a biological component by Scanning the exci 50 low many different patterns. For instance, FIG. 4 illustrates a
tation beam through an objective lens. The detection system support structure 16 where the biological components 12, 14
images the emission from the excited point on the detector at sites or features on the first and second surfaces 18, 20 are
without “descanning the retrobeam. This occurs since the distributed evenly in a spatially ordered pattern 66 of biologi
retrobeam is collected by the objective lens and is split off the cal component sites 68. For example, certain types of
excitation beam optical path before returning back through 55 microarrays may be used where the location of individual
the scan means. Therefore, the retrobeam will appear on the biological component sites 68 may be in a regular spatial
detector 36 at different points depending on the field angle of pattern. The pattern can include sites at pre-defined locations.
the original excitation spot in the objective lens. The image of In contrast, in other types of biological imaging arrays, bio
the excitation point, at the detector 36, will appear in the logical components attach to Surfaces at sites that occur in
shape of a line as the excitation point is scanned across the 60 random or statistically varying positions such that imaging
sample. This architecture is useful, for example, if the scan the microarray is used to determine the location of each of the
means cannot for Some reason accept the retrobeam from the individual biological component features. Thus, the pattern of
sample. Examples are holographic and acoustic optic scan features need not be pre-determined despite being the product
means that are able to Scan a beam at very high speeds but of a synthetic or manufacturing process.
utilize diffraction to create the scan. Therefore, the scan prop 65 For instance, FIG. 5 illustrates a support structure 16 where
erties are a function of wavelength. The retrobeam of emitted the sites on the first and second surfaces 18, 20 are located in
radiation is at a different wavelength from the excitation a random spatial distribution 70 of biological component sites
 US 8,039,817 B2
11 12
72. However, with both fixed arrays 66 and random distribu of the upper plate 42 plus 100 microns of the fluid within the
tion 70 of biological sample sites, imaging of multiple Sur internal volume 46 of the support structure 46.
faces 18, 20 of the support structure 16 may be possible. In In certain embodiments, the objective 92 may be designed
addition, it should be noted that in both instances, the bio for diffraction-limited focusing and imaging on only one of
logical components at the individual sites may constitute the first or second surfaces 18, 20 of the support structure 16.
either a population of identical molecules or a random mix of For example throughout the present description of FIGS. 7
different molecules. Furthermore, the density of biological through 14, the objective 92 may be designed for pre-com
samples may vary and may be at least 1,000 sites per square pensation of the 300 microns of the upper plate 42 plus the
millimeter. 100 micron read buffer of the fluid within the internal volume
The present techniques accommodate Such varied physical 10 46 of the support structure 16. In such a scenario, diffraction
arrangements of the multiple surfaces within the Support limited performance may only be possible on the second
structure 16, as well as the varied disposition of the sites surface 20. Furthermore, the spherical aberration introduced
by the 100 micron read buffer may severely impact the imag
within components on the Surfaces. As discussed above with ing quality when imaging from the first Surface 18. However,
reference to FIGS. 2 and 3, in the embodiments with a support 15 reducing the lane thickness of the internal volume 46 of the
structure 16 having a first surface 18 and a second surface 20, Support structure 16 might increase the amount of surface-to
a first source of excitation radiation 58 may irradiate sites of surface “crosstalk.” Therefore, perhaps the most appropriate
biological component 12 on the first surface 18, and return a Solution is to correct the aberration. As such, it may be nec
first fluorescent emission 60, while a second source of exci essary to use a compensator capable of achieving diffraction
tation radiation 62 may irradiate sites of biological compo limited imaging performance on both the first and second
nent 14 on the second surface 20 and return a second fluores surfaces 18, 20 of the support structure 16.
cent emission 64 source, as illustrated in FIG. 3. Thus, It should be noted that the need for a compensator may be
components of the Volume of sample between two Surfaces more pronounced when using objectives 92 with high
need not be detected and can be rejected. Selective detection numerical aperture (NA) values. Exemplary high NA ranges
of a Surface of a Support structure provides preferential detec 25 for which the invention is particularly useful include NA
tion of the surface compared to the volume of the support values of at least about 0.6. For example, the NA may be at
structure adjacent the Surface and compared to one or more least about 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or higher.
other Surfaces of the Support structure. Those skilled in the art will appreciate that NA, being depen
In more complex configurations, it may be useful to irra dent upon the index of refraction of the medium in which the
diate more than two surfaces. For instance, FIG. 6 illustrates 30 lens is working, may be higher including, for example, up to
a Support structure 16 having N number of plates including a 1.0 for air, 1.33 for pure water, or higher for other media such
first plate 42, a second plate 44, ..., an N-2 plate 74, an N-1 as oils. The compensator may also find use in objectives
plate 76, and an N plate 78. These plates define M number of having lower NA values than the examples listed above. In
Surfaces including a first Surface 18, a second Surface 20, ... general, the NA value of an objective 92 is a measure of the
, an M-3 surface 80, an M-2 surface 82, an M-1 surface 84, 35 breadth of angles for which the objective 92 may receive light.
and an M surface 86. In the illustrated embodiment, not only The higher the NA value, the more light that may be collected
the first surface 18 and the second surface 20 of the support by the objective 92 for a given fixed magnification. This is
structure 16 may be irradiated but, rather, all M number of because the collection efficiency and the resolution increase.
