U.S. patent application number 12/638770 was filed with the patent office on 2010-06-24 for dynamic autofocus method and system for assay imager.
This patent application is currently assigned to ILLUMINA, INC. Invention is credited to JOHN A. MOON, HONGJI REN, DARREN R. SEGALE.
Application Number | 20100157086 12/638770 |
Document ID | / |
Family ID | 42265470 |
Filed Date | 2010-06-24 |
United States Patent
Application |
20100157086 |
Kind Code |
A1 |
SEGALE; DARREN R. ; et
al. |
June 24, 2010 |
DYNAMIC AUTOFOCUS METHOD AND SYSTEM FOR ASSAY IMAGER
Abstract
A method and system are provided for controlling focus
dynamically of a sample imager. The method comprises scanning a
sample with an optical assembly that apportions the sample into
regions based on a scan pattern. The optical assembly has a focal
setting with respect to the sample. The method further comprises
shifting the focal setting of the optical assembly during scanning
of the sample, and detecting one or more images representative of
one of the regions from the sample. The one or more images have
associated degrees of focus corresponding to the focal setting of
the optical assembly. The method analyzes the image(s) to obtain a
focus score or scores corresponding thereto, where the focus scores
represent a degree to which the optical assembly was in focus when
detecting the images. The method adjusts the focus setting based on
the focus score(s).
Inventors: |
SEGALE; DARREN R.; (SAN
DIEGO, CA) ; MOON; JOHN A.; (SAN DIEGO, CA) ;
REN; HONGJI; (SAN DIEGO, CA) |
Correspondence
Address: |
THE SMALL PATENT LAW GROUP LLP
225 S. MERAMEC, STE. 725T
ST. LOUIS
MO
63105
US
|
Assignee: |
ILLUMINA, INC
SAN DIEGO
CA
|
Family ID: |
42265470 |
Appl. No.: |
12/638770 |
Filed: |
December 15, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61122550 |
Dec 15, 2008 |
|
|
|
Current U.S.
Class: |
348/222.1 ;
348/E5.024; 382/255 |
Current CPC
Class: |
H04N 5/232122 20180801;
G01N 2015/1452 20130101; G01N 21/6458 20130101; G02B 26/10
20130101; H04N 5/23212 20130101; G01B 11/24 20130101; G02B 21/244
20130101; C12Q 1/6818 20130101; G01N 21/6428 20130101; C12Q 1/6874
20130101; G02B 26/127 20130101; G01B 9/02063 20130101 |
Class at
Publication: |
348/222.1 ;
382/255; 348/E05.024 |
International
Class: |
H04N 5/225 20060101
H04N005/225; G06K 9/40 20060101 G06K009/40 |
Claims
1. A method for controlling focus dynamically of a sample imager,
comprising: scanning a sample with an optical assembly by
apportioning the sample into a plurality of regions defined by a
scan pattern, the optical assembly having a focal setting with
respect to the sample, thereby obtaining images of the plurality of
regions; shifting the focal setting of the optical assembly during
scanning of the sample, whereby the images have an associated
degree of focus corresponding to the focal setting of the optical
assembly; analyzing the images to obtain at least two focus scores,
the focus scores representing a degree to which the optical
assembly was in focus when detecting the images; and adjusting the
focus setting based on a function of the at least two focus
scores.
2. The method of claim 1, wherein the shifting operation introduces
an error signal into a focal position of the optical assembly, the
error signal being monitored as a function of the focal position of
the optical assembly, the adjusting operation reducing the error
signal by adjusting the focal position.
3. The method of claim 1, wherein the regions of the sample are
non-overlapping such that the detecting operation detects a series
of adjacent images that are separate and distinct from one
another.
4. The method of claim 1, wherein the regions comprise rows and
columns, the scanning operation comprising scanning an incident
beam along the columns in the regions in a raster manner.
5. The method of claim 1, wherein the image comprises an array of
pixels and the analyzing operation calculates the focus score based
on at least one of contrast, spot size, a signal-to-noise ratio,
and a mean-square-error between pixel values for the at least one
image being analyzed.
6. The method of claim 1, wherein the image contains at least one
of an emission pattern and a transmission pattern produced by the
sample, the method further comprising identifying the at least one
emission pattern and transmission pattern.
7. The method of claim 6, wherein the identifying and analyzing
operations operate upon the same image to identify the focus score
and the at least one of the emission pattern and transmission
pattern.
8. The method of claim 1, wherein the analyzing operation includes
calculating a coefficient of variation in contrast for the image,
the coefficients of variation in contrast representing the focus
score.
9. The method of claim 1, wherein the analyzing operation includes
calculating the full width half maximum (FWHM) measure for a
Gaussian spot derived from the image, the FWHM representing the
focus score.
10. The method of claim 1, wherein the optical assembly includes a
focus lens, the shifting operation including modulating a
z-position of the focus lens repeatedly with respect to the
sample.
11. The method of claim 1, wherein the shifting operation includes
periodically adding a focal offset to the focal setting.
12. The method of claim 1, wherein the shifting, analyzing and
adjusting operations are continuously updated during a time delay
integration scan using real time information in the image to
control a focal position of the optical assembly.
13. The method of claim 1, wherein the shifting, analyzing and
adjusting operations are continuously repeated in a control loop to
lock in on a desired focal position of the optical assembly.
14. The method of claim 1, wherein the sample emits fluorescence
that is captured in the image as a fluorescence spatial emission
pattern, the focus score being based on contrast or spot size
within the fluorescence spatial emission pattern.
15. The method of claim 1, wherein the sample comprises multiple
microparticles that have at least one of first and second labels
that emit fluorescence at different first and second wavelengths,
the image containing a fluorescence spatial emission pattern of
fluorescence emitted at the first and second wavelengths, the
method further comprising detecting the first and second labels
from the same image utilized to obtain the focus score.
16. The method of claim 1, wherein the sample comprises multiple
microparticles that have optically detectable codes that are
captured in the image as a coded spatial transmission pattern, the
focus score being based on contrast or spot size within the coded
spatial transmission pattern.
17. The method of claim 1, wherein the sample comprises multiple
microparticles that have optically detectable codes, the
microparticles having chemical probes attached thereto, each of the
chemical probes being associated with a corresponding one of the
codes, the image containing optically detectable codes spatially
distributed across the image, the method further comprising
detecting the codes from the same image utilized to obtain the
focus score.
18. An optical imaging system, comprising: a sample holder to
receive a sample; an optical assembly to scan the sample, the
optical assembly apportioning the sample into regions defined by a
scan pattern, the optical assembly having a focal setting with
respect to the sample; a focus control module to introduce a shift
by a predetermined extent into the focal setting of the optical
assembly; a detector to detect images representative of at least
two regions from the sample, the images each having an associated
degree of focus corresponding to the focal setting of the optical
assembly; and an image analysis module to analyze the images to
obtain at least two focus scores, each of the focus scores
representing a degree to which the optical assembly was in focus
when detecting the image, and to determine a desired focal setting
based on a function of the at least two focus scores, wherein the
focus control module adjusts the focus setting based on the desired
focal setting.
19. A method for controlling focus dynamically of a sample imager,
comprising: (a) detecting a first region of a sample with an
optical assembly, the optical assembly having a first focal setting
with respect to the sample, thereby obtaining a first image; (b)
analyzing the first image to obtain a first focus score; the first
focus score representing a degree to which the optical assembly was
in focus when detecting the first image; (c) shifting the focal
setting of the optical assembly by a predetermined extent to a
second focal setting; (d) detecting a second region of the sample
with the optical assembly at the second focal setting, thereby
obtaining a second image; (e) analyzing the second image to obtain
a second focus score; the second focus score representing a degree
to which the optical assembly was in focus when detecting the
second image; (f) determining a desired focal setting for the
optical assembly based on a function of at least the first focus
score and the second focus score; and (g) repeating steps (a)
through (e) under conditions wherein the first focal setting is
adjusted based on the desired focal setting.
20. The method of claim 19, wherein the function comprises a
difference between the first focus score and the second focus
score.
21. The method of claim 20, wherein the first focus score and the
second focus score are based on contrast or spot size within the
images.
22. The method of claim 19, wherein the first region and the second
region are adjacent regions of the sample.
23. The method of claim 22, wherein the first image and the second
image are obtained by continuous scanning.
24. The method of claim 19, further comprising performing a second
repetition of the method, wherein the first focal setting is
adjusted to a lesser extent in the second repetition.
25. The method of claim 22, further comprising changing the
relative locations of the optical assembly and the sample prior to
the shifting of the focal setting.
26. A method for controlling focus dynamically of a sample imager,
comprising: (a) detecting a plurality of images of a sample, the
plurality of images including information relating to detected
light signals from the sample, the plurality of images including
first and second images; (b) analyzing the first and second images
to obtain respective focus scores, the focus scores representing a
degree to which the optical assembly was in focus when detecting
the first and second images, wherein the focus scores of the first
and second images are different; (c) comparing the focus scores of
the first and second images; and (d) relatively shifting the sample
with respect to the optical assembly based upon said comparison of
the focus scores.
27. The method of claim 26 wherein the first image corresponds to
light signals within a first spectral band that are emitted from a
first label in the sample, and the second image corresponds to
light signals within a second spectral band that are emitted from a
second label in the sample, wherein the first and second spectral
bands are different, the optical assembly having different optimal
focal planes for the first and second labels.
28. The method of claim 26 wherein the first and second images are
of adjacent scan regions, the focal setting of the optical assembly
being shifted a predetermined extent before obtaining the second
image.
29. A method for controlling focus dynamically of a sample imager,
comprising: (a) obtaining first and second images of a scan region
of a sample, the sample being positioned relative to an optical
assembly, the first and second images including information
relating to detected light signals from first and second labels in
the sample, respectively; (b) analyzing the first and second images
to obtain first and second focus scores; the focus scores
representing a degree to which the optical assembly was in focus
when detecting the first and second images; (c) comparing the first
and second focus scores; and (d) relatively shifting the sample
with respect to the optical assembly based upon said comparison of
the first and second focus scores, the sample having a modified
position relative to the optical assembly.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/122,550, filed Dec. 15, 2008 and having the same
title, which is hereby incorporated by reference in the
entirety.
BACKGROUND OF THE INVENTION
[0002] Embodiments of the present invention relate generally to
sample imaging, and more specifically to dynamic autofocus methods
and systems for assay imaging.
[0003] A wide variety of optical systems exist that capture images
of an area of interest on assays for subsequent analysis. Each
image may be obtained by detecting light produced across an entire
area of interest on an assay substrate at one point in time.
Alternatively, each image may be obtained by scanning an
illumination source across the area of interest while detecting
light produced at the current illuminated spot. For example, a
series of successive line scans of a tightly focused illumination
beam may be directed across the area of interest, such as in a
raster manner, to build up a two-dimensional detected image.
[0004] Optical systems exist that image microarrays of silica beads
that self assemble in microwells on substrates (e.g., fiber optic
bundles or planar silica slides). When randomly assembled on the
substrate, the beads have a uniform spacing of .about.5.7 microns.
Each bead is covered with hundreds of thousands of copies of a
specific oligonucleotide that act as the capture sequences in
assays. Imaging of microparticles provides a robust detection
method to multiplex assays requiring high precision, accuracy, and
speed. Microbeads are useful for numerous bioassays including
genotyping, gene expression, and protein-based assays.
