U.S. patent application number 15/641134 was filed with the patent office on 2018-05-10 for method and system for multiplex genetic analysis.
The applicant listed for this patent is APPLIED BIOSYSTEMS, LLC. Invention is credited to Eric S. NORDMAN, Mark F. OLDHAM, Timothy WOUDENBERG.
Application Number | 20180127818 15/641134 |
Document ID | / |
Family ID | 37532840 |
Filed Date | 2018-05-10 |
United States Patent
Application |
20180127818 |
Kind Code |
A1 |
NORDMAN; Eric S. ; et
al. |
May 10, 2018 |
METHOD AND SYSTEM FOR MULTIPLEX GENETIC ANALYSIS
Abstract
The present disclosure provides apparatus, systems and method
for detecting separately and substantially simultaneously light
emissions from a plurality of localized light-emitting analytes. A
system according to exemplary embodiments of the present disclosure
comprises a sample holder having structures formed thereon for
spatially separating and constraining a plurality of light-emitting
analytes each having a single nucleic acid molecule or a single
nucleic acid polymerizing enzyme, a light source configured to
illuminate the sample holder, an optical assembly configured to
collect and detect separately and substantially simultaneously
light emissions associated with the plurality of light emitting
analytes. The system may further include a computer system
configured to analyze the light emissions to determine the
structures or properties of a target nucleic acid molecule
associated with each analyte.
Inventors: |
NORDMAN; Eric S.; (Palo
Alto, CA) ; OLDHAM; Mark F.; (Emerald Hills, CA)
; WOUDENBERG; Timothy; (San Francisco, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
APPLIED BIOSYSTEMS, LLC |
Carlsbad |
CA |
US |
|
|
Family ID: |
37532840 |
Appl. No.: |
15/641134 |
Filed: |
July 3, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15230687 |
Aug 8, 2016 |
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15641134 |
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11423403 |
Jun 9, 2006 |
9410889 |
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15230687 |
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60689692 |
Jun 10, 2005 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 21/6486 20130101;
G01N 21/648 20130101; C12Q 1/6874 20130101; G01N 21/6428 20130101;
G01N 2021/6441 20130101; C12Q 1/6869 20130101; C12Q 2563/107
20130101; C12Q 1/6869 20130101; G01N 21/6452 20130101 |
International
Class: |
G01N 21/64 20060101
G01N021/64 |
Claims
1.-60. (canceled)
61. An apparatus for sequencing a plurality of target nucleic acid
molecules, comprising: a sample holder configured to separate and
confine a plurality of source points each comprising a single one
of the target nucleic acid molecules, a fraction of a nucleic acid
molecule, or a nucleic acid polymerizing enzyme molecule; a light
source configured to direct excitation light toward the sample
holder at an angle with respect to a normal of the sample holder,
the excitation light illuminating the source points and causing the
source points to fluoresce; at least one detector; and an optical
assembly configured to collect fluorescent signals from illuminated
source points to form images of the source points on the at least
one detector.
62. The apparatus of claim 61 wherein the at least one detector has
multiple pixel elements and the optical assembly is configured to
form spatially resolved images of the source points on the at least
one detector.
63. The apparatus of claim 62 wherein the angle is within
10.degree. of a Brewster's angle associated with the sample
holder.
64. The apparatus of claim 63 wherein the excitation light is
linearly polarized with an electric field vector parallel to a
plane of incidence upon the sample holder.
65. The apparatus of claim 61 wherein the sample holder comprises a
transparent substrate and the excitation light enters the
transparent substrate at an angle selected to cause total internal
reflection when a reflected portion of the excitation light
impinges upon an internal surface of the transparent substrate.
66. The apparatus of claim 65 wherein the sample holder further
comprises a transparent member in direct or fluidic contact with
the transparent substrate and the light source is configured to
direct the excitation light toward the sample holder such that the
excitation light enters the transparent substrate through the
transparent member.
67. The apparatus of claim 65 wherein the sample holder further
comprises a prism, grism or grating formed on or attached to a
portion of a bottom surface of the transparent substrate, and
wherein the excitation light is coupled into the transparent
substrate through the prism, grism or grating.
68. The apparatus of claim 65 wherein the excitation light is
directed toward a side of the transparent substrate.
69. The apparatus of claim 68 wherein the side has a beveled
surface forming an angle with a bottom surface of the transparent
substrate, the bottom surface being opposite to a top surface for
supporting the plurality of source points.
70. The apparatus of claim 62 wherein the at least one detector is
a CCD-based detector selected from a group consisting of EMCCD,
ICCD, and EBCCD.
71. The apparatus of claim 62, wherein the optical assembly
comprises at least one spectrum dispersing device and each image of
a source point on the at least one detector is spectrally
resolved.
72. The apparatus of claim 71 wherein the spectrum-dispersing
device is a grating, prism, or grism.
73. The apparatus of claim 72 wherein the optical assembly
comprises a plurality of dichroic, highpass, lowpass, notch, or
bandpass filters configured to direct fluorescent light of
different ranges of wavelengths from each source point to different
pixel elements of the at least one detector or to different ones of
the at least one detector.
74. The apparatus of claim 61 wherein the sample holder comprises a
metallic film over a transparent substrate, the metallic film
having patterns configured to spatially confine and separate the
plurality of source points.
75. The apparatus of claim 74 wherein the patterns form channels in
the metallic film, each channel having a width that is smaller than
a wavelength of the excitation light and a length sufficiently
large to allow an oligonucleotide molecule to be stretched along
the channel.