Surfaces may be irradiated. For instance, a source of excita As a result, multiple objects may be distinguished more
tion radiation 88 may be used to irradiate biological compo 40 readily when using objectives 92 with higher NA values
nent sites on the M" surface 86 of the support structure 16 and because a higher feature density may be possible. Therefore,
return a fluorescent emission 90 from the irradiated biological in general, a higher NA value for the objective 92 may be
component. For Support structures having a plurality of Sur beneficial for imaging. However, as the NA value increases,
faces it may be desirable to excite upper layers from the top its sensitivity to focusing and imaging-through media thick
and lower layers from the bottom to reduce photobleaching. 45 ness variation also increases. In other words, lower NA objec
Thus, components on layers that are closer to a first exterior tives 92 have longer depth of field and are generally not as
side of a support structure can be irradiated from the first side, sensitive to changes in imaging-through media thickness.
whereas irradiation from the opposite exterior side can be FIG. 8 is an exemplary chart 94 of spherical aberration (in
used to excite components present on layers that are closer to waves) vs. thickness (in microns) of the upper plate 42 of the
the opposite exterior side. 50 support structure 16 of FIG. 7 in accordance with the present
FIG. 7 illustrates an objective 92 through which radiation invention. Specifically, the upper line 96 of the graph depicts
from emissive biological components 12, 14 on first and the amount of spherical aberration of an image taken from
second surfaces 18, 20, respectively, of the support structure biological components 12 on the first surface 18 of the Sup
16 may be detected. The objective 92 may be one of the port structure 16 while the lower line 98 of the graph depicts
components of the focusing optics 32 described above. 55 the amount of spherical aberration of an image taken from
Although not drawn to scale, FIG. 7 illustrates exemplary biological components 14 on the second surface 20 of the
dimensions between the objective 92 and the support struc support structure 16. In the illustrated embodiment, the
ture 16. For instance, the objective 92 may typically be spaced spherical aberration generated by the 100 micron read buffer
approximately 600 or more microns from the upper plate 42 is around 4 waves, which is much higher than the diffraction
of the Support structure 16. The biological sample imaging 60 limited performance requirement of less than 0.25 waves, for
system 10 may be configured to detect emitted radiation from instance. As illustrated, at 300 microns (i.e. the thickness of
biological components 12 on the first surface 18 through 300 the upper plate 42), the spherical aberration for the first sur
microns of the upper plate 42 which may, for instance, be face 18 is around -13.2 waves (e.g., point 100) while the
made of glass and may have a refractive index N. of 1.472. In spherical aberration for the second surface 20 is around -17.2
addition, the biological sample imaging system 10 may also 65 waves (e.g., point 102). FIG.9A illustrates exemplary images
be configured to detect emitted radiation from biological expected for the first and second surfaces 18, 20 of the support
components 14 on the second surface 20 through 300 microns structure 16 corresponding to the thickness of the upperplate
 US 8,039,817 B2
13 14
42 (i.e., 300 microns) in accordance with the present inven from the first surface 18 of the support structure 16 under
tion, where the imaging system is optimized for the second similar conditions to that of its design point for the second
surface 20 (pre-compensated for-17.2 waves). As shown, the surface 20 of the support structure 16. Therefore, by detecting
imaging system is capable of providing high image quality on images for the second Surface 20 without the compensator
the second surface 20 since, according to the present scenario, 110 and detecting images for the first surface 18 with the
it was designed to do so. However, the images taken for the compensator 110, the objective 92 may be capable of detect
first surface 18 contain aberrations. ing images on both Surfaces with diffraction-limited perfor
To balance out the spherical aberration, it is beneficial to mance similar to the design of the objective 92.
introduce an additional thickness (e.g., by introducing an The chromatic shift curve may be limited to wavelength
additional coverslip) between the objective 92 and the Sup 10 ranges of between 530 nm to 780 nm. Chromatic shifts of
port structure 16. For instance, returning now to FIG. 8, if an different color wavelength bands may be compensated for by
additional thickness of approximately 40 microns were to be focusing the focusing optics 32 in each band. The compen
introduced between the objective 92 and the first and second sator 110 should preferably be “invisible' to the focusing
surfaces 18, 20 of support structure 16, the difference optics 32. In other words, the compensator 110 should correct
between the spherical aberrations at the design thickness (i.e., 15 the spherical aberration difference of the read buffer but
300 micron upper plate plus 100 microns offluid) may be split should maintain the chromatic shift curve in the wavelength
such that the image produced for both the first and second range of 530-780 nm. More specifically, the chromatic shift
Surfaces 18, 20 may have similar quality. For instance, as relationships among the peak wavelengths of 560 nm, 610
illustrated, at 340 microns (i.e. the thickness of the upper plate nm, 687 nm, and 720 nm should be maintained. In addition,
42 plus an additional 40 micron thickness), the spherical other specifications, including NA, field curvature, field dis
aberration for the first surface 18 is around -15.2 waves (e.g., tortion, detection magnification, and so forth, should also be
point 104) while the spherical aberration for the second sur maintained. Furthermore, the compensator 110 package
face 20 is around -19.2 waves (e.g., point 106), splitting the should be relatively small (e.g., no more than 10 mm of total
difference of -17.2 waves (e.g., point 108) which may be thickness). Moreover, insensitivity to positioning error of the
characterized as the design point for the objective 92. FIG.9B 25 compensator 110 may be preferred.