[0005] Imaging systems exist that are used in DNA sequencing that
uses parallel analysis of unamplified, or amplified single
molecules, either in the form of planar arrays or on beads. The
methodology used to analyze the sequence of the nucleic acids in
such sequencing techniques is often based on the detection of
fluorescent nucleotides or oligonucleotides. The detection
instrumentation used to read the fluorescence signals on such
arrays may be based on either epifluorescence or total internal
reflection microscopy. One detection instrument has been proposed
that use an optical sequencing-by-synthesis (SBS) reader. The SBS
reader includes a laser that induces fluorescence from a sample
within water channels of a flowcell. The fluorescence is emitted
and collected by imaging optics which comprises one or more
objective lens and tube lens. As the fluorescence travels along an
optics path within the imaging optics, but prior to reaching a
detection camera, the fluorescence propagates through an
interference emission filter.
[0006] Optical imagers include, among other things, a light source
to illuminate a sample in the region of interest, one or more
detectors, and optical components to direct light from the region
of interest to the detector(s). The optical imagers also include a
focus mechanism that maintains focus of the optical components on
the region of interest in order that light received at the
detectors is received in focus.
[0007] However, conventional optical imagers have experienced
certain limitations. Conventional focus mechanisms are often
implemented as a separate sub-system including a separate focus
light source and focus detector. The focus light is directed onto
the sample and reflected to the focus detector. The light received
at the focus detector is analyzed and used to adjust the optical
components to maintain focus. However, conventional focus
mechanisms utilize components separate and part from the optical
components that are used to capture images of the region of
interest, thereby increasing the cost, complexity, and number of
parts that may potentially fail.
[0008] Further, in certain optical systems, the image cannot be
captured until after the focus mechanism first performs focus
measurements and adjusts the optical components relative to the
area of interest. Optical systems that first measure and adjust the
focus, before capturing images exhibit increased time between
capture of images. Cycle time represents the rate at which images
may be acquired (either through line scan or through snap-shot type
detection). The image acquisition rate is slower for systems that
must first ascertain the focal position prior to image
acquisition.
[0009] Moreover, conventional optical systems that use separate
focus mechanisms adjust focus based on reflectance measurements by
the focus mechanism. The reflectance measurement is derived from a
focus light beam and focus detector that are separate and distinct
from the actual data image captures for the area of interest.
Therefore, the reflectance measurement represents an indirect
estimate of the correct focal position for the actual data image.
When the focus mechanism loses calibration with the optical
components, the focal plane of the focus mechanism may become
mis-aligned with, or slightly differ from, the actual or true focal
plane associated with the actual image. Thus, the focus mechanism
may adjust the focal plane in a manner that is incomplete or
inaccurate.
[0010] It is desirable to provide improved methods and systems to
focus dynamically sample imaging systems.
BRIEF DESCRIPTION OF THE INVENTION
[0011] In accordance with one embodiment, a method is provided for
controlling focus dynamically of a sample imager. The method
includes scanning a sample with an optical assembly by apportioning
the sample into a plurality of regions defined by a scan pattern.
The optical assembly has a focal setting with respect to the
sample. The method also includes shifting the focal setting of the
optical assembly during scanning of the sample whereby the images
have an associated degree of focus corresponding to the focal
setting of the optical assembly. The method further includes
analyzing the images to obtain at least two focus scores. The focus
scores represent a degree to which the optical assembly was in
focus when detecting the images. The method also includes adjusting
the focus setting based on a function of the at least two focus
scores.
[0012] In another embodiment, an optical imaging system is
provided. The imaging system includes a sample holder to receive a
sample and an optical assembly to scan the sample. The optical
assembly apportions the sample into regions defined by a scan
pattern. The optical assembly has a focal setting with respect to
the sample. The imaging system also includes a focus control module
to introduce a shift by a predetermined extent into the focal
setting of the optical assembly. The imaging system also includes a
detector to detect images representative of at least two regions
from the sample. The images each have an associated degree of focus
corresponding to the focal setting of the optical assembly. The
imaging system also includes an image analysis module to analyze
the images to obtain at least two focus scores. Each of the focus
scores represents a degree to which the optical assembly was in
focus when detecting the image. The image analysis module may also
determine a desired focal setting based on a function of the at
least two focus scores. The focus control module adjusts the focus
setting based on the desired focal setting.
[0013] In a further embodiment, a method is provided for
controlling focus dynamically of a sample imager. The method
includes detecting a first region of a sample with an optical
assembly thereby obtaining a first image. The optical assembly has
a first focal setting with respect to the sample. The method also
includes analyzing a first image to obtain a first focus score. The
first focus score represents a degree to which the optical assembly
was in focus when detecting the first image. The method further
includes shifting the focal setting of the optical assembly by a
predetermined extent to a second focal setting and detecting a
second region of the sample with the optical assembly at the second
focal setting thereby obtaining a second image. The method also
includes analyzing the second image to obtain a second focus score.
The second focus score represents a degree to which the optical
assembly was in focus when detecting the second image. Furthermore,
the method includes determining a desired focal setting for the
optical assembly based on a function of at least the first focus
score and the second focus score.
[0014] In a further embodiment, a method for controlling focus
dynamically of a sample imager is provided. The method includes
detecting a plurality of images of a sample. The plurality of
images include information relating to detected light signals from
the sample. The plurality of images include first and second
images. The method also includes analyzing the first and second
images to obtain respective focus scores. The focus scores
represent a degree to which the optical assembly was in focus when
detecting the first and second images. The focus scores of the
first and second images are different. The method also includes
comparing the focus scores of the first and second images and
relatively shifting the sample with respect to the optical assembly
based upon said comparison of the focus scores.
[0015] Optionally, the first image may correspond to light signals
within a first spectral band that are emitted from a first label in
the sample and the second image may correspond to light signals
within a second spectral band that are emitted from a second label
in the sample. Furthermore, the optical assembly may have different
optimal focal planes for the first and second labels.
[0016] Also optionally, the first and second images may be of
adjacent scan regions of the sample. The focal setting of the
optical assembly may be shifted a predetermined extent before
obtaining the second image.
[0017] In yet another embodiment, a method for controlling focus
dynamically of a sample imager is provided. The method includes
obtaining first and second images of a scan region of a sample. The
sample is positioned relative to an optical assembly. The first and
second images include information relating to detected light
signals from first and second labels in the sample, respectively.
The method also includes analyzing the first and second images to
obtain first and second focus scores. The focus scores represent a
degree to which the optical assembly was in focus when detecting
the first and second images. The method further includes comparing
the first and second focus scores and relatively shifting the
sample with respect to the optical assembly based upon said
comparison of the first and second focus scores. The sample has a
modified position relative to the optical assembly.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 shows an exemplary optical imaging system formed in
accordance with an embodiment.
[0019] FIG. 2 illustrates a block diagram of an imaging subsystem
formed in accordance with an embodiment.
[0020] FIG. 3 illustrates a graph plotting a relation between the
focus score and a defocus spread.
[0021] FIG. 4 illustrates a method for controlling focus
dynamically of an optical imaging system in accordance with an
embodiment.
[0022] FIG. 5 illustrates a further embodiment for controlling
focus dynamically of an optical imaging system.
[0023] FIG. 6 illustrates a graphical representation of a dynamic
focus control operation that may be carried out by the method of
FIG. 4 or 5.
[0024] FIG. 7 illustrates an alternative scan arrangement of
regions that may be scanned in accordance with an embodiment.
[0025] FIG. 8 illustrates an alternative arrangement in which the
focus score may be obtained.
[0026] FIG. 9 illustrates an imaging system that is formed in
accordance with an alternative embodiment.
[0027] FIG. 10 illustrates a graph plotting relations between focus
scores that are associated with light signals of two different
spectral bands.
[0028] FIG. 11 illustrates a graphical representation of a dynamic
focus control operation in accordance with various embodiments.
[0029] FIG. 12 is a block diagram illustrating a further embodiment
for controlling focus dynamically of an optical imaging system.
[0030] FIGS. 13 and 14 display an exemplary embodiment of a
flowcell that may be utilized to carry samples in accordance with
various embodiments.
[0031] FIG. 15 illustrates an imaging system for detecting
bioassays that is formed in accordance with an alternative
embodiment.
DETAILED DESCRIPTION OF THE INVENTION
[0032] FIG. 1 illustrates an optical imaging system 10 that is
formed in accordance with an embodiment. By way of example, the
imaging system 10 may be constructed to include various components
and assemblies as described in PCT application PCT/US07/07991,
titled "System and Devices for Sequence by Synthesis Analysis",
filed Mar. 30, 2007 and/or to include various components and
assemblies as described in PCT application PCT/US2008/077850,
titled "Fluorescence Excitation and Detection System and Method",
filed Sep. 26, 2008, for both of which the complete subject matter
are incorporated herein by reference in their entirety. In
particular embodiments, the imaging system 10 can include various
components and assemblies as described in U.S. Pat. No. 7,329,860,
of which the complete subject matter is incorporated herein by
reference in its entirety.
[0033] As can be seen in FIG. 1, a fluid delivery module 12 directs
the flow of reagents (e.g., fluorescent nucleotides, buffers,
enzymes, cleavage reagents, etc.) to (and through) flowcell 14 and
waste valve 16. The flow cell 14 may represent a substrate having
one or more samples provided on or in the substrate. In particular
embodiments, the flowcell 14 comprises clusters of nucleic acid
sequences (e.g., of about 200-1000 bases in length) to be sequenced
which are optionally attached to the substrate of the flowcell 14,
as well as optionally to other components. The flowcell 14 may also
comprise an array of beads, where each bead optionally contains
multiple copies of a single sequence.
[0034] The imaging system 10 also comprises temperature station
actuator 18 and heater/cooler 20, which can optionally regulate the
temperature of conditions of the fluids within the flowcell 14. The
flowcell 14 is monitored, and sequencing is tracked, by detection
assembly 22 which can interact with focusing assembly 24.
Excitation assembly 26 (e.g., one or more excitation lasers within
an assembly) acts to illuminate fluorescent sequencing reactions
within the flowcell 14 via laser illumination through fiber optic
28 (which can optionally comprise one or more re-imaging lenses, a
fiber optic mounting, etc.). Low watt lamp 30 (optional), mirror
32, and reverse dichroic beam splitter 34 are also presented in the
embodiment shown. Additionally, mounting stage 36, allows for
proper alignment and movement of the flowcell 14, temperature
station actuator 18, detection assembly 22, etc. in relation to the
various components of the system. Focus (z-axis) component 38 can
also aid in manipulation and positioning of various components such
as lens 40 and source emitter 42. The emitter 42 scans one or more
samples provided in the flow cell 14 based on a scan pattern. The
focus component 38 causes the emitter 42 to move an excitation
laser 44 in a raster scan pattern. The detection assembly 22
apportions the sample into regions. The regions can be in the form
of blocks or any other shape appropriate to the imaging optics in
use. The sample produces at least one of an emission pattern and a
transmission pattern that is conveyed along detection path 46 to
the detection assembly 22.
[0035] The detection assembly 22 includes a label detector 48, a
code detector 50, a dichroic beam splitter 34, and mirror 52. A
system controller 54 controls overall operation of the imaging
system 10. Such components are optionally organized upon a
framework and/or enclosed within a housing structure. It will be
appreciated that the illustrations herein are of exemplary
embodiments and are not necessarily to be taken as limiting. Thus,
for example, different embodiments can comprise different placement
of components relative to one another (e.g., embodiment A comprises
a heater/cooler as in FIG. 1, while embodiment B comprises a
heater/cooler component beneath the flowcell, etc.).