76. The apparatus of claim 75 wherein the light source is
configured to direct linearly polarized excitation light toward the
sample holder, and wherein an electric field vector in the
excitation light is oriented along a width direction of the
channels.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims a priority benefit under 35 U.S.C.
.sctn. 119(e) from U.S. Provisional Application No. 60/689,692
filed Jun. 10, 2005, which is incorporated herein by reference.
FIELD
[0002] The present application relates to molecular analysis, and
more particularly to methods and systems for multiplex genetic
analysis of single molecule nucleic acid synthesis.
INTRODUCTION
[0003] The information stored in a DNA molecule depends on
particular sequences of nucleotides, which are bases or building
blocks of the DNA molecule. DNA sequencing allows the determination
of the nucleotide sequence of a particular DNA segment. A
conventional method of DNA sequencing starts with a defined
fragment of a DNA molecule as a template. Based on this template, a
population of molecules differing in size by one base of a known
composition is generated. The population of molecules is then
fractioned based on size using, for example, acrylamide or agarose
gel electrophoresis of single-stranded DNA molecules. The base at
the truncated end of each of the fractionated molecules is
thereafter determined to establish the nucleotide sequence.
[0004] A sequencing method called dideoxy sequencing was developed
by Fred Sanger. His method is based on DNA synthesis in the
presence of dideoxy nucleotides (ddNTP), which differ from normal
deoxynucleotides (dNTP) in that they lack a 3'-hydroxyl group so
that once a dideoxy nucleotide is incorporated, it will terminate
strand synthesis. The procedure for dideoxy sequencing starts with
setting up four reactions each in a different tube containing the
single strand DNA to be sequenced, labeled (tagged) primer, DNA
polymerase, normal dNTPs, and a different ddNTP (i.e. for A, T, C,
or G). A dideoxy nucleotide will be incorporated, randomly, at each
point the corresponding nucleotide occurs in the template strand.
Each time a dideoxy nucleotide is incorporated, it will stop
further DNA replication. This will generate a set of fragments of
various lengths, each fragment corresponding to the point at which
there is a nucleotide complementary to the dideoxy nucleotide. The
fragments are then separated based on their length by
electrophoresis. With the smaller fragments migrating faster, the
sequence can be determined by associating the base composition with
each fragment.
[0005] The above technique for DNA sequencing suffer from the
disadvantage that sample preparation is relatively complex in order
to ensure that the tubes contain the same DNA molecules or
fragments of the same DNA molecules to be sequenced. This leads to
increased costs and the possibility of error. A simpler method
results if molecule-based investigation techniques are used to
observe the synthesis of a single DNA molecule. Because only one
molecule is being observed, there is no need to ensure that all of
the surrounding molecules are the same.
[0006] Specialized tools for imaging and spectroscopy have been
developed to characterize nanomaterials and nanomaterials-related
phenomenon. Techniques for constructing these tools comprise
near-field scanning optical microscopy (NSOM) and single molecule
spectroscopy (SMS). These techniques offer unique capabilities for
investigating properties at the molecular level owing to their high
spatial resolution, chemical sensitivity, and their ability to
determine dynamical properties such as molecule binding/unbinding
kinetics and the structural dynamics of polymers. For example, a
sample-scanning confocal fluorescence microscope using SMS
developed by McNeil et al. has demonstrated spatial resolution of
.about.400 nm, and single molecule sensitivity. It uses a detector
system having a single-photon avalanche diode and a sensitive
TE-cooled CCD spectrometer, permitting the ability to monitor
fluorescence in the range of 400 to 1100 nm at a resolution of 20
nm and the ability to conduct time-lapse fluorescence spectroscopy
with single molecule sensitivity.
[0007] The single-molecule techniques described above, however,
often employ femtoliter-scale observation volumes and require the
use of picomolar to nanomolar sample concentrations to ensure that
on average only one molecule will be present in the sample volume.
These concentrations are far lower than those that normally occur
in nature. Thus, molecule dynamics that are affected by
concentration cannot be suitably tested using the techniques. To
overcome the deficiencies of NSOM and SMS techniques, other
developments have been proposed. For example, Levene et al.
describes a device for single molecule analysis employing a sample
plate, which has 50 nm-diameter holes in a 100 nm thick aluminum
film on a fused silica coverslip. When the holes are illuminated
from under the fused silica coverslip, the holes act as zero-mode
waveguides prohibiting the light from going through the aluminum
film because the diameter of the holes are much smaller than the
wavelength of the light, which is about 400-700 nm. The light,
however, does generate an evanescent field that extends about 10 nm
into the cavity of each illuminated hole producing a
zeptoliter-scale effective observation volume near the opening of
the hole. See Levene et al., US Patent Application Publication
Number 2003/0174992 A1, which is incorporated herein by
reference.
[0008] The small observation volume provided by the zero-mode
waveguides described by Levene, however, raises other challenges
spanning from sample preparation, signal detection, noise or
background suppression, data collection and data analysis
algorithms. Accordingly, significant further developments are
needed.
[0009] The present teaching in one aspect comprises an affordable
high-sensitivity and high-throughput system and method for
single-molecule analysis that performs at a lower cost relative to
conventional systems used in sequencing, resequencing, and SNP
detection. These and other features of the present teaching are set
forth herein.