illustrates exemplary images expected for the first and second Several various designs may be implemented to introduce
surfaces 18, 20 of the support structure 16 corresponding to the corrective optics of the compensator 110 into the optical
the thickness of the upper plate 42 plus an additional thick train of the imaging optics of the biological sample imaging
ness (i.e., 300 microns plus 40 microns) in accordance with system 10. For example, FIG. 11 is an exemplary compensa
the present invention, illustrating how the additional thick 30 tor 110 design, incorporating a first objective 92 and a second
ness may allow for balance between images taken for the first objective 112 which may replace the first objective 92 in the
and second surfaces 18, 20 of the support structure 16. optical train in accordance with the present invention. In the
However, merely introducing an additional thickness illustrated embodiment, each respective objective 92, 112
between the objective 92 and the support structure 16 may not may contain the optics required to image respective Surfaces,
be desired for all uses of the imaging system set forth herein. 35 such as the first and second surfaces 18, 20 of the support
For instance, as illustrated in FIGS. 9A and 9B, by simply structure 16. For instance, the first objective 92 may contain
introducing the additional 40 micron thickness between the the imaging optics necessary to focus on and image emissive
objective 92 and the support structure 16, images from both biological components 14 on the second surface 20 of the
the first and second surfaces 18, 20 may still experience support structure 16 while the second objective 112 may
residual aberration from the design point 108 of the objective 40 contain the imaging optics plus the corrective optics neces
92. Therefore, a more precise solution may be to only intro sary to focus on and image emissive biological components
duce the additional thickness when detecting radiation from 12 on the first surface 18 of the support structure 16. In
biological components 12 on the first surface 18 of the Sup operation, the first objective 92 may detect images from the
port structure 16. In Such a scenario, the spherical aberration second surface 20 of the support structure 16. The first objec
corresponding to the design point 108 of the objective 92 may 45 tive 92 may be replaced by the second objective 112 in the
generally be achieved for both the first and second surfaces optical train, at which point the second objective 112 may
18, 20. It should be noted that the particular dimensions and detect images from the first surface 18 of the support structure
measurements (e.g., thicknesses, spherical aberration values, 16. An advantage of the embodiment illustrated in FIG. 11 is
and so forth) described with respect to FIGS. 9A and 9B are that the optics may be decoupled and may operate indepen
merely intended to be exemplary of the manner in which the 50 dently. However, a disadvantage in Some situations is that
present invention functions. As such, these dimensions and having two entirely separate objectives 92, 112 may not be
measurements are not intended to be limiting. Indeed, the cost-effective since certain components may be duplicated
particular geometries and resulting measurement values may for each objective 92, 112. Furthermore, in embodiments
vary between implementations. where multiple images of an object are obtained, the use of
For example, FIG. 10A illustrates an exemplary objective 55 two objectives may increase the computational resources
92 imaging the second surface 20 of the support structure 16 required for registration between images. In particular
without the assistance of a compensator 110 in accordance embodiments, imaging of both Surfaces may occur through
with the present invention. Without the compensator 110, the the same objective to provide particular advantages as set
objective 92 may focus and detect images from the second forth below. In other words, the first objective 92 need not be
Surface 20 of the Support structure 16 according to its design 60 removed or replaced with the second objective 112 for imag
and experiencing the design spherical aberration. However, ing of the different surfaces.
FIG. 10B illustrates an exemplary objective 92 imaging the FIG. 12 is another exemplary compensator 110 design,
first surface 20 of the support structure 16 with the assistance incorporating a corrective device 114 which may be inserted
of a compensator 110 in accordance with the present inven between the objective 92 and the support structure 16 in
tion. By using the compensator 110 (e.g., similar to the addi 65 accordance with the present invention. The corrective device
tional 40 micron thickness described above with respect to 114 may, for instance, be a coverslip or other thin layer of
FIGS. 8 and 9), the objective 92 may focus and detect images glass. As illustrated, the corrective device 114 may simply be
 US 8,039,817 B2
15 16
inserted into and removed from the optical path between the be inserted into and extracted from the fluidic corrector
objective 92 and the support structure 16 depending on the depending on which Surface is imaged.
particular Surface 16 being imaged. For instance, the correc Regardless of the particularembodiment selected, all of the
tive device 114 may be removed from the optical path when embodiments disclosed herein are characterized by repeat
the objective 92 is used to focus on and image emissive ability and the ability to automate the use of the embodiments.
biological components 14 on the second surface 20 of the These are important considerations in that the embodiments
support structure 16. Conversely, the corrective device 114 allow for the detection of images from biological components
may be inserted into the optical path when the objective 92 is 12, 14 on multiple surfaces 18, 20 of the support structure 16
used to focus on and image emissive biological components in an automated fashion. This may allow not only for
12 on the first surface 18 of the support structure 16. An 10 increased imaging production but may also allow for greater
advantage of the embodiment illustrated in FIG. 12 is that it is flexibility in Switching between the multiple surfaces,
relatively straightforward. The required additional compen depending on the particular imaging needs.
sator thickness may simply be inserted into the optical path. As described in greater detail above, a support structure 16
Typically, the corrective device 114 may be placed such that useful in the apparatus or methods set forth herein can have
it does not physically contact the support structure 16 or the 15 two or more surfaces upon which a biological component is
objective 92. attached. In particular embodiments, the Surface is a fabri
FIG. 13 is another exemplary compensator 110 design, cated Surface. Any of a variety of surfaces known in the art can
incorporating a correction collar 116 in accordance with the be used including, but not limited to, those used for making
present invention. In the illustrated embodiment, the correc arrays as set forth above. Examples include, glass, silicon,
tion collar 116 may be adjusted between binary states. For polymeric structures, plastics, and the like. Surfaces and flow
instance, the first state 118 may correspond to the situation cells that are particularly useful are described in PCT Publi
where the objective 92 is focused on and detecting images cation No. WO 2007/123744, which is incorporated herein by
from the second surface 20 of the support structure 16 while reference. The Surfaces of a Support structure can have the
the second state 120 may correspond to the situation where same or different properties. For example, in the embodiment
the objective 92 is focused on and detecting images from the 25 shown in FIG. 3, plate 42 can be transparent to the excitation
first surface of the support structure 16. As such, when the and emission wavelengths used in a detection method,
correction collar 116 is in the first state 118, the imaging whereas plate 44 can optionally be transparent or opaque to
optics within the objective 92 may not include the corrective the excitation or emission wavelengths. Accordingly, the Sur
optics within the optical path. Conversely, when the correc faces can be made of the same material or the two or more
tion collar 116 is in the second state 120, the imaging optics 30 surfaces can be made of different materials.