[0036] Optionally, the imaging system 10 may be utilized for
detection of samples on microarrays. A microarray is a population
of different probe molecules that is attached to one or more
substrates such that the different probe molecules can be
differentiated from each other according to relative location. An
array can include different probe molecules, or populations of the
probe molecules, that are each located at a different addressable
location on a substrate. Alternatively, a microarray can include
separate substrates, such as beads, each bearing a different probe
molecule, or population of the probe molecules, that can be
identified according to the locations of the substrates on a
surface to which the substrates are attached or according to the
locations of the substrates in a liquid. Exemplary arrays in which
separate substrates are located on a surface include, without
limitation, a Sentrix.RTM. Array or Sentrix.RTM. BeadChip Array
available from Inc. (San Diego, Calif.) or others including beads
in wells such as those described in U.S. Pat. Nos. 6,266,459,
6,355,431, 6,770,441, and 6,859,570; and PCT Publication No. WO
00/63437, each of which is hereby incorporated by reference. Other
arrays having particles on a surface include those set forth in US
2005/0227252; WO 05/033681; and WO 04/024328, each of which is
hereby incorporated by reference.
[0037] Further examples of commercially available microarrays that
can be used include, for example, an Affymetrix.RTM. GeneChip.RTM.
microarray or other microarray synthesized in accordance with
techniques sometimes referred to as VLSIPS.TM. (Very Large Scale
Immobilized Polymer Synthesis) technologies as described, for
example, in U.S. Pat. Nos. 5,324,633; 5,744,305; 5,451,683;
5,482,867; 5,491,074; 5,624,711; 5,795,716; 5,831,070; 5,856,101;
5,858,659; 5,874,219; 5,968,740; 5,974,164; 5,981,185; 5,981,956;
6,025,601; 6,033,860; 6,090,555; 6,136,269; 6,022,963; 6,083,697;
6,291,183; 6,309,831; 6,416,949; 6,428,752 and 6,482,591, each of
which is hereby incorporated by reference. A spotted microarray can
also be used in a method according to an embodiment of the
invention. An exemplary spotted microarray is a CodeLink.TM. Array
available from Amersham Biosciences. Another microarray that is
useful is one that is manufactured using inkjet printing methods
such as SurePrint.TM. Technology available from Agilent
Technologies.
[0038] The systems and methods set forth herein can be used to
detect the presence of a particular target molecule in a sample
contacted with the microarray. This can be determined, for example,
based on binding of a labeled target analyte to a particular probe
of the microarray or due to a target-dependent modification of a
particular probe to incorporate, remove, or alter a label at the
probe location. Any one of several assays can be used to identify
or characterize targets using a microarray as described, for
example, in U.S. Patent Application Publication Nos. 2003/0108867;
2003/0108900; 2003/0170684; 2003/0207295; or 2005/0181394, each of
which is hereby incorporated by reference.
[0039] Exemplary labels that can be detected in accordance with
embodiments of the invention, for example, when present on a
microarray include, but are not limited to, a chromophore;
luminophore; fluorophore; optically encoded nanoparticles;
particles encoded with a diffraction-grating;
electrochemiluminescent label such as Ru(bpy).sup.32+; or moiety
that can be detected based on an optical characteristic.
Fluorophores that are useful in the invention include, for example,
fluorescent lanthanide complexes, including those of Europium and
Terbium, fluorescein, rhodamine, tetramethylrhodamine, eosin,
erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green,
Cy3, Cy5, stilbene, Lucifer Yellow, Cascade Blue.TM., Texas Red,
alexa dyes, phycoerythin, bodipy, and others known in the art such
as those described in Haugland, Molecular Probes Handbook, (Eugene,
Oreg.) 6th Edition; The Synthegen catalog (Houston, Tex.),
Lakowicz, Principles of Fluorescence Spectroscopy, 2nd Ed., Plenum
Press New York (1999), or WO 98/59066, each of which is hereby
incorporated by reference.
[0040] Any of a variety of microarrays known in the art, including,
for example, those set forth previously herein, can used in
embodiments of the invention. A typical microarray contains sites,
sometimes referred to as features, each having a population of
probes. The population of probes at each site is typically
homogenous having a single species of probe, but in some
embodiments the populations can each be heterogeneous. Sites or
features of an array are typically discrete, being separated with
spaces between each other. The size of the probe sites and/or
spacing between the sites can vary such that arrays can be high
density, medium density or lower density. High density arrays are
characterized as having sites separated by less than about 15
.mu.m. Medium density arrays have sites separated by about 15 to 30
.mu.m, while low density arrays have sites separated by greater
than 30 .mu.m. An array useful in the invention can have sites that
are separated by less than 100 .mu.m, 50 .mu.m, 10 .mu.m, 5 .mu.m,
1 .mu.m, or 0.5 .mu.m. An apparatus or method of an embodiment of
the invention can be used to image an array at a resolution
sufficient to distinguish sites at the above densities or density
ranges.
[0041] Optionally, the imaging system 10 may be utilized for
sequencing-by-synthesis (SBS). In SBS, a plurality of fluorescently
labeled modified nucleotides are used to sequence dense clusters of
amplified DNA (possibly millions of clusters) present on the
surface of a substrate (e.g., a flowcell). The flowcells 14 may
contain nucleic acid samples for sequencing where the flowcells 14
are placed within the appropriate flowcell holders. The samples for
sequencing can take the form of single nucleic acid molecules,
amplified populations of a nucleic acid molecule template in the
form of clusters, or beads comprising one or more molecules of
nucleic acid. The nucleic acids are prepared such that they
comprise an oligonucleotide primer adjacent to an unknown target
sequence. To initiate the first SBS sequencing cycle, one or more
differently labeled nucleotides, and DNA polymerase, etc., are
flowed into/through the flowcell by the fluid flow subsystem
(various embodiments of which are described herein). Either a
single nucleotide can be added at a time, or the nucleotides used
in the sequencing procedure can be specially designed to possess a
reversible termination property, thus allowing each cycle of the
sequencing reaction to occur simultaneously in the presence of
labeled nucleotides (e.g. A, C, T, G). Where the four nucleotides
are mixed together, the polymerase is able to select the correct
base to incorporate and each sequence is extended by a single base.
One or more lasers may excite the nucleic acids and induce
fluorescence. The fluorescence emitted from the nucleic acids is
determined by the fluorophores of the incorporated base, and
different fluorophores may emit different wavelengths of emission
light. Exemplary sequencing methods are described, for example, in
Bentley et al., Nature 456:53-59 (2008), which is incorporated
herein by reference.
[0042] Although embodiments of the invention have been exemplified
above with regard to detection of samples on a microarray or
flowcell, it will be understood that other samples having features
or sites at the above densities can be imaged at the resolutions
set forth above. Other exemplary samples include, but are not
limited to, biological specimens such as cells or tissues,
electronic chips such as those used in computer processors, or the
like. Examples of some of the applications of the invention include
microscopy, satellite scanners, high-resolution reprographics,
fluorescent image acquisition, analyzing and sequencing of nucleic
acids, DNA sequencing, sequencing-by-synthesis, imaging of
microarrays, imaging of holographically coded microparticles and
the like.
[0043] The heating/cooling components 20 of the imaging system 10
regulate the reaction conditions within the flowcell channels and
reagent storage areas/containers (and optionally the camera,
optics, and/or other components), while the fluid flow components
allow the substrate surface to be exposed to suitable reagents for
incorporation (e.g., the appropriate fluorescently labeled
nucleotides to be incorporated) while unincorporated reagents are
rinsed away. During laser excitation by the excitation assembly 26,
the image/location of emitted fluorescence from the nucleic acids
on the substrate is captured by the detection assembly 22, thereby,
recording the identity, in the computer component, of the first
base for each single molecule, cluster, or bead.
[0044] FIG. 2 illustrates a more detailed block diagram of an
imaging subsystem 100 that may be utilized in the imaging system 10
of FIG. 1. The imaging subsystem 100 includes a computer 102 that
receives information and data from, and controls operation of, the
other components of the subsystem 100. The imaging subsystem 100
includes one or more excitation source 104, an optical assembly
106, combinations of lenses and filters 114, 112, a code (and/or
transmission light) detector 110 and a label detector 116. The
imaging subsystem 100 includes a sample holder 118 that is
configured to receive a sample 120. For example, the sample 120 may
represent micro-particles flowing within a flow cell (e.g. flow
cell 14 in FIG. 1) and the holder 118 may represent a flowcell
holder. The optical assembly 106 is controlled by the computer 102
to scan the sample 120. During the scanning operation, the optics
assembly 106 apportions the sample 120 into regions, for which
separate images are detected based on a scan pattern. An x-y
controller 117 is mechanically and electrically coupled to the
holder 118. The x-y controller 117 moves the holder 118 in the y
direction as denoted by arrow y in FIG. 2 during a scanning
operation. The x-y controller 117 also moves the holder 118 in the
x direction which is orthogonal to the y and z directions. The x
direction is shown in FIG. 6.
[0045] Settings of at least a portion of the components within the
optical assembly 106 may affect a degree to which images are in
focus when detected. The position and settings of the optical
assembly 106 relative to the sample 120 affect a degree to which
images are detected in focus. For example, the optical assembly 106
includes focus components 108 that are focused on an actual focal
plane 119. The sample 120 has a preferred focal plane 121, for
example, at or below the surface of the sample 120. The focus
components 108 are adjusted intermittently or continuously in a
control feedback loop in an effort to align and overlay the actual
and preferred focal planes 119 and 121. When not aligned, a defocus
spread 123 is introduced between the actual and preferred focal
planes. As the defocus spread 123 increases, the degree of focus of
the image decreases. The focus control module 126 seeks to remove
or minimize the defocus spread 123.
[0046] Focus related parameters of the focus components 108 in the
optical assembly 106 are adjusted (e.g., in position, in
orientation) based on focal settings of the optical assembly 106.
By way of example, a focal setting may adjust the position of the
optical assembly 106 in a z-direction (denoted by arrow z) with
respect to the holder 118 and sample 120. As another example, the
focus components 108 may include a focus lens 122 that is moved in
the z-direction (along the axis denoted by arrow z) toward or away
from the sample 120. By moving the lens 122 in the z-direction, the
imaging subsystem 100 adjusts a degree of focus of the optical
assembly 106 with respect to the sample 120.
[0047] Alternatively, the focus setting may adjust the
inter-relation between components within the focus components 108,
such as by moving one or more lens toward or away from one another.
The focus components may include a z-motor that is controlled to
modulate focus. The motor may be positioned to pivot the lens 122
in an oscillating manner. Optionally, optical path modulation may
be achieved with a piezo on a mirror, LCD or electro optics and the
like. Any of a variety of methods for adjusting the z position in
an optical system can be controlled in accordance with the methods
and apparatus set forth herein.
[0048] The excitation source 104 generates an excitation light 105
that is directed onto the sample 120. The excitation source 104 may
generate one or more laser beams at one or more predetermined
excitation wavelengths. The light may be moved in a raster pattern
across groups of a sample, such as groups in columns and rows of
the sample 120. Alternatively, the excitation light 105 may
illuminate one or more entire regions of the sample 120 at one time
and serially stop through the regions in a "step and shoot"
scanning pattern. Line scanning can also be used as described, for
example, in U.S. Pat. No. 7,329,860, of which the complete subject
matter is incorporated herein by reference in its entirety. The
sample 120 produces at least one of emission light 134 and
transmission light 136 that is directed toward the lens 122.
Emission light 134 may be generated in response to illumination of
a fluorescent component in the sample 120 responsive to excitation
source 104. Alternatively, the emission light 134 may be generated,
without illumination, based entirely on emission properties of a
material within the sample 120 (e.g., a radioactive or
chemiluminescent component in the sample).