SUMMARY
[0010] The present disclosure provides apparatus, systems and
method for analyzing a plurality of molecules by detecting
separately and substantially simultaneously light emissions from a
plurality of localized light-emitting analytes each including a
single one of the plurality of molecules. The detected light
emissions, after being properly analyzed, can be used to deduce the
structure or properties of each of the plurality of molecules. In
some embodiments, the apparatus, systems and methods can be used
for nucleic acid sequencing, nucleic acid resequencing, and/or
detection and/or characterization of single nucleotide polymorphism
(SNP analysis) including gene expression.
[0011] In various embodiments, the present invention can provide an
apparatus for sequencing a plurality of target nucleic acid
molecules including a sample holder configured to separate and
confine a plurality of source points each including a single one of
the target nucleic acid molecules, a fraction of a nucleic acid
molecule, or a nucleic acid polymerizing enzyme molecule, a light
source configured to direct excitation light toward the sample
holder at an angle with respect to a normal of the sample holder,
the excitation light illuminating the source points and causing the
source points to fluoresce, at least one detector, and an optical
assembly configured to collect fluorescent signals from illuminated
source points to form images of the source points on the at least
one detector.
[0012] In various embodiments, the present invention can provide a
method for sequencing a plurality of target nucleic acid molecules,
including subjecting a plurality of source points of a sample
holder to nucleic acid polymerization reactions, wherein the source
points each include fluorescence-labeled bases, primers, and at
least one nucleic acid polymerizing enzyme molecule, and wherein
the plurality of source points each has a single one of the target
nucleic acid molecules, directing excitation light toward the
sample holder at an angle with respect to a normal of the sample
holder to illuminate the source points and to cause the source
points to fluoresce, and collecting fluorescent signals from the
illuminated source points and focusing the fluorescent signals onto
at least one detector to form images of the source points on the at
least one detector to determine time sequences of base
incorporations in the polymerization reactions.
[0013] In various embodiments, the present invention can provide a
method for sequencing a plurality of target nucleic acid molecules,
including subjecting a plurality of source points of a sample
holder to nucleic acid polymerization reactions, wherein the source
points each include fluorescence-labeled bases, primers, and at
least one of the target nucleic acid molecules, and wherein the
plurality of source points each has a single nucleic acid
polymerizing enzyme molecule, directing excitation light toward the
sample holder at an angle with respect to a normal of the sample
holder to illuminate the source points and to cause the source
points to fluoresce, and collecting fluorescent signals from the
illuminated source points and focusing the fluorescent signals onto
at least one detector to form images of the source points on the at
least one detector to determine time sequences of base additions in
the polymerization reactions.
[0014] In various embodiments, the present invention can provide a
method for sequencing a plurality of target nucleic acid molecules,
including enriching a sample holder with a plurality of source
points each having a single one of the target nucleic acid
molecules and/or a single nucleic acid polymerizing enzyme
molecule, subjecting the plurality of source points to nucleic acid
polymerization reactions, (1) wherein when the source points have a
single one of the target nucleic acid molecules, the source points
each further include fluorescence-labeled bases, primers, and at
least one nucleic acid polymerizing enzyme molecule, (2) wherein
when the source points have a single nucleic acid polymerizing
enzyme molecule, the source points each further include
fluorescence-labeled bases, primers, and at least one of the target
nucleic acid molecules, and (3) wherein when the source points have
a single one of the target nucleic acid molecules and a single
nucleic acid polymerizing enzyme molecule, the source points each
further include fluorescence-labeled bases and primers, directing
excitation light toward the sample holder at an angle with respect
to a normal of the sample holder to illuminate the source points
and to cause the source points to fluoresce, and collecting
fluorescent signals from the illuminated source points and focusing
the fluorescent signals onto at least one detector to form images
of the source points on the at least one detector to determine time
sequences of base incorporations in the polymerization
reactions.
[0015] A system according to exemplary embodiments of the present
disclosure comprises a sample holder having structures formed
thereon for spatially separating and constraining a plurality of
light-emitting analytes each having a single one of the plurality
of molecules to be analyzed. In exemplary embodiments, each of the
plurality of molecules is a single nucleic acid molecule, a
fraction of the nucleic acid molecule, an oligonucleotide molecule,
or a single nucleic acid polymerizing enzyme. The system further
comprises a light source configured to illuminate the sample
holder, an optical assembly configured to collect and detect
separately and substantially simultaneously light emissions
associated with the plurality of light emitting analytes. The
system may further include a computer system configured to analyze
the light emissions to determine the structures or properties of a
target nucleic acid molecule associated with each analyte.
[0016] In one exemplary embodiment of the present invention, the
light source is configured to produce excitation light that is
directed toward the sample holder at an angle with respect to a
normal of a plane associated with the sample holder. In further
embodiments, excitation light is directed toward the sample holder
such that total internal reflection occurs and the excitation light
is recycled multiple times before exiting the sample holder.
[0017] In one exemplary embodiment of the present invention, the
optical assembly comprises at least one pixilated sensor device
such as a charge coupled device (CCD) detector or CMOS detector
configured to detect substantially simultaneously light emissions
from the multiple localized light-emitting analytes. In further
embodiments, the optical assembly is configured to disperse
spectrally the light emitted from the multiple localized
light-emitting analytes onto the detector(s) so that different
frequency bands of the emitted light are detected by different
areas of the detector(s).
[0018] These and other features of the present teaching are set
forth herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] The skilled artisan will understand that the drawings,
described below, are for purposes of illustration only, and are not
intended to limit the scope of the present teaching in any way.
[0020] FIG. 1 is a block diagram of an exemplary embodiment of a
high throughput system for single molecule analysis.
[0021] FIG. 2 is a top view of a sample holder in the system.
[0022] FIG. 3 is a cross-sectional view of a portion of the sample
holder.