within the objective 92 may include the corrective optics A Support structure having two or more Surfaces can be
within the optical path. Although illustrated as consisting of formed by adhering the surfaces to each other or to other
binary states 118, 120, the correction collar 116 may, in fact, Supports. For example, an adhesive material. Such as epoxy
include multiple states. For instance, when more than two resin, can be dispensed in the form of a paste onto a planar
Surfaces of the Support structure 16 are used for imaging, the 35 Substrate in a pattern forming one or more channel character
correction collar 116 may be configured to adjust between istics of a flow cell. An exemplary flow cell 124 is shown in
multiple states such that the imaging and corrective optics FIG. 15. Utilizing a programmable, automated adhesive dis
vary for each respective surface of the support structure 16. penser, such as the Millennium(R) M-2010 from Asymtek
An advantage of the embodiment illustrated in FIG. 13 is that Corp., Carlsbad Calif., a desired pattern of adhesive 126 can
it may be relatively easy to operate. For instance, the correc 40 be designed and laid down onto the Surface of a planar lower
tion collar 116 may simply be adjusted between states when substrate 128. The thickness of the flow cell (and cross sec
ever different surfaces of the support structure 16 are being tional height in the fluidic channels) can be set by means of
imaged. precision mechanical spacers 130 placed between the lower
FIG. 14 is another exemplary compensator 110 design, substrate 128 and an upper substrate 132. Another exemplary
incorporating an infinite space compensator 122 in accor 45 flow cell 134 is shown in FIG. 16. To create a multi-layer cell,
dance with the present invention. This embodiment is some an interim transparent substrate layer 136, shorter in length
what similar to the corrective device 114 embodiment of FIG. than the lower and upper substrate layers 128, 132 can be
12 in that the infinite space compensator 122 may be inserted included. The shorter length allows fluidic access to both/all
into and removed from the optical path. However, a main layers from ports 138 passing through only one Substrate.
difference between the embodiments is that, in the embodi 50 This intermediate layer 136 bifurcates the flow cell cavity
ment of FIG. 14, there may be more space available (e.g., up horizontally and nearly doubles the available surface area for
to 10 mm, as opposed to 600 microns in the embodiment of the attachment of biologically interesting molecules.
FIG. 12) within which to insert the infinite space compensator An exemplary method 140 for fabricating such a flow cell
122 into the optical path. Therefore, the embodiment of FIG. is shown in FIG. 17. A planar substrate acting as the structural
14 may allow for greater flexibility than the corrective device 55 base of the cell is provided (block 142). Desired canalizing
114 embodiment of FIG. 12. features of the cell are designed, for example, using a com
In addition to the embodiments presented in FIGS. 11 puter assisted design program (block 144). A pattern designed
through 14, there may be other compensator 110 designs in this way can be exported to a file compatible with driving
which may prove beneficial. For instance, a fluidic corrector an automatic adhesive dispensing system (block 146). A pro
may be inserted between the objective 92 and the support 60 gram can be executed to dispense the adhesive in the desired
structure 16. In this fluidic corrector design, the fluidic cor pattern onto the substrate (block 148). Precision mechanical
rector may be filled with a fluid, which may effectively act as spacers can be placed onto the base substrate before or after
the compensator 110. The optics may be configured such that the adhesive is dispensed (block 150). A second transparent
the fluid matches the upper surface of the support structure 16 Substrate can then be placed onto the adhesive pattern, press
and, in the absence of fluid air, matches the bottom surface of 65 ing downward until the lower surface is in full contact with
Support structure 16. This design may prove beneficial in that the mechanical spacers (block 152). A weight or other force is
it may make automation easier since the fluid would simply applied to the top substrate to hold it in full contact with the
 US 8,039,817 B2
17 18
adhesive. The spacers will typically have a height that is combination thereof, in order to repeat these steps of scanning
equivalent or slightly less than the height of the adhesive layer individual lines. Alternatively, entire regions of the first sur
Such that bonding can occur without causing undesirable face 18 may be scanned before regions of the second surface
aberrations in the shape of the canalized features. The steps 20 are scanned. The individual processing steps taken may
for adhering substrates may be repeated for any number of 5 depend upon several variables including the particular con
layers desired. Optionally, the assembly can be heat treated, figuration of the biological component sites 12, 14 on the
for example, in an oven or exposed to UV light, depending surfaces 18, 20 as well as other variables, including environ
upon the cure requirements of the adhesive (block 154). mental and operating conditions.