[0049] The transmission light 136 may result when the excitation
source 104 directs excitation light 105 from a location above the
sample 120 onto an upper surface of the sample 120 and the sample
120 reflects the transmission light 136. Alternatively,
transmission light 136 may result when the excitation light 105 is
located below the sample 120 and directs upward through the holder
118 through the sample 120. The transmission and emission light 136
and 134 may result from a common excitation light 105 or separate
excitation lights 105. The emission light 134 and transmission
light 136 are conveyed through the lens 122, along the optical
assembly 106 and are directed onto corresponding code and label
detectors 110 and 116.
[0050] In the example of FIG. 2, the emission light 134 and the
transmission light 136 are separated at a beam splitter 141 along
separate orthogonal optical paths 137 and 139. The emission light
134 may include label information produced by labels within the
sample 120. The emission light 134 is conveyed along optical path
137 through the lens/filter assembly 114 onto the label detector
116. The transmission light 136 may include code information
associated with optically detectable codes within or on the sample
120. The transmission light 136 may also include information
associated with at least one of reflection or refraction of an
optical substrate(s) (e.g., flow cell, sample holder,
microparticles, and the like). The transmission light 136 is
directed along optical path 139 through the lens/filter assembly
112 onto the code detector 110. Optionally, the code and label
detectors 110 and 116 may be combined into a common detector to
detect both emission light 134 and transmission light 136.
Alternatively, the sample 120 may have only one of code information
and label information therein. Thus, only the corresponding one of
the label detector 116 and code detector 110 would be provided and
utilized.
[0051] In some embodiments, the label information or signals from
the emission light 134 includes first fluorescent signals emitted
in response to a first excitation wavelength and also second
fluorescent signals emitted in response to a second excitation
wavelength. The first and second fluorescent emissions typically
include light at different wavelengths. The first and second
fluorescent signals may be emitted by, for example, a
carboxyfluorescein (FAM) label and a near-infrared (NIR) label.
[0052] The optical assembly 106 may have different actual focal
planes for the different spectral bands. For example, due to
inherent properties, arrangement, or alignment of the focus
components in an optical assembly, an optimal focal plane for
detecting a first spectral band may be spaced apart or separated
from an optimal focal plane of a second spectral band for the same
optical assembly. However, the optical assembly may also be adapted
to have different focal planes for different spectral bands. For
example, the optical assembly 106 may be configured such that first
and second light emissions are conveyed along different optical
paths. The first and second light emissions may be conveyed to an
objective lens located adjacent to the sample and along a common
optical path therefrom. The first and second light emissions may
then be filtered and/or reflected such that the first and second
light emissions are directed along different optical paths and
detected by different cameras or detectors. The focus components
along the different optical paths may be arranged to form different
focal planes. Accordingly, the focal plane of the optical assembly
106 for the first light emissions may be different than the focal
plane of the optical assembly 106 for the second light
emissions.
[0053] In alternative embodiments, the first and second light
emissions are separately conveyed along generally the same optical
path, but the focus components 108 or other components of the
imaging subsystem 100 are adjusted to affect the degree of focus
for the different light emissions. For example, in one embodiment,
a first excitation wavelength is first incident upon the sample.
The emission light 134 may include first fluorescent signals that
are conveyed along an optical path. The emission light 134 may then
be detected by a detector or camera. After excitation of the sample
with the first excitation wavelength, the focus components 108, the
sample holder 118, and/or other components of the imaging subsystem
100 are adjusted in a manner that moves the actual focal plane 119.
The second excitation wavelength is incident upon the sample and
the emission light 134 includes second fluorescent signals that are
conveyed along the same optical path. The emission light 134 is
then detected by the same camera or detector. However, the actual
focal planes 119 associated with the first and second fluorescent
emission signals are different.
[0054] The code and label detectors 110 and 116 and any other
detectors of the imaging subsystem 100 may detect images from the
sample 120. The label detector 116 may include multiple label
detectors or cameras. Each image comprises an array of pixels,
values for which are dependent upon the intensity of the light
including corresponding code and label information. The information
may also be associated with the reflection and/or refraction of
light from an optical substrate. The images are representative of
one or more regions into which the sample 120 has been apportioned
during the imaging operation. The images detected at code and label
detectors 110 and 116 or other detectors of the imaging subsystem
100 are passed to the computer 102. Each detected image has an
associated degree of focus that corresponds to, and is dependent
upon, the focal settings of the optical assembly 106.
[0055] The label and code detectors 116 and 110 may be, for example
photodiodes or cameras. In some embodiments herein, the detection
camera can comprise a 1 mega pixel CCD-based optical imaging system
such as a 1002.times.1004 CCD camera with 8 .mu.m pixels, which at
20.times. magnification can optionally image an area of
0.4.times.0.4 mm per tile using an excitation light 105 that has a
laser spot size of 0.5.times.0.5 mm (e.g., a square spot, or a
circle of 0.5 mm diameter, or an elliptical spot, etc.). The
detection cameras can optionally have more or less than 1 million
pixels, for example a 4 mega pixel camera can be used. In many
embodiments, it is desired that the readout rate of the camera
should be as fast as possible, for example the transfer rate can be
10 MHz or higher, for example 20 or 30 MHz. More pixels generally
mean that a larger area of surface, and therefore more sequencing
reactions or other optically detectable events, can be imaged
simultaneously for a single exposure. In particular embodiments,
the CCD camera/TIRE lasers may collect about 6400 images to
interrogate 1600 tiles (since images are optionally done in 4
different colors per cycle using combinations of filters, dichroics
and detectors as described herein). For a 1 Mega pixel CCD, certain
images optionally can contain between about 5,000 to 50,000
randomly spaced unique nucleic acid clusters (i.e., images upon the
flowcell surface). At an imaging rate of 2 seconds per tile for the
four colors, and a density of 25000 clusters per tile, the systems
herein can optionally quantify about 45 million features per hour.
At a faster imaging rate, and higher cluster density, the imaging
rate can be improved. For example, a readout rate of a 20 MHz
camera, and a resolved cluster every 20 pixels, the readout can be
1 million clusters per second. A detector can be configured for
Time Delay Integration (TDI) for example in line scanning
embodiments as described, for example, in U.S. Pat. No. 7,329,860,
of which the complete subject matter is incorporated herein by
reference in its entirety.
[0056] The images detected at the code detector 110 are stored as
code images 140 in code image sets 130 in memory 128. A code image
set 130 stores code information (ID and position for) a sample 120
and comprises a series of code images 140 associated with the
adjacent individual regions into which a sample 120 is apportioned
(as explained below in more detail). The memory 128 also stores
label image sets 132 that comprise label images 142 which are
detected at the label detector 116. A label image set stores label
information (type and position) for a sample 120. For example, for
each type of label, the label image set 132 may comprise a series
of label images 142 associated with adjacent individual regions of
the sample 120. The label image set 132 may also include label
images 142 for different labels (e.g., FAM, NIR) for reach
region.
[0057] The computer 102 includes, among other things, an image
analysis module 124, a focus control module 126, and the memory
128. The analysis module 124, among other things, analyzes images
obtained at one or both of the code and label detectors 110 and 116
in order to identify the ID and type of labels and codes within the
sample 120. The analysis module 124 also analyzes the images to
obtain the position of the labels and codes within the sample 120.
The images 140, 142 may contain an emission pattern and/or a
transmission pattern produced by the sample 120 and output as the
emission light 134 and/or transmission light 136. The analysis
module 124 analyzes the emission pattern and/or transmission
pattern to identify the ID, type, and position of codes and labels
within the sample 120.
[0058] The analysis module 124 also analyzes the same code and
label images 140 and 142 to determine a degree to which the optical
assembly 106 is focused in a desirable manner on the sample 120.
The analysis module 124 calculates a focus score associated with
the code and/or label image(s) 140 and 142. The focus score
represents the degree to which the optical assembly 106 was in
focus when the code and label detectors 110, 116 captured the code
and/or label image 140, 142. The analysis module 124 may calculate
the focus score based on one or more image quality parameters.
Examples of image quality parameters include image contrast, spot
size, image signal to noise ratio, and the mean-square-error
between pixels within the image. By way of example, when
calculating a focus score, the analysis module 124 may calculate a
coefficient of variation in contrast within the image. The
coefficient of variation in contrast represents an amount of
variation between intensities of the pixels in an image or a select
portion of an image. As a further example, when calculating a focus
score, the analysis module 124 may calculate the size of a spot
derived from the image. The spot can be represented as a Gaussian
spot and size can be measured as the full width half maximum
(FWHM), in which case smaller spot size is typically correlated
with improved focus. The image quality parameters are measured
directly from the actual sample image(s) that are scanned and also
analyzed to identify the codes and labels. The image quality
parameters are not necessarily obtained from a separate dedicated
focus image. The analysis module 124 continuously calculates real
time image quality parameters to achieve the desired degree of
focus. The computer 102 uses the image quality parameters in a
control loop to maintain or lock the optical assembly 106 at the
preferred focal plane 121.
[0059] Emission light 134 is captured in a label image 142 as a
fluorescence spatial emission pattern. The analysis module 124 may
calculate the focus score based on image quality parameters (e.g.,
contrast or spot size) of the fluorescence spatial emission
pattern. Optionally, the sample 120 may comprise multiple
microparticles that have one or more different labels that emit
fluorescence at one or more different wavelengths. The label image
142 contains a fluorescence spatial emission pattern emitted by the
different wavelengths associated with the labels. The analysis
module 124 identifies the individual labels based on the
fluorescence spatial emission pattern within the image. The
analysis module 124 may perform both determination of the focus
score and identification of the type and position of the labels
from the single common label image 142. In some embodiments, the
captured emission light 134 is limited to a spectral band. In such
images where the fluorescence spatial emission pattern is known,
the analysis module 124 may determine only the position of the
labels in the label images 142.
[0060] Optionally, the sample 120 may comprise multiple
microparticles that have optically detectable codes therein or
thereon. The codes produce the transmission light 136 that is
detected at the code detector 110 and stored in memory 128 as code
images 140. The code detector 110 captures the codes in the code
image 140 as a coded spatial transmission pattern. The analysis
module 124 calculates the focus score based on image quality
parameters (e.g., contrast or spot size) within the coded spatial
transmission pattern. The microparticles within the sample 120 may
have chemical probes attached thereto, where each of the chemical
probes is associated with a corresponding one of the codes. The
code image 140 captured by code detector 110, and containing the
optically detectable codes spatially distributed there across, is
then analyzed by the analysis module 124. The analysis module 124
identifies the codes, both for ID and position, from the same
common code image 140 as used to obtain the focus score.
[0061] Furthermore, the transmission light 136 may include at least
one of reflection and refraction information about the sample or
about an optical substrate that holds the sample. For example, the
transmission light 136 may include reflection and/or refraction
information regarding a surface of a flow cell. The reflection
and/or refraction information may be associated with microparticles
that have the biomolecules immobilized thereon. The reflection
and/or refraction information may be associated with a sample
holder.
[0062] FIG. 3 illustrates a graph 170 plotting a relation between
the focus score (on the vertical axis) and a defocus spread (along
the horizontal axis). The focus score may correspond to the
coefficient of variation in contrast, spot size, or another image
quality parameter as discussed herein. The defocus spread, as shown
at 123 in FIG. 2, represents a difference between the actual focal
plane 119, to which the focus components 108 are set, and a
preferred focal plane 121, at which images should be obtained with
a preferred degree of focus.