[0023] FIG. 4 is a flowchart illustrating an exemplary embodiment
of a method useful for enriching the sample holder with a plurality
of spatially restrained source points.
[0024] FIG. 5 is a diagram illustrating further embodiments of a
method useful for enriching the sample holder with a plurality of
spatially constrained source points.
[0025] FIG. 6 is a 3-dimensional view of a portion of the sample
holder according to further embodiments of the present
teaching.
[0026] FIG. 7 is a diagram illustrating a DNA sequencing process
along a channel on the sample holder according to exemplary
embodiments of the present teaching.
[0027] FIGS. 8A and 8B are flowcharts illustrating further
embodiments of a method useful for placing a plurality of source
points on the sample holder in FIG. 6.
[0028] FIGS. 8C and 8D are diagrams illustrating embodiments of a
setup for stretching each of a plurality of oligonucleotide
molecules along a bottom surface of a channel on the sample
holder.
[0029] FIG. 9 is a block diagram illustrating exemplary embodiments
of an optical arrangement useful for illuminating the source points
placed on the sample holder.
[0030] FIGS. 10A-10E are block diagrams illustrating further
embodiments of an optical arrangement useful for illuminating the
source points placed on the sample holder.
[0031] FIG. 11 is top view of a sample holder showing a plurality
of source points.
[0032] FIG. 12 is a top view of an image plane in a detector in the
system in FIG. 1 according to exemplary embodiments.
[0033] FIG. 13 is a diagram illustrating an optical assembly for
detecting light signals from the source points on the sample holder
according to exemplary embodiments.
[0034] FIGS. 14A-14C are diagrams illustrating further embodiments
of an optical assembly for detecting light signals from the source
points on the sample holder.
[0035] FIG. 15A is a block diagram illustrating a frame transfer
CCD array in a detector in the system in FIG. 1 according to
exemplary embodiments of the present teaching.
[0036] FIG. 15B is a block diagram illustrating an interline CCD
array in the detector in the system in FIG. 1 according to
exemplary embodiments of the present teaching.
[0037] FIG. 16A are examples of normalized fluorescent spectra
corresponding to four different fluorescent dyes.
[0038] FIG. 16B is a block diagram illustrating full spectrum
data.
[0039] FIG. 16C is a block diagram of the full spectrum data with
the most informative wavelengths distinguished from less
informative wavelengths.
[0040] FIG. 17A is a flowchart illustrating a method for reading
out CCD data associated with a plurality of source points according
to exemplary embodiments.
[0041] FIGS. 17B-17C are each a spreadsheet for estimating the
throughput of reading out data from a CCD array using the method in
FIG. 17A.
[0042] FIG. 18 is a block diagram of a computer system used in the
system in FIG. 1 according to exemplary embodiments of the present
teaching;
[0043] FIG. 19 is a histogram illustrating the number of photons in
different spectral bins detected from a single incorporation
event;
[0044] FIG. 20 is a flowchart of an exemplary embodiment of a
method useful for base determination according to the present
teaching; and
[0045] FIG. 21 is a plot of composite data over a plurality of time
bins according to exemplary embodiments of the present
teaching.
DESCRIPTION OF VARIOUS EMBODIMENTS
[0046] It is to be understood that both the foregoing summary and
the following description of various embodiments are exemplary and
explanatory only and are not restrictive of the present teachings.
In this application, the use of the singular comprises the plural
unless specifically stated otherwise. Also, the use of "or" means
"and/or" unless stated otherwise. Similarly, "comprise,"
"comprises," "comprising," "comprise," "comprises," and "including"
are not intended to be limiting.
[0047] Additionally, while certain embodiments are described in
detail herein, particularly embodiments suitable for analysis of
single molecule nucleic acid synthesis, it is to be understood the
apparatus, systems and methods of the present disclosure may be
employed in other applications for analysis of single molecules,
such as but not limited to directed resequencing, SNP detection,
and gene expression.
[0048] Furthermore, the figures in this application are for
illustration purposes and many of the figures are not to scale with
corresponding hardware. Many parts of the features in the figures
in this application are drawn out of scale purposefully for ease of
illustration.
[0049] Systems according to some embodiments of the present
disclosure generally comprise a sample holder configured to hold a
plurality of localized light-emitting analytes each comprising a
single one of a plurality of molecules to be analyzed, a light
source configured to illuminate the sample holder, and an optical
assembly configured to collect and detect light emitted from the
source points.
FIG. 1 is a block diagram of an exemplary embodiment of a system
100 for detecting and analyzing light emitted from the plurality of
light-emitting analytes. As shown in FIG. 1, system 100 comprises a
sample holder 110, a light source 120, and an optical assembly 130.
System 100 may further comprise a host computer system 140 (see
also FIG. 18) configured to analyze optical data detected by
optical assembly 130. System 100 may also comprise one or more
digital signal processors (DSP) or field programmable gate arrays
(FPGA) 150 coupled between optical assembly 130 and host computer
system 140. DSPs or FPGAs 150 can be used to execute algorithms for
base determination, as explained in more detail below.
[0050] System 100 may optionally comprise an index-matching prism
108. A space between the sample holder 110 and the optical assembly
130 may be filled with a fluid 104. The utility of the
index-matching prism 108 and the fluid 104 is discussed below.
Although for reasons discussed below, it may be advantageous to
direct the excitation light from the light source 120 to the sample
holder 110 at an angle, as shown by the solid line 121 in FIG. 1,
system 100 is not limited to such use, and the excitation light can
be directed via a dichroic filter 125 toward the sample holder
along a normal N of the sample holder 110, as shown by the dashed
lines in FIG. 1.