Another exemplary method for fabricating a flow cell is to Particular embodiments may allow for simultaneous exci
use an intermediate layer that is cut to a desired pattern in 10 tation of multiple surfaces of the support structure 16. For
place of an adhesive layer. A particularly useful material for instance,
the intermediate layer is silicone. The silicone layer can be radiation FIG. 20 illustrates an embodiment utilizing dual
heat bonded to the lower substrate 128 and upper substrate embodiment, the firstandsurface
sources dual detectors. However, in this
18 and the second surface 20 of
132. Exemplary methods utilizing Bisco Silicone HT 6135 as
an intermediate layer are described, for example, in Groveret 15 the Support structure 16 may be simultaneously scanned. This
al., Sensors and Actuators B 89:315-323 (2003). may be accomplished using focusing lenses 164, 166, 168,
Still further, FIG. 18 illustrates an embodiment utilizing 170 and a dichroic mirror 172 along the excitation path in
one radiation source and dual detectors. Radiation from the order to switch surfaces and filters 158, 160 to achieve mul
radiation source 22 is directed by the directing optics 30 tiple color channels. Again, this illustrated embodiment may
toward the focusing optics 32. From the focusing optics 32. also be extended to any number of detectors to improve
the excitation radiation 58 irradiates a biological component throughput, Scanning efficiency, and to reduce movement of
12 on a first surface 18 of the support structure 16. The the filters and other system components.
biological component 12 emits a fluorescent emission 60 FIG. 21 illustrates another embodiment utilizing dual
back through the focusing optics 32 toward the directing radiation sources and dual detectors which allows for simul
optics 30. This retrobeam is allowed to pass through the 25 taneous scanning of the first and second surfaces 18, 20 of the
directing optics 30 to the detection optics 34 which, in this support structure 16. In this illustrated embodiment, however,
illustrated embodiment, may include a wavelength filter 156 not only are focusing lenses 164, 166, 168,170 and a dichroic
or some other device for separating the retrobeam, and first mirror 172 used in the excitation path but focusinglenses 174,
and second color filters 158, 160 for achieving multiple color 176 may be used just upstream of the first and second detec
channels. The wavelength filter 156 may split the retrobeam 30 tors 36, 162 in conjunction with the filters 158, 160 along the
into two beams with one beam directed toward the first detec emission pathin order to Switch Surfaces and achieve multiple
tor 36 via the first color filter 158 and the other beam directed color channels. Once again, this illustrated embodiment may
toward a second detector 162 via the second color filter 160. also be extended to use any number of detectors to increase
In this manner, the biological sample imaging system 10 may throughput and Scanning efficiency.
sequentially scan the first and second surfaces 18, 20, first 35 For instance, FIG. 22 illustrates an embodiment utilizing
scanning the first Surface 18 of the Support structure 16 using multiple radiation Sources and multiple detectors which are
the first excitation radiation 58 from the radiation source 22 capable of simultaneously outputting multiple channels with
and the returned first fluorescent emission 60 (as depicted in few moving parts. In the illustrated embodiment, radiation
the left portion of FIG. 18), and next scanning the second sources 22 and 24 have been replaced by radiation source
surface 20 of the support structure 16 using the second exci 40 groups 178 and 180 which are capable of outputting multiple
tation radiation 62 from the same radiation source 22 and the radiation sources and varying wavelengths. In addition,
returned second fluorescent emission 64 (as depicted in the detectors 36 and 38 have been replaced by detector groups
right portion of FIG. 18). 182 and 184 in the illustrated embodiment. These detector
Alternatively, FIG. 19 illustrates an embodiment utilizing groups 182, 184 are similarly capable of detecting multiple
dual radiation sources and dual detectors. Again, the two 45 color channels. This embodiment therefore illustrates the
surfaces 18, 20 of the support structure 16 may be scanned considerable adaptability of the present techniques to a range
sequentially. However, in this embodiment, the first surface of configurations capable of imaging components on multiple
18 of the support structure 16 is first scanned using the first Surfaces of the Support.
radiation source 22 which generates the first excitation radia In the embodiments described above where scanning of the
tion58 and the first fluorescent emission 60 (as depicted in the 50 first and second surfaces 18, 20 of the support structure 16
left portion of FIG. 19) and, the second surface 20 of the may be performed simultaneously, focusing of the excitation
Support structure 16 is scanned using the second radiation radiation 58 source may be accomplished in several various
Source 24 which generates the second excitation radiation 62 ways. For instance, it may be possible to focus the excitation
and the second fluorescent emission 64 (as depicted in the radiation 58 on one of the surfaces preferentially over the
right portion of FIG. 19). This embodiment may also be 55 other surface. In fact, due to the nature of the configuration of
extended to use any number of detectors in order to reduce the first surface 18 with respect to the second surface 20, it
movement of the filters. may be necessary to do so. However, alternate focusing tech
In the embodiments described above where scanning of the niques may be employed depending on the specific configu
first and second surfaces 18, 20 of the support structure 16 ration of the support structure 16. Moreover, it may be advan
may be performed sequentially, the individual steps of scan 60 tageous in these various configurations to first image the
ning the first and second surfaces 18, 20 of the support struc upper Surface (i.e., the Surface closer to the radiation source)
ture 16 may be performed in a number of ways. For instance, in order to reduce photobleaching of the components on that
it may be possible to scan a single line of the first surface 18, Surface that could result from first imaging the lower Surface
then scan a single line of the second Surface 20, then gradually (i.e., the surface farther from the radiation source). Such
move the first and second surfaces 18, 20 relative to the 65 selection of which Surface to image may apply both when the
excitation radiation 58, 62 by translating the support structure Surfaces are imaged sequentially as well as when they are
16, the directing optics 30, the focusing optics 32, or some imaged simultaneously.