[0063] The relation in graph 170 includes a local maximum 171 where
the defocus spread approaches zero and the optical assembly 106
obtains images having the preferred degree of focus. At the local
maximum 171, the actual focal plane 119 is co-located with the
preferred focal plane 121. The actual focal plane 119 may be spaced
from the preferred focal plane 121 by a positive or negative
distance. For example, the actual focal plane 119 may be positioned
-1 micrometers (.mu.m), -5 .mu.m, -10 .mu.m, +1 .mu.m, +2 .mu.m,
etc. from the preferred focal plane 121. In graph 170, the tail 172
represents the range of the defocus spread in which the actual
focal plane 119 is moved in a negative direction from (e.g., below)
the preferred focal plane 121. In graph 170, the tail 173
represents the range of the defocus spread in which the actual
focal plane 119 is moved in a positive direction from (e.g., above)
the preferred focal plane 121. As the defocus spread increases in
the negative or positive direction, the focus score decreases in a
predetermined manner represented by the graph 170. The graph 170 is
merely illustrative. The shape of the graph will vary based upon
the type and properties of the optical system, the properties of
the sample, the content of the images, and the like.
[0064] In accordance with at least one embodiment, the focus
control module 126 compares the current change in the focus score
to the shift dz and to past focus scores in order to identify a
direction and an amount to change the focal offset. To understand
how the focus control module 126 may determine direction and
amounts to adjust the focal offset, attention is directed to FIG.
3.
[0065] FIG. 3 also illustrates an exemplary shift dz plotted at 175
that may be introduced into the z-distance between the lens 122 and
the sample 120. Below the shift dz 175, three alternative focus
score plots 176-178 are presented. The focus score plots 176-178
illustrate alternative patterns that may be exhibited by the focus
score in response to the shifts dz in the actual focal plane 119.
The alternative focus score plots 176-178 are associated with three
separate points along the graph 170. Plots 176-178 illustrate that,
depending upon the state of the optical assembly 106 along the
graph 170, the focus score will change by a different amount and
with a different phase for each shift dz in the defocus spread.
[0066] Plot 178 provides an example of how the focus score may
change when the state of the optical assembly 106 is in a low/poor
degree of focus, such as where the defocus spread is -5 .mu.m. In
plot 178, the focus score begins (at 180) with a low/poor value
corresponding to a defocus of -5 .mu.m. The shift dz is added to
the focus setting at 181. For example, the shift dz may be 0.5
.mu.m. Thus, when the shift dz=0.5 .mu.m is added to the z-distance
of the optical assembly 106, the defocus spread is reduced to -4.5
.mu.m. As the defocus spread is reduced, the focus score improves
at 182. The transition (between 180 and 182) in the focus score
(e.g., changing from a low value to a better value) is in phase
with the shift dz to 181 which also changed from a low value to a
better value.
[0067] Plot 176 provides an example of how the focus score may
change when the state of the optical assembly 106 is in a low/poor
degree of focus, such as where the defocus spread is +4 .mu.m. In
plot 176, the focus score begins (at 185) with a medium value
corresponding to a defocus of 6 .mu.m. The shift dz is added to the
focus setting at 181. For example, the shift dz may be 0.5 .mu.m.
Thus, when the shift dz=0.5 .mu.m is added to the z-distance of the
optical assembly 106, the defocus spread is increased to 4.5 .mu.m.
As the defocus spread moves further away from zero, the focus score
deteriorates or worsens at 186. The transition between 185 and 186
in the focus score, namely changing from a medium value to a low
value, is 180 degrees out of phase with the shift dz to 181 which
transitioned from a low value to a high value.
[0068] In connection with plots 176 and 178, the focus control
module 126 analyzes the phase of each change in the focus score
relative to the phase of the shift dz to make a determination
regarding whether the defocus spread is positive or negative. For
example, when the change in focus score is in phase with the shift
dz, then the defocus spread is positive. Alternatively, when the
change in the focus score is out of phase with the shift dz, then
the defocus spread is negative. Once the sign of the defocus spread
is determined, the focus control module 126 determines whether to
adjust the focus setting by increasing or decreasing the z-distance
138. Thus, the phase relation of the focus score and defocus spread
is utilized to determine a direction in which to adjust the focus
setting.
[0069] Plot 177 provides an example of how the focus score may
change when the state of the optical assembly 106 is already in a
high/good degree of focus, such as where the defocus spread is near
or at 0 .mu.m. In plot 177, the focus score begins (at 183) with a
high/good value corresponding to a defocus of 0 .mu.m. The shift dz
is added to the focus setting at 181. When the shift dz=0.5 .mu.m
is added to the z-distance of the optical assembly 106, the defocus
spread is increased to 0.5 .mu.m. As the defocus spread moves
slightly away from zero, the focus score deteriorates or worsens
slightly at 184. The transition between 183 and 184 in the focus
score is 180 degrees out of phase with the shift dz transition to
181.
[0070] In connection with plot 177, the focus control module 126
analyzes the amplitude of the change in the focus score for one
step in the shift dz to make a determination regarding whether the
defocus spread is near or far from a preferred focus value (e.g.,
zero). For example, when the change in focus score for one step in
the shift dz is small, then the defocus spread is small.
Alternatively, when the change in the focus score is large for one
step in the shift, then the defocus spread is large. Once the
amplitude of the defocus spread is determined, the focus control
module 126 determines an amount of the adjustment to the focus
setting. Thus, the amplitude and phase of the transition in the
focus score is utilized to determine an amount and direction to
adjust the focus setting.
[0071] FIG. 4 illustrates a method for controlling focus
dynamically for an optical imager in accordance with an embodiment.
At 190, a sample is scanned with the optical assembly 106 that
apportions the sample 120 into regions based on a scan pattern. The
optical assembly 106 has a focal setting with respect to the sample
120. As illustrated in FIG. 2, the regions may be arranged adjacent
to one another in a non-overlapping manner.
[0072] At 192, the focal setting of the optical assembly 106 is
shifted during scanning of the sample 120. The shifting operation
may include modulating a z-position of the focus lens repeatedly
with respect to the sample 120. The shifting operation may include
periodically adding a focal offset (e.g., a dz) to the focal
setting. The shifting operation introduces an error signal into a
focal position of the optical assembly. The error signal is
monitored as a function of the focal position of the optical
assembly. By way of example only, the optical path length (e.g.,
z-distance 138) may be modulated by 0.5 .mu.m every 5 .mu.m of
physical scan distance in the x direction across the sample. The
shift may be introduced at a predetermined periodic rate (e.g., 125
Hz, 12.5 Hz, and the like). For example, it may be desirable to
vary the shift at 125 Hz. Thus, the focus control module 126
introduces the shift every 10 scan lines (e.g., columns) and
maintains the shift for 10 scan lines before removing the shift for
10 scan lines. Columns in a region may be scanned at a rate of 1.25
mm/sec with 0.5 .mu.m resolution. Alternatively, it may be
desirable to vary the shift at 12.5 Hz. Thus, the focus control
module 126 introduces the shift every 100 scan lines (e.g.,
columns) and maintains the shift for 100 scan lines before removing
the shift for 100 scan lines.
[0073] At 194, the system detects one or more images representative
of one of the regions from the sample 120. The image(s) has an
associated degree of focus corresponding to the focal setting of
the optical assembly 106. The image(s) may contain at least one of
an emission pattern and a transmission pattern produced by the
sample 120. At 195 the system identifies emission and/or
transmission patterns. For example, a region may have a width of 10
columns or scan lines, a width of 100 columns or scan lines, a
width of 5 .mu.m, 20 .mu.m and the like.
[0074] At 196, the system analyzes the one or more images to obtain
the focus score or scores corresponding thereto. The analyzing
operation calculates the focus score(s) based on at least one of
contrast, spot size, a signal-to-noise ratio, and a
mean-square-error between pixel values for the at least one image
being analyzed. The analyzing operation includes calculating a
coefficient of variation in contrast for the image, the
coefficients of variation in contrast representing the focus score.
As a further example, the analyzing operation can include
calculating the size of a spot derived from the image. The spot can
be represented as a Gaussian spot and the full width half maximum
(FWHM) can represent the focus score. The identifying operation at
195 and the analyzing operation at 196 operate upon the same image
to identify the focus score and the emission and/or transmission
patterns. When the sample emits fluorescence that is captured in
the image as a fluorescence spatial emission pattern, the focus
score can be based on contrast or spot size within the fluorescence
spatial emission pattern. When the sample has at least one of first
and second labels that emit fluorescence at different first and
second wavelengths, the variation in contrast or spot size may be
calculated for only one or for both of the first and second
wavelengths. The system detects the first and second labels from
the same image utilized to obtain the focus score.
[0075] At 198, the focus control module 126 adjusts the focus
setting based on the focus score or focus scores. As part of the
adjustment operation, the amplitude and phase of the focus score is
analyzed. It is determined whether the focus score is in phase, or
out of phase, with the shift. It is also determined whether the
focus score changed by a large amount or a small amount during the
most recent shift. Based on the phase and amplitude changes of the
focus score, the focus control module 126 determines a direction
and an amount to change the focus shift. The adjusting operation
reduces the error signal by adjusting the focal position. By way of
example, the focal setting at 198 may be calculated using a PI
("proportional/integral") feedback loop. The focus score is first
calculated using the following equation (1):
CV ( y i ) = .sigma. ( y i ; z ) .mu. ( y i ; z ) ##EQU00001##
[0076] In the equation (1), the focus score is calculated for an
image at a particular z distance. The variable y, represents pixel
values along the y-axis for a given z-distance. The focus score
equals the ratio of the standard deviation for the variable y, over
the mean for the variable y, for a current group in the image. The
CV is integrated over N.sub.COL columns 9 or other groups) for an
entire image. Once the CV value is known for the image, an error
signal can be calculated based on equation (2) below:
e ( y i ) = [ CV ( y i ; z + dz ) - CV ( y i - 1 ; z ) ] N col
##EQU00002##
[0077] The error signal e(y.sub.i) in equation (2) represents a
difference between the CV value for the current group y.sub.i and
the CV value for the next group y.sub.i-1. Once the error signal
e(y.sub.i) is known, the correction to the focus offset may be
chosen based on the following equation (3):
.DELTA. z i + 1 = g P e ( y i ) + g I j = i - N i e ( y j )
##EQU00003##
[0078] In equation (3), "g.sub.p" and "g.sub.I" represent gain
factors, while "e(y.sub.i)" represents a proportion, and
".SIGMA.e(y.sub.i)" represents an integral. The shifting,
analyzing, and adjusting operations are continuously updated during
a time delay integration scan using real time information in the
image to control a focal position of the optical assembly. The
shifting, analyzing, and adjusting operations are continuously
repeated in a control loop to lock in on a desired focal position
of the optical assembly.
[0079] Optionally, the amount of correction made at each iteration
through 190-198 in FIG. 4 may be limited to a maximum incremental
change in focus offset. This limitation may be the same or
different for various regions as the imaging system steps across
the sample. At 199, the process determines whether the current
region is the last region on a sample. When the currently scanned
region is the last region, the process is done. When the currently
scanned region is not the last region, flow returns to the
beginning above 190 and the next region is scanned.
[0080] FIG. 5 illustrates a method in accordance with another
embodiment of the invention. At 1901, a method for controlling
focus dynamically for an optical imager is initiated. At 1902 an
image for a region of a sample is obtained with the optical
assembly 106. The optical assembly 106 has a focal setting with
respect to the sample 120. As illustrated in FIG. 2, the regions
may be arranged adjacent to one another in a non-overlapping
manner.