[0051] In exemplary embodiments of the present teaching, sample
holder 110 is configured to support and confine the plurality of
light-emitting analytes. For ease of discussion, each localized
light-emitting analyte will hereafter be referred to as a dye or a
"source point". In various embodiments, a dye or a source point
comprises a single nucleic acid molecule, a fraction of a nucleic
acid molecule, an oligonucleotide molecule, or a single nucleic
acid polymerizing enzyme. The dye or source point may also comprise
one or more other molecules, constituents, or reactants. The
emitted light from the complex can be used to deduce the structure
or properties of a target nucleic acid molecule.
[0052] In one exemplary embodiment, in applications employing
nucleic acid sequencing, each source point is a complex of a single
nucleic acid polymerizing enzyme, a target nucleic acid molecule,
and at least one incorporated or incorporating fluorescence-labeled
nucleotide analog. The source point is localized or spatially
constrained in at least one dimension that is less than the
wavelength of the excitation light. The fluorescent label on the
nucleotide analog emits fluorescent light upon illumination by
light source 120. In exemplary embodiments of the present teaching,
four different nucleotide analogs are labeled with four different
fluorescent dyes each having a unique emission spectrum. The four
different fluorescent dyes can also be associated with four
different frequency bands each corresponding to a peak in emission
intensity according to the respective spectrum. The four different
frequency bands are hereafter referred to as first, second, third,
and fourth frequency bands.
[0053] Thus, the time sequence of base incorporation can be
observed by detecting fluorescent signals from sequentially
incorporated nucleotide analogs associated with a source point. The
fluorescent light signals from different source points on the
sample holder 110 are substantially simultaneously collected and
detected by optical assembly 130 and are analyzed by computer
system 140 to determine the identities of the incorporated nucleic
acid molecule in each of the source points. To reduce or eliminate
interference between fluorescent signals associated with
consecutive incorporation events on a same source point, after
detection of an incorporation event, fluorescent label on the newly
incorporated nucleotide can be bleached, cleaved or otherwise
removed with a known technique. Photo-cleavable linkers may be
utilized to facilitate efficient and consistent removal of the
fluorescent labels.
[0054] In some embodiments, the source points are localized or
spatially constrained at different locations on sample holder 110
by immobilizing the single nucleic acid molecule or the single
nucleic acid polymerizing enzyme in each source point at one of the
locations. This allows separate and substantially simultaneous
detection of fluorescent emission from the plurality of source
points. A conventional method or one of the methods discussed below
can be used to immobilize the enzymes or the template nucleic acid
molecules.
[0055] FIG. 2 is a block diagram of a top-down view of sample
holder 110 according to exemplary embodiments. As shown in FIGS. 1
and 2, in exemplary embodiments, sample holder 110 comprises a
substrate 112 made of a material transparent to the excitation
light from light source 120 and to the fluorescent emissions from
the source points. A metallic film 114 is formed on a top surface
of substrate 112. Depending on specific applications, for reasons
discussed below, metallic film 114 may extend to the side surfaces
and edge portions of a bottom surface of the substrate 112, as
shown in FIG. 1. Sample holder 110 may further comprise a sealer
115 and a cover 116 for evaporation control. A space 118 is formed
between the cover 116 and the substrate 112, which space serves as
a sample chamber for holding a sample fluid that supplies at least
one of the constituents or reactants in each source point. In
various embodiments, in applications of nucleic acid sequencing,
the sample fluid comprises a fluorophore solution of different
types of fluorescent-labeled nucleotides. Sample holder 110 may
further comprise a fill hole 230 for filling the sample chamber 118
with the sample fluid and a drain hole 240 for draining the sample
fluid from the sample chamber. Fill hole 230 and drain hole 240 are
preferably located near two opposite corners of sample holder 110,
as shown in FIG. 2, for more complete draining and washing away of
sample fluid.
[0056] As shown in FIG. 2, sample holder 110 is configured to hold
a plurality of spatially separated and constrained source points
210 in a field of view 220 of the optical assembly 130. The spatial
separation and confinement of the source points 210 help in one
aspect to detect light signals from the source points 210
separately and substantially simultaneously. Although FIG. 2 shows
that the source points 210 on the sample holder 110 are arranged in
an array having two rows and a number of columns, such arrangement
is not necessary as long as the source points are sufficiently
spaced from each other so that the light signals from them can be
effectively resolved by optical assembly 130 When the source points
are arranged in an array, the array may be perfect, meaning each
array element site has an immobilized functional source point, or
imperfect, meaning at least one array element site is missing a
source point, has a source point that is not functional, or has
multiple source points that are too close together to allow
resolution by the optical assembly 130.
[0057] In various embodiments, the metallic film 114 on the top
surface of the substrate 112 has etched patterns forming cavities
for housing the plurality of source points and separating the
plurality of source points to allow resolution by the optical
assembly 130. In some embodiments, zero-mode waveguides, such as
those described in Patent Application Number US 2003/0174992 by
Levene et al, which is incorporated herein by reference, are formed
in metallic film 114, as shown in a cross-sectional view in FIG. 3.