 US 8,039,817 B2
19 20
In addition, the embodiments disclosed above have illus structure 188 before and after imaging. In addition, a control/
trated an epifluorescent imaging scheme wherein the excita processing system 212 may be used to control operation of the
tion radiation is directed toward the surfaces of the support radiation source 194, the focusing light source 206, and the
structure 16 from a top side, and returned fluorescent radia heating/cooling station 210, movement and focusing of the
tion is received from the same side. However, the techniques focusing optics 198, the translation system 208, and the
of the present invention may also be extended to alternate detection optics 202, and acquisition and processing of sig
arrangements. For instance, these techniques may also be nals from the detector 204.
employed in conjunction with TIR imaging whereby the Sur As discussed above, the TIR method of imaging may be
faces of the support structure are irradiated from a lateral side used to direct the radiation beam 196 from a lateral side of the
with radiation directed at an incident angle within a range of 10
support structure 188, as illustrated in FIG. 24. Each flow lane
critical angles so as to convey the excitation radiation within 190 of the support structure 188 may include a bottom surface
the Support or into the Support from a prism positioned adja
cent to it. TIR techniques can be carried out as described, for 214 and a top surface 216 and emissive components can
example, in U.S. Patent Application Publication No. 2005/ optionally be attached to either or both surface. In the illus
0057798, which is hereby incorporated by reference. Such 15 trated embodiment, the radiation beam 196 is directed toward
techniques cause fluorescent emissions from the components a bottom surface 214 of one of the flow lanes 190 of the
that are conveyed outwardly for imaging, while the reflected support structure 188. Part of the radiation beam 196 may be
excitation radiation exits via aside opposite from that through reflected off the bottom surface 214 of the flow lane 190, as
which it entered. Here again, biological components on the depicted by reflected light beam 218. However, as long as the
multiple Surfaces may be imaged sequentially or simulta incidentangle of the radiation beam 196 is within the range of
neously. critical angles, a separate fluorescent emission beam 220 may
For example, in FIG. 23, a TIR biological sample imaging be emitted from emissive components toward the focusing
system 186 is illustrated diagrammatically. A Support struc optics 198 which in the illustrated embodiment is a lens
ture 188 may be used which includes multiple flow lanes 190 objective 222. Indeed, directing the radiation beam 196 at a
containing biological components. For example, the Support 25 bottom surface 214 of a flow lane 190 of the support structure
structure 188 may be a flow cell through which reagents, 188 is a typical implementation of the TIR imaging method.
flushes, and other fluids may be introduced using the flow However, in doing so, imaging data which may be collected
lanes 190 to contact emissive components attached to the from a top surface 216 of a flow lane 190 of the support
surface of the flow cell. The support structure 188 may be structure 188 may be overlooked.
supported by a prism 192. In the TIR biological sample imag 30 Therefore, the orientation of the radiation source 194 and/
ing system 186, the radiation source 194 may output a radia or the support structure 188 and prism 192 may be adjusted in
tion beam 196 through the prism 192 from a lateral side of the order to allow the radiation beam 196 to not be directed at a
support structure 188. The radiation beam 196 may, for bottom surface 214 of a flow lane 190 of the support structure
instance, be directed toward a bottom surface of one of the 188, as illustrated in FIG. 25. In the illustrated embodiment,
flow lanes 190 of the support structure 188, thereby exciting 35 the radiation beam 196 is oriented so that the radiation beam
emissive components attached to the Surface. 196 passes through the prism 192 and support structure 188
As discussed in further detail below, as long as the incident until contacting an air/glass interface 224 of the Support struc
angle of the radiation beam 196 is within the range of critical ture 188 at which point the radiation beam 196 is redirected
angles (as described, for example, in US 2005/0057798), a toward a top surface 216 of a flow lane 190 of the support
portion of the radiation beam 196 will be reflected off the 40 structure 188. At this point, part of the radiation beam 196
bottom Surface whereas a separate fluorescent emission beam may be reflected back toward another air/glass interface 224
from surface-bound emissive components will be directed of the support structure 188. However, a separate fluorescent
toward focusing optics 198. Typically, a well collimated emission beam 220 may be emitted from an emissive com
radiation beam is used to prevent spread of angles within the ponent on the top surface 216 toward the lens objective 222.
beam, thereby preventing unwanted hindrance of total inter 45 Using this technique, top surfaces 216 of the flow lanes 190 of
nal reflectance. The fluorescent emission beam may propa the Support structure 188 may be imaged using TIR imaging
gate back through the focusing optics 198, directing optics methods. This, in effect, may allow for double the imaging
200, and detection optics 202 which may direct the beam data output for cluster based sequencing applications while
toward a detector 204. The focusing optics 198, directing keeping other variables, such as Surface coating, cluster cre
optics 200, detection optics 202, and detector 204 may oper 50 ation, and sequencing, the same.