[0081] At 1903, the image of the region obtained in 1902 is
analyzed to obtain a first focus score. The analyzing operation
calculates the focus score, for example using methods set forth
above in regard to 195 of FIG. 4. Thus, focus score can be based on
at least one of contrast, spot size, a signal-to-noise ratio, and a
mean-square-error between pixel values for the at least one image
being analyzed. The analyzing operation can include calculating a
coefficient of variation in contrast for the image, the
coefficients of variation in contrast representing the focus score,
or the analyzing operation can include calculating the size of a
spot derived from the image.
[0082] At 1904 the focal setting of the optical assembly 106 is
shifted. The shifting operation may be carried out as set forth
above in regard to 192 of FIG. 4. Thus, shifting can include
modulating a z-position of the focus lens with respect to the
sample 120. The shifting operation may include adding a focal
offset (e.g., a dz) to the focal setting to a known or
predetermined extent. The extent of the shift can be characterized
in terms of magnitude, such as the size of dz, and direction, such
as the sign (+/-) of dz. The extent of the offset can be determined
at any step prior to 1904 including for example, prior to 1903,
1902 or 1901. The shifting operation introduces an error signal
into a focal position of the optical assembly. The error signal can
be monitored as a function of the focal position of the optical
assembly as set forth below in the context of the following
steps.
[0083] At 1905, the system obtains an image representative of a
second region from the sample 120 at the focal setting to which the
optical assembly was shifted in 1904. The image has an associated
degree of focus corresponding to the focal setting of the optical
assembly 106. The image may contain at least one of an emission
pattern and a transmission pattern produced by the sample 120.
Returning to the example of FIG. 2, the second region can be
adjacent to or overlapping with the region that was imaged at
1902.
[0084] At 1906 the image of the region obtained in 1905 is analyzed
to obtain a second focus score. The analyzing operation is carried
out as set forth above in regard to 1903 and a focus score of a
similar type is obtained.
[0085] At 1907, a desired focal setting is determined based on a
function of the first focus score and the second focus score. For
example, the amplitude and phase of the focus scores can be
analyzed. Thus, it can be determined whether the difference in the
first focus score determined at 1903 and the second focus score
determined at 1906 is in phase, or out of phase, with the shift at
1904. It can also be determined whether the focus score changed by
a large amount or a small amount as a result of the shift. By way
of example, the desired focal setting may be calculated using a PI
("proportional/integral") feedback loop. The focus score is first
calculated using equations (1), (2) and (3) as set forth above in
regard to 198 of FIG. 4.
[0086] In some embodiments, the desired focal setting may be
determined based on multiple focus scores for each focal setting.
For example, steps 1902-1906 may be repeated one or more times to
obtain a plurality of first focus scores corresponding to one focal
setting and a plurality of second focus scores corresponding to
another focal setting. The plurality of first focus scores may be
determined from different images along the scan region, and the
plurality of second focus scores may be determined from different
images along the scan region. More specifically, the optical
assembly 106 may alternate between first and second focal settings
as the optical assembly moves along the scan region and obtain a
plurality of focus scores for each focal setting. The determining,
at 1907, may be based on at least one of a function of the first
focus scores and a function of the second focus scores. For
example, the determining operation may be based on a function of an
average of the first focus scores and an average of the second
focus scores.
[0087] At 1908 the focal setting is adjusted based on the desired
focal setting. The focus control module 126 adjusts the focus
setting based on the focus desired focal setting. The desired focal
setting can be communicated to the focus control module as a
particular setting or as an extent of change from a current or
otherwise known focal setting. For example, based on the phase and
amplitude changes of the focus score, the focus control module 126
can determines a direction and an amount to change the focus shift.
The adjusting operation will typically reduce the error signal by
adjusting the focal position. It is possible that the desired focal
setting is the same as the current focal setting and little to no
change is necessary or desired. In such a situation the focus
control module can be instructed to make little or no change to the
current focal setting.
[0088] At 1909, the process determines whether the current region
is the last region on a sample for which an image is desired. When
the currently scanned region is the last region, the process is
done 1911. When the currently scanned region is not the last
region, the system proceeds to 1910 where the relative location of
the optical assembly and the sample is changed such that another
region of the sample is positioned for imaging. Flow then returns
to 1902 and the other region is scanned.
[0089] FIG. 6 illustrates a graphical representation of a dynamic
focus control operation that may be carried out by the imaging
subsystem 100 of FIG. 2 in connection with the method of FIGS. 4
and 5. In FIG. 6, a top plan view of a first portion of the sample
120 is shown, such as from the view point of the lens 122 of FIG.
2. A series of regions 152 are overlaid on the sample 120 to
demonstrate a potential step-wise scan pattern that apportions the
sample 120. Separate images are captured for each region 152 of the
sample 120. In the example of FIG. 6, the sample 120 is comprised
of an array of micro-particles 154 arranged in groups such as rows
and columns. When an excitation source 104 (FIG. 2) is used, a
light source 104 may be controlled to move in a raster scan pattern
along each column of the region 152. The raster motion may be
achieved by moving the light source 104 with respect to the sample
120, or by moving the holder 118 in the x and y directions. The
raster scan pattern may move from top to bottom downward, or from
bottom to top upward, along each column. As a column is scanned by
the light source 104, a corresponding column of the image is
captured at detectors 110, 116. Once a column is scanned (and the
corresponding column of the image is captured), the light source
may be moved to the next column and the process repeated for
consecutive columns until an image is captured for the entire
region 152.
[0090] Optionally, multiple groups may be scanned in one pass (at
the same time). As a further option, an entire region 152 may be
scanned by the light source in one pass from top to bottom, or
bottom to top. As yet a further option, each region 152 need not be
illuminated with a moving light source. Instead, a complete region
152 may be illuminated at once by the light source and the image
obtained for the entire region 152 instantaneously as a snap-shot
to capture an image associated with the region 152 at one point in
time. After the image is captured for the first region 152, the
process is repeated for the second region 152 in a step and shoot
manner. With each of the foregoing techniques for capturing an
image for one region 152, the overall sample 120 is "scanned" by
repeating the capture process sequentially for multiple regions
152, regardless of whether the excitation light 105 is moved
relative to a current region, rastering or otherwise.
[0091] In FIG. 6, the regions 152 are labeled A-E for purposes of
illustration. The sample 120 is apportioned such that regions A-E
are arranged in a non-overlapping manner. When images are captured
for each of the regions A-E, the images form a series of adjacent
images that are separate and distinct from one another. In the
example of FIG. 6, an image set 156 is shown to include images A-E
which correspond to the regions A-E from the sample 120. The
analysis module 124 (FIG. 2) analyzes one or more of the images A-E
to identify and locate codes and/or labels, and to calculate values
for image quality parameters associated with the focus score. The
analysis module 124 (FIG. 2) may also analyze one or more of the
images A-E to identify and locate areas of reflection and/or
refraction and calculate corresponding values for image quality
parameters.
[0092] FIG. 6 also illustrates a series of graphs 160-163 that are
referenced in connection with explaining an application of the
focus control process implemented in accordance with at least one
embodiment. The horizontal axis in each graph corresponds to the
x-position across the bottom 155 of the sample 120. In graph 160,
the vertical axis corresponds to the z-distance 138 (FIG. 2)
between the lens 122 and the preferred focal plane 121 of the
sample 120. Graph 160 plots an example of how the focus control
module 126 may adjust the distance between the focus lens 122 and
the sample 120 as a scan steps across the sample 120.
[0093] In graph 161, the vertical axis represents the degree of
focus for each image. Graph 161 plots an example of how the degree
of focus changes from the first region A to the last region E
during a scanning process. In graph 162, the vertical axis
represents an image quality parameter, such as coefficient of
variation (CV) that is calculated by the analysis module 124 for
images A-E. Graph 162 plots exemplary CV values calculated by the
analysis module 124 for each of images A-E. In graph 163, the
vertical axis represents a shift that is introduced by the focus
control module 126 into the z-distance 138. Graph 163 plots a
series of focal offsets that are periodically added. It should be
recognized that FIG. 6 is illustrative only and that the sizes,
inter-relation, and number of focal offsets, CV calculations, and
changes in the focus setting and degree of focus are narrative, not
actual.
[0094] First, the region A is scanned. During capture of at least a
first portion (e.g., one or more of the columns) of the first image
A for region A, the optical assembly 106 has an initial focal
setting. For example, the initial focal setting may be set to a
z-distance 138 that is denoted at focal setting Az in graph 160.
During the scan of region A, the focus control module 126
introduces at least one temporary shift (as denoted at shift Adz)
into the focal setting Az of the optical assembly 106. For purposes
of simplification, the focus process is described in connection
with one shift during scan of a complete region. However, it should
be recognized that multiple shifts may be performed during scan of
a single region and a corresponding number of multiple adjustments
to the focal setting may be made during scan of the same
region.
[0095] During the scan of region A, the lens 122 is located a
z-distance 138 from the sample 120 that is determined by the focal
setting Az and in addition by the shift Adz. The label and/or code
detectors 116, 110 detect an image A, representative of region A.
The image A has an associated degree of focus corresponding to the
focal setting Az and shift Adz. The analysis module 126 analyzes
the image A to obtain the focus score CVa. The focus score CVa
represents a coefficient of variation in the contrast of the pixels
in image A. Next, the focus control module 126 adjusts the focus
setting based on the focus score as discussed above in connection
with FIGS. 3 and 4. In the example of FIG. 6, the focus setting is
adjusted to Bz (graph 160). During the scan of region B, the lens
122 is located a z-distance 138 from the sample 120 determined by
the focal setting Bz. The shift Bdz is introduced during scan of
region B and, once image B is captured, the focus score CVb is
determined. Based on focus score CVb, the focal setting is adjusted
to Cz. The process is repeated for regions C, D, and E, utilizing
shifts Cdz, Ddz, and Edz. Focus scores CVc, CVd and CVe are
calculated by the analysis module 124 and used to adjust the focal
setting to Cz, Dz, and Ez. As shown in graph 161, the degree of
focus improves/increases as the scanning process steps across the
sample 120. Hence, the image E will have a higher degree of focus
than the image A. The system can return to obtain an image of
region A at a focal setting determined from the image of region E.
However, the system need not return to scan a previous region that
was obtained at a lower degree of focus. Rather, the region having
a lower degree of focus can be ignored or discarded when evaluating
the image of the sample.
[0096] FIG. 7 illustrates an alternative scan arrangement for
regions that may be obtained at 190 in FIG. 4 in accordance with an
embodiment. In FIG. 7, a portion of a sample 320 is illustrated
with a lead-in sub-region 322 that is scanned to obtain a reference
focus score. For example, the sub-region 322 may be smaller (e.g.,
have less width) than the following regions 324-327. An excitation
light 105 illuminates a beam spot 328 that is moved in the
direction of arrow 329 to scan region 324. Optionally, the beam
spot 328 may be smaller to cover fewer groups or columns of the
region 324. The sub-region 322 is imaged and analyzed to obtain a
focus score which is then used to adjust the focus offset. The
regions 324-327 are arranged in an overlapping arrangement. For
example, the overlap may correspond to one a few groups or
columns.
[0097] FIG. 8 illustrates an alternative arrangement in which the
focus score may be obtained at 196 in FIG. 4. FIG. 8 illustrates an
example of an image 330 that is obtained for a region of a sample.
The image 330 is analyzed by the analysis module 124 to identify
codes and labels as explained above. A subset of the columns in of
the image 330 is also analyzed to obtain the focus score. For
example, a portion of the image 330 may be designated as focal test
regions 331-333. In the example of FIG. 8, the focal test regions
331-333 are defined as elongated strips that extend from the top
335 to the bottom 336 of the image 330 and are spaced apart from
one another. The regions 337 and 338 between the focal test regions
331-333 are not analyzed to obtain focus scores. By using spatially
distributed focal test regions 331-333, the system reduces the
number of columns to analyze in each image 330 to obtain a focus
score.