Zero-mode waveguides are known in the art and can be created using
a variety of materials and methods. As a specific, non-limiting
example, substrate 112 is a fused silica substrate, metallic film
114 is an aluminum film formed on the fused silica substrate, and
an array of holes 310 are formed by masking and plasma etching the
aluminum film to create holes 310 in the aluminum film. Each hole
310 has a diameter that is substantially smaller than a wavelength
of the excitation light from light source 120 and a depth that is
sufficient to block transmission of the excitation light through
the hole. Thus, each hole 310 acts as a zero-mode waveguide for the
excitation light from light source 120, allowing the excitation
light, which comes to the waveguides from the substrate side, to
penetrate only a bottom portion 312 of the hole 310. At the same
time, the zero-mode waveguides also serves to block light emitted
or scattered from the sample fluid on the sample holder 110 except
emissions coming from any light emitting agents immobilized in the
bottom portions 312 of the waveguides or diffusing past the bottom
portions 312 of the waveguides.
[0058] Thus, in some embodiments, to allow the detection and
analysis of light emitted from the source points 210, each source
point 210 is immobilized in the bottom portion 312 of a zero-mode
waveguide 310, so that light emitted from the source point can
escape the hole 310, pass through substrate 112 and be collected by
optical assembly 130. Preferably, only one source point should be
present in the bottom portion 310 of a hole 310 because it would be
difficult for the optical assembly to distinguish the emitted light
from more than one source point in a single hole 310 considering
the size of the hole. Therefore, in the exemplary embodiment, holes
310 that either do not have any source point immobilized in the
bottom portion 312 or have more than one source point immobilized
in the bottom portion 312 do not contribute to the analysis and are
considered as empty sites in an array of source points 210.
[0059] For ease of discussion, the description hereafter will be
illustrated in the context of nucleic acid sequencing, while the
methods, systems and apparatus of the present teaching can be
applied to other types of molecular analysis. Methods of
immobilizing molecules involved in a genetic assay in waveguides
310 are described in detail in US Patent Application Number US
2003/0044781 by Korlach et al., which is incorporated herein by
reference. Using the methods described by Korlach, some of the
array of holes 310 can each contain a single DNA molecule or enzyme
immobilized in the bottom portion 312, while a large percentage of
the holes may contain none or multiple molecules in each of them
and are thus useless in the analysis.
[0060] FIG. 4 illustrates a flowchart of one embodiment of a method
400 for enriching the sample holder 110 with source points. Method
400 increases the efficiency and throughput of system 100 by
maximizing the percentage of holes 310 that have in each of them a
single source point in the bottom portion 312. As shown in FIG. 4,
according to exemplary embodiments of the present teaching, method
400 comprises the following steps: step 410 in which uncovered
portions of the substrate 112 are coated with streptavidin, step
420 in which a dilute solution including a plurality of molecules
each being a nucleic acid molecule or a nucleic acid polymerizing
enzyme is applied to the waveguides 310. Each of the plurality of
molecules has a photoactivatable biotin attached to it. The
concentration of the plurality of molecules in the solution is
selected to be lower than the optimal Poisson distribution so that
when the solution is applied to the waveguides 310 on the sample
holder 110, most of the waveguides 310 would be populated by zero
number of the molecules and that statistically few of the
waveguides 310 would be occupied by more than one of the
molecules.
[0061] Still referring to FIG. 4, method 400 further comprises step
430 in which a first group of waveguides are identified as each
being occupied by at least one nucleic acid molecule or a nucleic
acid polymerizing enzyme. The first group of waveguides can be
identified by using, for example, a simplified sequencing assay.
Method 400 further comprises step 440 in which light is shown on
each of the first group of waveguides. The light activates the
biotin attachment in those waveguides and thus immobilizes the
molecules using the biotin-streptavidin bound. Step 440 is followed
by an optional step 450 in which the solution is removed from the
sample holder by washing or inactivation, leaving only those
molecules bonded to the bottom of the waveguides. Step 450 is
followed by an optional step 460 in which another dilute solution
of the biotin attached molecules are applied to the waveguides 310
on sample holder 110. Method 400 further comprises step 470 in
which an additional group of waveguides are identified as each
being occupied by at least one nucleic acid molecule or a nucleic
acid polymerizing enzyme. In one embodiment, the additional group
of waveguides do not overlap with previously identified group(s) of
waveguides. Method 400 further comprises step 480 in which light is
shown on each of the additional group of waveguides and thus
immobilizes the nucleic acid molecules or enzymes in the additional
group of waveguides. Method 400 then repeats steps 450-480 until
most of the waveguides are populated by bound molecules. Note that
steps 450 and 460 are optional because, instead of carrying out
steps 450 and 460, one can simply wait for a period of time to
allow more of the plurality of molecules to diffuse into some of
the waveguides.
[0062] Optionally, after populating the waveguides with polymerase
molecules, a primer is attached to each polymerase molecule by a
flexible linker. Attaching the primer to the polymerase molecule
helps the analysis because the DNA template would be tethered and
not float away, allowing subsequent synthesis to occur on the same
template. In one aspect, this benefits the analysis by increasing
read lengths and throughput. Longer read lengths help to simplify
any fragment assembly problem.
[0063] In some embodiments, a method for enriching the sample
holder involves the use of nanobeads. As shown in FIG. 5, the
sample fluid comprise nanobeads 510, and the enzyme or nucleic acid
molecule 520 is attached to a nanobead 510 by a cleavable linker
515, in a manner that most of the nanobeads in the sample fluid
each have at most one enzyme or nucleic acid molecule attached. The
nanobeads are sized such that only one nanobead is likely to fit in
a waveguide 310. The enzyme or nucleic acid molecule 520 has a
photoactivatable linker 525 that allows attachment of the composite
including the nanobead, the nucleic acid molecule or enzyme, and
the cleavable linker 515 to an attachment site 530 at the bottom of
a waveguide 310. The attachment is activated by shining light from
the bottom of substrate 112. Since only those nanobeads each having
an enzyme or nucleic acid molecule with the photoactivatable linker
can bind to the substrate 312, and the presence of a nanobead in a
waveguide 310 excludes other enzyme or nucleic acid molecules from
diffusing into the same waveguide, there is no need to determine
which waveguides are occupied by the nanobeads before shining light
on the waveguides to activate the linkers 525. The shining of light
can be repeated later when more nanobeads with enzyme or nucleic
acid molecules attached thereon diffuse into other waveguides.