ate in much the same manner as with the epifluorescent tech In order to accomplish this TIR imaging oftop surfaces 216
niques discussed above. In the TIR biological sample imag of the flow lanes 190 of the support structure 188, the radia
ing system 186, the focusing light source 206 may be used as tion beam 196 reaches the air/glass interface 224 of the Sup
a separate light source from the radiation source 194 to focus port structure 188 unperturbed. To do so, the radiation beam
the optics on a particular surface to be imaged. For instance, 55 196 does not first come into contact with emissive compo
the focusing light source 206 may be directed to the directing nents in adjacent flow lanes 190. To do so, either the radiation
optics 200 where it is redirected toward the focusing optics beam 196 may be directed around the adjacent flow lanes 190
198which are used to focus the system on a particular surface or the adjacent flow lanes 190 may be index matched with the
of the support structure 188. support structure 188 material. In some embodiments, the
The TIR biological sample imaging system 186 may also 60 flow lanes 190 may be spaced within the support structure
include a translation system 208 for moving the Support struc 188, leaving sufficient room between the flow lanes 190 for
ture 188 and prism 192 in one or more dimensions. The the radiation beam 196 to pass. However, spacing the flow
translation system 208 may be used with focusing, redirecting lanes 190 in this manner may ultimately reduce the amount of
the radiation source 194 to different areas of the support emissive components which may be imaged. Therefore, in
structure 188, as well as for moving the support structure 188 65 other embodiments, it may be possible to accomplish the
and prism 192 to a heating/cooling station 210. The heating/ same effect by temporarily filling alternate flow lanes 190
cooling station 210 may be used to heat and cool the Support with index matching fluid. Doing so may allow for easier
 US 8,039,817 B2
21 22
direction of the radiation beam 196 toward a top surface 216 2007/0166705; and PCT Publication Nos. WO 05/065814,
of a flow lane 190 of the support structure 188. WO 06/064199, and WO 07/010251; each of which is incor
It may also be possible to direct the radiation beam 196 in porated herein by reference.
such a way that it bounces off multiple top surfaces 216 of In particular uses of the apparatus and methods herein, flow
flow lanes 190 of the support structure 188, as illustrated in 5 cells containing arrayed nucleic acids are treated by several
FIG. 26. In order to accomplish this, the spacing of the flow repeated cycles of an overall sequencing process. The nucleic
lanes 190 can be matched with the angle of radiation beam acids are prepared Such that they include an oligonucleotide
196 such that the radiation beam 196 is able to pass by the primer adjacent to an unknown target sequence. To initiate the
flow lanes 190, such that it reaches the air/glass interface 224 first SBS sequencing cycle, one or more differently labeled
of the support structure 188 unperturbed, while also being 10 nucleotides and a DNA polymerase are flowed into the flow
able to bounce back and forth between top surfaces 216 of cell. Either a single nucleotide can be added at a time, or the
flow lanes 190 and the air/glass interface 224 of the support nucleotides used in the sequencing procedure can be specially
designed to possess a reversible termination property, thus
structure 188. As described above, in certain embodiments, allowing each cycle of the sequencing reaction to occur
some of the flow lanes 190 may be filled with an index 15 simultaneously in the presence of all four labeled nucleotides
matching fluid, such that these index-matched flow lanes 190 (A, C, T. G). Following nucleotide addition, the features on
effectively become “invisible” to the radiation beam 196. In the surface can be imaged to determine the identity of the
other words, the radiation beam 196 may be allowed to pass incorporated nucleotide (based on the labels on the nucle
through the index-matched flow lanes 190. By allowing the otides). Then, reagents can be added to the flow cell to remove
radiation beam 196 to pass through the index-matched flow the blocked 3' terminus (if appropriate) and to remove labels
lanes 190, the support structure 188 may be used in multiple from each incorporated base. Such cycles are then repeated
configurations without the need of varying the spacing of the and the sequence of each cluster is read over the multiple
flow lanes 190. chemistry cycles.
In some embodiments, mirrors 226 or other suitable reflec Other sequencing methods that use cyclic reactions
tive material may be used within certain flow lanes 190, 25 wherein each cycle includes steps of delivering one or more
facilitating this multi-bounce technique. In any event, assum reagents to nucleic acids on a Surface and imaging the Surface
ing N number of flow lanes 190, it may only be possible to bound nucleic acids can also be used Such as pyrosequencing
image N-2 number of top surfaces 216 of the flow lanes 190 and sequencing by ligation. Useful pyrosequencing reactions
in this manner due to the fact that the outer flow lanes 190 on are described, for example, in U.S. Pat. No. 7,244.559 and
either side of the support structure 188 may not be accessible 30 U.S. Patent Application Publication No. 2005/0191698, each
using these techniques. However, modification of the prism of which is incorporated herein by reference. Sequencing by
192 and/or support structure 188 may allow for imaging of the ligation reactions are described, for example, in Shendure et
top surfaces 216 of these outermost flow lanes 190. For al. Science 309: 1728-1732 (2005); and U.S. Pat. Nos. 5,599,
instance, the support structure 188 may be designed to fit 675 and 5,750,341, each of which is incorporated herein by
within the prism 192, allowing the radiation beam 196 to 35 reference.
propagate into a lateral side of the support structure 188. The methods and apparatus described herein are also use
In some embodiments, as discussed above briefly with ful for detection of features occurring on Surfaces used in
respect to FIG. 23, the support structure 188 may be moved to genotyping assays, expression analyses and other assays
a heating/cooling station 210, for example, by the action of known in the art such as those described in U.S. Patent Appli
the translation system 208. The heating/cooling station 210 40 cation Publication Nos. 2003/0108900, US 2003/0215821,
may be configured to both heat and cool the Support structure and US 2005/0181394, each of which is incorporated herein
188 before and after imaging. The heating/cooling station 210 by reference.
may, in fact, be configured to heat and cool both a top Surface While only certain features of the invention have been
228 and a bottom surface 230 of the support structure 188, as illustrated and described herein, many modifications and
illustrated in FIG. 27. Indeed, all surfaces of the support 45 changes will occur to those skilled in the art. It is, therefore, to
structure 188 may be heated or cooled at the heating/cooling be understood that the appended claims are intended to cover
station 210. In this manner, it may further be possible to heat all Such modifications and changes as fall within the true spirit
and cool both the top surfaces 216 and bottom surfaces 214 of of the invention.