[0098] FIG. 9 illustrates an imaging subsystem system 202 that is
formed in accordance with an alternative embodiment. The subsystem
202 generally includes an excitation assembly 204, and a detection
assembly 220. The excitation assembly 204 is optically coupled to a
sample 212 that is, in turn, optically coupled to the detection
assembly 220. The sample 212 is provided on a substrate 213. For
example, the sample 212 may represent a plurality of nucleic acid
clusters/beads or other features, with multiple fluorescent labels,
which are attached to a surface of the substrate 213 (e.g., a flow
cell or microarray). The excitation assembly 204 illuminates the
same or common active area, or tile, in a temporally multiplexed
manner with one or more different excitation wavelengths during
successive excitation events. The excitation assembly 204 performs
temporal multiplexing by generating one or more excitation
wavelengths sequentially, such as through the use of multiple
alternating sources or lasers 206 and 208, or multiple exposures of
the same lasers. The lasers 206 and 208 are coupled through an
excitation light guide 210 to illuminate a common area, or tile, on
the substrate 213 and sample 212. In response thereto, the sample
212 emits fluorescence which is collected by an objective lens
223.
[0099] In the example of FIG. 9, a dashed line generally denoted at
214 illustrates an excitation beam that is channeled from the laser
206, through the light guide 210 and onto the sample 212 at a
desired angle of incidence with respect to the surface or a
reference plane on or within the substrate 213 holding the sample
212. A dashed line generally denoted at 215 illustrates an
excitation beam that is channeled from the laser 208, through the
light guide 210 and onto the sample 212 at a desired angle of
incidence with respect to the surface or a reference plane on or
within the substrate holding the sample 212. The control module 211
controls the excitation assembly 204 to generate an excitation
light pattern. By way of example, the control module 211 may
instruct the lasers 206 and 208 to generate excitation light at
successive, non-overlapping periods of time. The laser 206 may
supply a first pulse or burst of light as excitation beam 214
(e.g., at 532 nm) for a predetermined pulse duration, terminate the
excitation beam 214, after which the laser 208 may supply a second
pulse or burst of light as excitation beam 215 for a pulse duration
and then terminate the excitation beam 215. In order to record two
fluorophores with different wavelength emissions, each laser may be
used once, or more than once on a single area (tile). For example,
the sequence to record four different images in a single substrate
tile may be a: wavelength one; filter one, b: wavelength one;
filter two; c: wavelength two; filter three; d: wavelength two;
filter four. The exposure time may be the same for each wavelength
emission channel, or may be altered to control the intensity of the
fluorescent signal recorded in the different channels. The exposure
time may be the same for every cycle of sequencing, or may be
increased throughout the sequencing run to compensate for any
diminishing of the signal intensity as the cycles are
performed.
[0100] The lasers 206 and 208 generate excitation light at
different wavelengths that are chosen based on the wavelength
spectrum of the fluorescent bases of interest that will potentially
be present in the sample 212. In general, a number of bases may be
labeled with a plurality of dyes or combinations of dyes, where
each dye emits a corresponding known unique spectral pattern when
illuminated with excitation light at a predetermined wavelength.
For example, a number of bases (e.g., one or more) may be used that
are each labeled with one or more dyes, where the dyes produce
spectral patterns that are separately distinguishable along the
wavelength spectrum. In a particular embodiment of the invention,
each of the four bases is labeled with an individual fluorophore,
such that the four bases can be spectrally distinguished, for
example as described in PCT/GB2007/01770 or Bentley et al, supra
(2008), the contents of which are incorporated herein by reference
in their entirety.
[0101] The emission light 244 (e.g., fluorescence, luminescence,
chemiluminescence, etc.) is generated at the sample 212, such as in
response to the excitation beams 214 and 215, or in response to a
chemical reaction when no excitation beams are used. The emission
light 244 is comprised of multiple spectral bands denoted at
247-248. The spectral bands 247-248 generally differ from one
another and may have different center wavelengths, mean
wavelengths, median wavelengths, band widths, shapes, and the like.
The detection assembly 220 is located downstream. The detection
assembly 220 provides full field of view detection for the entire
area of each tile of the substrate 213 measured by the objective
lens 223.
[0102] The detection assembly 220 includes a dichroic member 225,
band pass filters 232 and 234, detection cameras 236 and 238, a
read out module 237 and a computer 250. The detection assembly 220
may include additional focus components that are not shown in FIG.
9. The detection assembly 220 is constructed entirely of non-moving
parts that remain stationary and fixed with respect to one another,
with respect to an axis of the optical system from the objective
lens 223, and with respect to reflective and transmissive detection
paths of the spectral bands 248 and 247, respectively. Accordingly,
the detection or optical paths for the spectral bands 248 and 247
may be different. In some embodiments, the focal plane of the
spectral bands 248 and 247 are also different.
[0103] The band pass filters 232 and 234 block high and low
spectral content of the incoming spectral bands 247 and 248,
respectively, and pass the portions of the spectral bands 247 and
248 within the upper and lower limits of the pass bands. The limits
of the pass bands may be set to sharpen edges of spectral patterns,
block noise, block scatter, block excitation light, and the like.
The passed portions of the spectral bands 247 and 248 are directed
onto corresponding detection cameras 236 and 238. The band pass
filters 232 and 234, and detection cameras 236 and 238 may be
oriented at various angles of incidence with respect to the
transmissive and reflective paths and with respect to one another.
For example, the detection cameras 236 and 238 may be oriented in a
perpendicular geometry or acute angular relation with one another
(e.g., 90.degree., etc.).
[0104] The detection cameras 236 and 238 detect the spectral bands
247 and 248, respectively, and provide electrical detection signals
241 and 243 to a readout module 237 to form images. The electrical
detection signals 241 and 243 may be analog or digital signals
representing an amount of emission energy (fluorescent or
otherwise) measured by the detection cameras 236 and 238. The
detection cameras 236 and 238 may output the detection signals 241
and 243 as continuous signals representative of an instantaneous
measurement. The readout module 237 records the detection signals
241 and 243 and provides a series of images 239 representative of
the emission light that was detected by each of the detection
cameras 236 and 238. The readout module 237 passes the images to
the computer 250.
[0105] The computer 250 includes an image analysis module 252, a
focus control module 254, and memory 256. The memory 256 stores the
images 258 and 260 captured by the detection cameras 236 and 238.
The analysis module 252 and the focus control module 254 perform
the shifting, analyzing and adjusting operations discussed above in
connection with the embodiment of FIGS. 2-6.
[0106] FIGS. 10-12 illustrate another method for dynamically
controlling a focus of an optical assembly or imaging subsystem
using focus scores from different images. Embodiments described
herein include obtaining images of detected light emissions from
different labels to control focus dynamically. For example, FIG. 10
illustrates a graph 702 that plots focus-score curves 704 and 706
for first and second spectral bands, respectively. The optical
assembly of the imaging subsystem may have different focal planes
for detecting different labels as described above. The first
spectral band may be associated with a first label (e.g., FAM-type
label), and the second spectral band may be associated with a
second label (e.g., NIR-type label). The first and second spectral
bands may be different. Curve 704 shows the relation between the
focus score and defocus spread of the first label. Curve 706 shows
the relation between the focus score and defocus spread of the
second label.
[0107] As shown in FIG. 10, the curves 704 and 706 have different
local maxima 708 and 710, respectively. The local maxima 708 and
710 have vertical axes 712 and 714 extending therethrough. The
vertical axes 712 and 714 may indicate where actual or optimal
focal planes for the corresponding labels are located for an
optical assembly of the imaging subsystem. Accordingly, the first
and second spectral bands are detected with a higher degree of
focus at different focal planes. The local maxima 708 and 710 (or
the vertical axes 712 and 714) may be separated from each other by
a distance D. The distance D represents the separation or spacing
between the focal planes of the respective labels for the optical
assembly. The distance D may be similar to a focal offset or shift
dz as described elsewhere.
[0108] Also shown in FIG. 10, the local maxima 708 and 710 have
approximately equal maximum focus scores FS.sub.MAX. However, in
some embodiments, the curves 704 and 706 may not have approximately
equal maximum focus scores FS.sub.MAX. In such embodiments, the
focus scores may be multiplied by a factor so that the maximum
focus scores FS.sub.MAX of the curves 704 and 706 are approximately
equal.
[0109] The curves 704 and 706 intersect each other at an
intersection point 728. A location of the intersection point 728 is
based upon a shape of the curves 704 and 706, but is generally
located at approximately half-way between the vertical axes 712 and
714 along the independent axis within a target region. The target
region may represent a range of acceptable z-positions of the
sample relative to the optical assembly. Furthermore, the
intersection point is generally located within a desired focus
score range FS.sub.RANGE. As will be described in greater detail
below, the imaging subsystem may control the focus so that the
focus scores for different images are about within the desired
focus score range FS.sub.RANGE. For example, the imaging subsystem
may move the z-position of the sample so that focus scores are
within the target region. The imaging subsystem may also move the
optical assembly relative to the sample so that the focus scores
are within the target region.
[0110] The desired focus score range FS.sub.RANGE and the target
region may be configured differently for different analysis
protocols. In some embodiments, the desired focus score range
FS.sub.RANGE is sufficient to enable the analysis module to
determine what local areas in a flow cell or microarray interacted
with a reagent or analyte and, optionally, to what degree. In other
embodiments, the desired focus score range FS.sub.RANGE is
generally sufficient to enable the analysis module to determine
which microbeads interacted with a target analyte and, optionally,
to what degree. In the illustrated embodiment, the desired focus
score range FS.sub.RANGE may be something less than the maximum
focus scores FS.sub.MAX associated with the first and second
labels.
[0111] FIG. 11 shows an exemplary situation that may be encountered
by an imaging subsystem when scanning a sample for different light
signals, such as the emission signals associated with different
first and second labels. In the exemplary embodiment, a common
region R is scanned for light emissions from first and second
labels in the sample. The dashed lines in FIG. 11 indicate focal
planes 720 and 722 of the different labels for the optical assembly
of the imaging subsystem. The focal planes 720 and 722 are
separated by the distance D. The solid lines of the regions R in
FIG. 11 represent a relative position of the sample regions with
respect to the focal planes 720 and 722.
[0112] After the region R is scanned to detect the images, a focus
score of each image is determined. In the exemplary embodiment, the
images of the first and second labels are acquired simultaneously.
However, the images may also be obtained sequentially. The focus
scores of the two images may be analyzed (e.g., compared) to
determine how to change or adjust the focal settings to dynamically
control the focus of the system. Changing or adjusting a focal
setting of the imaging subsystem includes relatively moving the
sample along the viewing axis 725 by moving the sample or moving
the optical assembly. Changing the focal setting of the imaging
subsystem may also include moving or reconfiguring the focus
components of the optical assembly to change the actual focal
plane(s).
[0113] By way of example and with respect to FIG. 11, a first
region R.sub.1 may be excited by light sources (e.g., lasers)
configured to excite the first and second labels within the sample.
After excitation, the imaging subsystem scans the region R.sub.1 of
the sample and obtains first and second images relating to the
first and second labels, respectively. The imaging subsystem
determines focus scores for each of the first and second images of
the scanned region R.sub.1. The focus scores may correspond to the
coefficient of variation in contrast, spot size, or another image
quality parameter as described herein. The focus score associated
with the first label is indicated in FIG. 10 as FS.sub.1 and is
located about within the desired focus score range FS.sub.RANGE.