Thus, the nanobeads can be used to increase the number of
waveguides each having a single nucleic acid molecule or enzyme
attached therein. After binding, the nanobeads are removed from the
enzyme or nucleic acid molecules by, for example, chemically
cleaving the linkers 515 or dissolving the nanobeads.
[0064] In alternative embodiments of the present teaching, sample
holder 110 comprises slots or channels to facilitate confining the
plurality of source points 210 on the sample holder 110. FIG. 6
illustrates a 3-dimensional view of a plurality of channels 610
formed in metallic film 114 on substrate 112. As a non-limiting
example, channels 610 are formed in an aluminum film over a fused
silica substrate. Each channel 610 has a width w that is smaller
than a wavelength associated with light source 120. In exemplary
embodiments of the present teaching, light from light source 120 is
linearly polarized and the polarization direction is oriented with
the electric field vector in the light wave along the width
direction of the channels. Thus, only a bottom portion 612 in each
channel 610 would be illuminated by the excitation light from light
source 120, as shown in FIG. 6. Channels 610 can be formed using
conventional techniques, such as conventional semiconductor
processing or integrated circuit (IC) fabrication techniques.
[0065] Sample holder 110 with channels 610 formed thereon has
multiple advantages over a sample holder with zero-mode waveguide
holes 310 formed thereon. Because the fluorescent emissions are
largely unpolarized, they would not be attenuated when they try to
exit the channels 610 as much as when they try to exit holes 310 of
sub-wavelength dimension. So, more emitted light from sample holder
110 can be collected and detected by optical assembly 130,
resulting in increased signal to noise ratio. In addition, each
channel 610 can house a larger DNA template molecule if the DNA
molecule is oriented parallel to the channel, as shown in a
top-down view of the channel in FIG. 7. This way, the polymerase
can migrate down the template for a much longer distance without
exiting the illuminated volume 612. The DNA molecule can be
tethered so that it can remain in one location while the
polymerase, having a finite processivity, may fall off the template
and be replaced by another polymerase. This can lead to longer read
lengths and thus significantly simplified assembly processes,
especially during denovo sequencing. Although FIG. 7 shows that
channel 610 is closed at both ends 701 and 702, the channels 610 on
sample holder can be open on either or both ends by extending all
the way to the edge(s) of the sample holder, as shown in FIG. 8C
below.
[0066] The polymerase or template molecules can be attached to
sample holder 110 using conventional photoactivatable linkers. In
exemplary embodiments of the present teaching, channels 610 may
house more than one polymerase or template molecules attached to
sample holder 110 by flexible linkers that are placed in the
channels 610. The molecules should be attached to the channels 610
in a resolvable fashion, meaning that they are sufficiently spaced
from each other to allow efficient resolution of the emissions
therefrom by the optical assembly 130.
[0067] FIG. 8A illustrates a method 800 for enriching the sample
holder 110 with source points by attaching the polymerase or
template molecules in channels 610 in a resolvable fashion
according to exemplary embodiments of the present teaching. As
shown in FIG. 8A, method 800 comprises step 810 in which exposed
portions of the top surface of the substrate 112 are coated with a
photoactivatable linking substance such as PHOTOACTIVATABLE
BIOTIN.TM. (PAB), step 820 in which a solution of enzyme or
template molecules is applied to sample holder 110, and step 830 in
which the PAB is exposed to a pattern of light shone from the
bottom surface of the substrate 112. The pattern of light may be
created by interference or refraction using grating or other
conventional techniques and has interleaving lighted and dark areas
in each channel 610. The distance between two neighboring lighted
areas is selected based on the resolution of the optical assembly
130 so that emissions from the two lighted areas can be separately
and substantially simultaneously detected by the optical assembly
130. The PAB in the lighted area will be activated causing the
template molecules to be attached to the sample holder in those
areas, while the PAB in the dark areas will not be activated so no
template molecules will be attached to those areas. Method 800
further comprises step 840 in which sample holder 110 is washed to
remove unbound template molecules, leaving the bound enzyme or
nucleic acid molecules in each channel 610 and allowing the
formation of optically resolvable source points 210 on sample
holder 110.
[0068] Referring to FIG. 8B, instead of enzyme or template
molecules, a solution of oligonucleotide (oligo) molecules can be
applied to the substrate in step 820, and additional steps can be
used to stretch an oligo along the bottom surface of each of a
plurality of channels 610 on the sample holder 110. As shown in
FIG. 8B, to attach an oligo to the bottom surface of a channel 610,
method 800 further comprises step 822 in which an end of the oligo
is attached at one end 701 of the channel using a chemical linker,
such as a biotin-streptavidin or PNA-PNA hybridization binding,
where one part of the linker is attached to the end of the oligo
and the other part of the linker is bound to the substrate. Method
800 further comprises step 824 in which the oligo is stretched
along the channel and held to the bottom surface of the channel.