the flow lanes 190 of the support structure 188 by directly
contacting one or more Surfaces of the flow cell with a heating 50 The invention claimed is:
or cooling device. This, of course, may facilitate the devel 1. A method for imaging a biological sample, comprising:
opment of biological components within the flow lanes 190 of (a) detecting radiation emitted from a first emissive com
the Support structure 188 and, therefore, facilitate imaging. ponent of a biological sample disposed on a first Surface
Although use of the heating/cooling station 210 has been of a flow cell using a detector, wherein the flow cell is
presented herein with respect to the TIR imaging methods, 55 mounted on an imaging station;
the heating/cooling station 210 may also be used to heat and (b) inserting corrective optics between the detector and the
cool multiple sides of a Support structure used in conjunction flow cell;
with the epifluorescent imaging methods discussed herein. (c) detecting radiation emitted from a second emissive
In particular embodiments, the current invention utilizes component of a biological sample disposed on a second
sequencing-by-synthesis (SBS). In SBS, four fluorescently 60 surface of the flow cell using the detector and the cor
labeled modified nucleotides are used to determine the rective optics, wherein the first and second surfaces are
sequence of nucleotides for nucleic acids present on the Sur in an arrangement whereby one of the Surfaces is dis
face of a support structure such as a flow cell. Exemplary SBS posed between the detector and the other surface,
systems and methods which can be utilized with the apparatus wherein the corrective optics reduce aberration of detec
and methods set forth herein are described in U.S. Pat. No. 65 tion at one of the Surfaces due to the arrangement; and
7.057,026: U.S. Patent Application Publication Nos. 2005/ (d) repeating steps (a)-(c) while maintaining the flow cell
0100900, 2006/0188901, 2006/0240439, 2006/0281 109, and on the imaging station.
 US 8,039,817 B2
23 24
2. The method of claim 1, wherein the first surface is 19. The method of claim 1, wherein a field distortion for
disposed between the detector and the second surface. detection in step (a) is the same as a field distortion for
3. The method of claim 2, wherein a fluid is present detection in step (b).
between the two surfaces and the corrective optics reduce 20. An imaging system for detecting radiation on a multi
aberration due to the fluid. 5 Surface flow cell, comprising:
4. The method of claim 3, wherein the detector is initially a multi-surface flow cell having first and second emissive
configured for diffraction-limited detection on the second components of a biological sample disposed on respec
surface and the corrective optics are configured for diffrac tive first and second surfaces of the flow cell;
tion-limited detection on the first surface. an optical train comprising an objective, imaging optics
5. The method of claim3, wherein the aberration comprises 10 configured to focus the optical train on the first emissive
spherical aberration. component via the objective, and corrective optics con
6. The method of claim 1, wherein the first surface is figured to focus the optical train on the second emissive
disposed between a source of radiation and the second Sur component and configured to reduce aberration of detec
face.
7. The method of claim 1, wherein step (b) comprises 15 tion at the first or second emissive component;
inserting a corrective device between an objective and the a radiation Source configured to direct excitation radiation
flow cell, wherein the objective is disposed between the towards the first and second emissive components;
detector and the flow cell. detection optics configured to capture emitted radiation
8. The method of claim 1, wherein step (b) comprises returned from the first and second emissive components
inserting a corrective lens between the detector and an objec- 20 via the optical train; and
tive, wherein the objective is disposed between the detector a detector for detecting the captured radiation.
and the flow cell. 21. The imaging system of claim 20, wherein components
9. The method of claim 1, wherein step (a) and (c) comprise of the corrective optics that are configured to focus the optical
obtaining an image of the Surfaces. train on the second emissive component are separate from
10. The method of claim 1, wherein steps (a) and (c) com- 25 components of the corrective optics that are configured to
prise focusing a line of radiation to an area on the first or reduce aberration of detection at the first or second emissive
second Surface comprising the first or second emissive com component.
ponent. 22. The imaging system of claim 20, wherein the corrective
11. The method of claim 1, wherein the emitted radiation is optics comprise a corrective device configured to be inserted
detected using epifluorescent excitation. 30 between the objective and the flow cell.
12. The method of claim 1, wherein an excitation radiation 23. The imaging system of claim 20, wherein the corrective
is directed towards the first and second surfaces by total optics comprise a corrective lens configured to be inserted
internal reflection excitation. between the detector and the objective.
13. The method of claim 12, wherein the excitation radia 24. The imaging system of claim 20, wherein the radiation
tion and flow cell are configured to produce a total internal 35 Source is configured to direct the excitation radiation toward
reflectance bounce that excites the first surface and the second the first and second emissive components at several different
Surface. wavelengths, and the detection optics and detector are con
14. The method of claim 1, wherein the first and second figured to capture and detect the emitted radiation returned in
emissive components comprise fluorescently tagged nucleic response to each wavelength.
acids of a nucleic acid sample. 40 25. The imaging system of claim 20, wherein the flow cell
15. The method of claim 1, wherein the first and second includes a fluid in an interior volume and in contact with the
emissive components comprise separate features in arrays of biological samples.
features on the first and second Surfaces, respectively. 26. The imaging system of claim 20, wherein the first and
16. The method of claim 15, wherein the separate features second emissive components comprise fluorescently tagged
each comprise a population of identical molecules. 45 nucleic acids from a nucleic acid sample.
17. The method of claim 1, whereinachromatic shift curve 27. The imaging system of claim 20, wherein the first
for the radiation detected in step (a) is the same as a chromatic Surface is disposed between the radiation source and the
shift curve for the radiation detected in step (b). second Surface.
18. The method of claim 1, wherein a field curvature for
detection in step (a) is the same as a field curvature for 50
detection in step (b).