The focus score associated with the second label is indicated in
FIG. 10 as FS.sub.2 and is not located about within the desired
focus score range FS.sub.RANGE.
[0114] After obtaining the focus scores FS.sub.1 and FS.sub.2 of
images corresponding to the different labels, the imaging subsystem
may analyze the focus scores FS.sub.1 and FS.sub.2. For example,
the imaging subsystem may compare the focus scores and determine
which focus score is greater than (or less than) the other and to
what degree. As shown in FIG. 10, if the focus score FS.sub.1 for
the first label is greater than the focus score FS.sub.2 for the
second label, then the sample is located to the left of the target
region (e.g., below the target region). The imaging subsystem may
also determine a difference in the focus scores FS.sub.1 and
FS.sub.2. The sample and/or optical assembly may be moved relative
to each other so that the sample is moved closer to the target
region between the focal planes 720 and 722. The amount of movement
may be based upon the difference between the focus scores FS.sub.1
and FS.sub.2. For example, the sample may be moved a shift Gz along
the viewing axis 725 closer to the target region. As shown in FIG.
11, the sample is moved to a position between the focal planes 720
and 722.
[0115] As another example, a second region R.sub.2 of the same or
different scan may be excited by light sources (e.g., lasers)
configured to excite the first and second labels within the sample.
After excitation, the imaging subsystem scans the region R.sub.2 of
the sample and obtains first and second images relating to the
first and second labels, respectively. The imaging subsystem
determines focus scores FS.sub.3 and FS.sub.4 for the first and
second images, respectively. The imaging subsystem may analyze the
focus scores FS.sub.3 and FS.sub.4 as described above and determine
that the focus score FS.sub.4 for the second label is greater than
the focus score FS.sub.3 for the first label. The imaging subsystem
may also determine a difference in the focus scores FS.sub.3 and
FS.sub.4. As such, the sample is located to the right of the target
region (e.g., above the target region) as shown in FIG. 10. The
sample and/or optical assembly may be moved relative to each other
so that the sample is moved closer to the target region. The amount
of movement may be based upon the difference between the focus
scores FS.sub.3 and FS.sub.4. For example, the sample may be moved
a shift Hz along the viewing axis 725 closer to the target region.
As shown in FIG. 11, the sample is moved to a position between the
focal planes 720 and 722.
[0116] As another example, the imaging subsystem may scan a region
R.sub.3 of the sample after excitation as described above. The
region R.sub.3 may be positioned within the target region of the
imaging subsystem. The focus scores FS.sub.5 and FS.sub.6 of the
images for the first and second labels, respectively, may both be
within the FS.sub.RANGE. The imaging subsystem may determine that
the focus score FS.sub.5 is greater than the focus score FS.sub.6.
The imaging subsystem may also determine a difference in the focus
scores FS.sub.5 and FS.sub.6. As such, the sample and/or optical
assembly may be moved relative to each other so that the sample is
moved upward. For example, the sample may be moved a shift Iz along
the viewing axis 725. However, the shift Iz may be smaller than the
shift Gz and Hz because the difference between the focus scores
FS.sub.5 and FS.sub.6 may be smaller than a predetermined
amount.
[0117] FIG. 12 illustrates a method 800 in accordance with another
embodiment. At 801, a method for controlling focus dynamically for
an optical imager is initiated. At 802, first and second images of
a scan region of a sample are obtained. The first image may include
detected light emissions of a spectral band or channel from a first
label, and the second image may include detected light emissions of
a spectral band or channel from a second label. By way of example,
the first label may be a FAM-type label and the second label may be
a NIR-type label. The optical assembly may have different focal
planes for the first and second labels.
[0118] In alternative embodiments, the first image may include
detected light emissions from a spectral band or channel, but the
second image may include detected light signals that were reflected
or refracted by an optical substrate in the scan region.
[0119] At 803, the first and second images are analyzed to
determine focus scores as described above. The focus scores of each
image may be plotted along a focus score curve. Optionally, at 804,
the focus score for at least one of the first and second labels is
multiplied by a factor so that local maxima of the focus score
curves are substantially equal. At 805, the first and second focus
scores are compared. By comparing the first and second focus
scores, the optical imager may determine whether the images
acquired by the optical imager have an acceptable degree of focus.
For example, if the focus score associated with the first label is
greater than the focus score associated with the second label by a
predetermined difference, then the optical imager may determine
that the sample is located below a target region. If the focus
score associated with the first label is less than the focus score
associated with the second label by a predetermined difference,
then the optical imager may determine that the sample is located
above the target region. In some embodiments, the predetermined
difference is any amount greater than zero.
[0120] At 806, the optical assembly and the sample are moved
relative to each other based upon the comparison of the focus
scores. The sample and the optical assembly may be moved relative
to each other by a predetermined amount. In some embodiments, the
predetermined amount is preset such that the optical assembly and
the sample are moved relative to each other regardless of the
difference between the focus scores. In other embodiments, a
difference between the focus scores may facilitate determining an
amount to move the sample and the optical assembly relative to each
other. Additionally or alternatively, a difference between the
focus score and the local maximum of the corresponding focus score
curve may facilitate determining an amount to move the sample and
the optical assembly relative to each other. At 807, the optical
imager queries whether the scan is done. If the scan is not done,
the optical imager may return to step 802 and repeat steps 802-806
for another scan region.
[0121] Before or during the method 800, the optical imager may be
trained to determine the distance separating the local maxima of
the focus curves. For example, the optical imager may determine the
focus curves for a red spectral band and a green spectral band and
also determine the optimal focal planes for both spectral bands.
Before, after, or during the method 800, the optical imager may
also be re-calibrated to facilitate maintaining the distance
separating the local maxima of the focus curves.
[0122] FIGS. 13 and 14 display one exemplary embodiment of a
flowcell. The flowcell may be held at holder 118 to convey samples,
such as sample 120. As can be seen, the particular flowcell
embodiment, flowcell 400, comprises base layer 410 (e.g., of
borosilicate glass 1000 .mu.m in depth), channel layer 420 (e.g.,
of etched silicon 100 .mu.m in depth) overlaid upon the base layer,
and cover, or top, layer 430 (e.g., 300 .mu.m in depth). When the
layers are assembled together, enclosed channels are formed having
inlets/outlets at either end through the cover. As will be apparent
from the description of additional embodiments below, some
flowcells can comprise openings for the channels on the bottom of
the flowcell.
[0123] It will be appreciated that while particular flowcell
configurations are presented herein, such configurations should not
necessarily be taken as limiting. Thus, for example, various
flowcells herein can comprise different numbers of channels (e.g.,
1 channel, 2 or more channels, 4 or more channels, or 6, 8, 10, 16,
or more channels, etc.). Additionally, various flowcells can
comprise channels of different depths and/or widths (different both
between channels in different flowcells and different between
channels within the same flowcell). For example, while the channels
formed in the cell in FIGS. 13-14 are 100 .mu.m deep, other
embodiments can optionally comprise channels of greater depth
(e.g., 500 .mu.m) or lesser depth (e.g., 50 .mu.m).
[0124] The imaging system 10 may be configured to utilize
diffraction grating based encoded optical identification elements
(such as microbeads). The microbeads have embedded codes therein or
thereon. The microbeads may be similar to or the same as those
described in pending U.S. patent application Ser. No. 10/661,234,
entitled Diffraction Grating Based Optical Identification Element,
filed Sep. 12, 2003, which is incorporated herein by reference in
its entirety, discussed more hereinafter. A bead cell may be
similar to or the same as that described in pending U.S. patent
application Ser. No. 10/661,836, entitled "Method and Apparatus for
Aligning Microbeads in Order to Interrogate the Same", filed Sep.
12, 2003, and U.S. Pat. No. 7,164,533, entitled "Hybrid Random
Bead/Chip Based Microarray", issued Jan. 16, 2007, as well as
pending US patent applications, Ser. No., 60/609,583, entitled
"Improved Method and Apparatus for Aligning Microbeads in Order to
Interrogate the Same", filed Sep. 13, 2004, Ser. No. 60610910,
entitled "Method and Apparatus for Aligning Microbeads in Order to
Interrogate the Same", filed Sep. 17, 2004, each of which is
incorporated herein by reference in its entirety.
[0125] FIG. 15 illustrates an imaging system 600 for detecting
bioassays implemented in accordance with an alternative embodiment.
The system 600 images encoded microparticles utilizing two CCD
cameras 602 and 604 for the simultaneous acquisition of a
reflectance and fluorescence image. The system 600 may be
configured as an inverted epi-fluorescence microscope. A well plate
606 includes multiple wells 608 that are imaged. The well plate 606
is placed on a microscope stage 610. The stages 610 may correspond
to holder 118 (FIG. 2) and the well plate may hold samples. The
stage may move in x and y directions. Particles that have been
dispensed into the well 608 in a fluid settle by gravity to the
bottom surface. Each well 608 or groups of wells 608 may represent
regions, for which images are acquired. Light coming from the light
source 612 goes through the excitation filter 614 which selects the
illuminating wavelength. The illuminating light reflects off the
beam splitter 616 and travels up through the objective 620. The
light returned to objective 620 may include emission and/or
transmission light. The objective 620 may be moved in the
z-direction to adjust the focal plane. The imaged area is referred
to as the "field" or "field area". Reflected, transmitted, or
emitted light (know together as the collection light) travels back
down the objective and passes through the first beam splitter 616.
The collection light then passes through the second beam splitter
622 which breaks it into the reflectance path and the fluorescence
path. The emission filter 624 is located in the fluorescence path
and selects the appropriate fluorescence emission wavelength. The
light in the fluorescence path is recorded with the fluorescence
CCD camera 602. The light in the reflectance path is recorded with
the reflectance CCD camera 604.
[0126] The system 600 also includes a computer 650 having an image
analysis module 652, a focus control module 654, and memory 656
that operate in the manner discussed above. The memory 656 stores
the images 658 and 660 captured by the detectors 602 and 604. The
analysis module 652 and the focus control module 654 perform the
shifting, analyzing and adjusting operations discussed above in
connection with the embodiment of FIGS. 2-6. For example, the focus
control module 654 controls the z-distance between the objective
620 and the well plate 606. The focus control module 654 introduces
shifts into the z-distance between the objective 620 and the well
plate 606. The analysis module 652 analyzes the images 658 and/or
660 to identify the focus score associated with a well 608 and the
focus control module 654 adjusts the z-distance before imaging the
next well 608.
[0127] It is to be understood that the above description is
intended to be illustrative, and not restrictive. For example, the
above-described embodiments (and/or aspects thereof) may be used in
combination with each other. In addition, many modifications may be
made to adapt a particular situation or material to the teachings
of the invention without departing from its scope. While the
specific components and processes described herein are intended to
define the parameters of the various embodiments of the invention,
they are by no means limiting and are exemplary embodiments. Many
other embodiments will be apparent to those of skill in the art
upon reviewing the above description. The scope of the invention
should, therefore, be determined with reference to the appended
claims, along with the full scope of equivalents to which such
claims are entitled. In the appended claims, the terms "including"
and "in which" are used as the plain-English equivalents of the
respective terms "comprising" and "wherein." Moreover, in the
following claims, the terms "first," "second," and "third," etc.
are used merely as labels, and are not intended to impose numerical
requirements on their objects. Further, the limitations of the
following claims are not written in means-plus-function format and
are not intended to be interpreted based on 35 U.S.C. .sctn.112,
sixth paragraph, unless and until such claim limitations expressly
use the phrase "means for" followed by a statement of function void
of further structure.
* * * * *