Many conventional techniques of stretching DNA molecules can be
used in step 824, including but not limited to hydrodynamic,
electrostatic, and magnetic manipulations. For example, the oligo
molecules can be stretched using dielectrophoresis, in which the
oligo is stretched by a direct current (DC) electric field or a
high-frequency (e.g., 1 MHz) and high-density (e.g., 1 MV/m)
alternating-current (AC) field applied between two electrodes.
[0069] In one embodiment, as shown in FIG. 8C, the sample holder
110 can be placed in a container 880 for holding the fluid
containing the oligonucleotides, an electrode 871 made of, for
example, Indium Ti, is placed above the end 701 of the channel, and
another electrode 872 made of the same or different conductive
material as electrode 871 is placed in the container 880 below the
bottom surface 612 of the channel 610 and near the other end 702 of
the channel. A field is provided between the two electrodes 871 and
872 such that the oligo 710 is stretched along the channel 610 and
held by the field along the bottom of the channel. Depending on the
relative lengths of the oligo 710 and the channel 610, the oligo
710 may extend beyond the channel toward the electrode 872, if the
channel is open at the end 702. Electrode 872 may also be placed
under the substrate 112, as shown in FIG. 8C.
[0070] With the electric field still on, step 830 is performed to
further attach the oligo 710 so that the field can be removed
later, preventing the field from interfering with sequencing
operation afterwards. The oligo in each of the plurality of
channels may be stretched and attached simultaneously using the
same or different electrodes.
[0071] After binding the enzyme, oligonucleotide, or target nucleic
acid molecules to the sample holder 110, the sample holder 110 is
placed in system 100. A fluorophore solution comprising
fluorescence labeled nucleotide analogs is applied to the sample
holder 110. In exemplary embodiments of the present teaching, the
speed of chemistry of incorporation can be altered by changing the
temperature, viscosity, and concentration of the fluorophore
solution, and/or by modifying the base chemistry. For example,
adding molecules such as dye molecules to the fluorophore solution
has been found to slow the rate of base incorporation. In addition,
the sample holder 110 in system 100 should ideally be under
temperature control to insure consistency. The temperature could be
changed during detection. For example, the temperature of the
sample holder 110 can be reduced to slow down or stop incorporation
activities until the rest of system 100 is ready to collect signals
from the sample holder 110, as discussed below.
[0072] To observe light emitted from the source points, excitation
light from light source 120 is directed towards the substrate side
of the sample holder 110, and signals from fluorescing nucleotides
are collected by optical assembly 130. The confinement of the
source points on sample holder 110 helps to distinguish the
fluorescent signals emitted by incorporated nucleotides in the
source points 210 from those emitted by freely diffusing
fluorescent ligands.
[0073] As explained in more detail below, multiple methods can be
used in exemplary embodiments for base determination. For example,
color, signal strength, bleaching life, fluorescent lifetime, and
incorporation time can be combined to gain better base
discrimination. The consistency of these measurements can be used
to predict a confidence value for the base determination.
Confidence values can be used to sort or weight the data and to
discard data of low quality, thus allowing automated consensus
generation from large amount of data. This can improve the quality
of the consensus as well as providing a measure of confidence.
[0074] Prior art systems, such as the one described by Levene et
al., 2003 in "Zero-Mode Waveguides for Single-Molecule Analysis at
High Concentrations," SCIENCE, Vol. 299:682, which article is
incorporated herein by reference, uses a confocal fluorescent set
up. The confocal fluorescent set up has multiple shortcomings.
First, the aluminum film reflects the excitation light directly
back into the collection optics. The reflected excitation light is
very intense compared to the fluorescent signals from incorporated
nucleotides. To attenuate the reflected light, multiple filters are
used, and each filter attenuates a significant percentage of the
already weak fluorescent signals. Furthermore, the excitation light
in the set up of Korlach and Levene, supra, can also excite
fluorescence in the optics. This unwanted fluorescence could pass
through the filters, increasing the background noise.
[0075] In exemplary embodiments of the present teachings,
excitation light from light source 120 is directed to the source
points in sample holder 110 in an off-axis manner such that
reflected excitation light, or a significant amount of it, could
not enter the optical assembly 130. In some embodiments, where
prism or wedge 108 is not provided, a light ray 901 from light
source 120 is directed to sample holder 110 at an angle .theta.
with respect to a normal direction N of substrate 112, as shown in
FIG. 9. As the substrate 112 is made of a transparent material,
such as fused silica, a relatively small first portion of the
incident light 901 is reflected by the bottom surface 910 of
substrate 112 and comes toward the optical assembly 103 as a first
reflected light ray 912, while a second portion of the incident
light enters the substrate at a different angle .theta.' with
respect to the normal N as a refracted light ray 914. Angle
.theta.' depends on angle .theta. and the refractive index n of the
substrate 112. The refracted light ray 914 impinges on the metallic
film at the angle .theta.' and a relatively large portion of the
refracted light ray 914 is likely to be reflected by the metallic
film 114 and comes toward the bottom surface 910 of substrate 112
as light ray 916. Light ray 916 when crossing the bottom surface
910 is refracted again and comes off the bottom surface 910 at the
angle .theta. as light ray 918. With the off-axis arrangement and a
proper selection of the angle .theta., little of the light ray 918
should enter the optical assembly 130 placed under sample holder
110, as shown in FIG. 9.
[0076] To eliminate or reduce reflection at the bottom surface 910
of substrate 112, 0 can be chosen to be within 10.degree. of the
Brewster's angle .theta..sub.B. Furthermore, to achieve zero or
near zero reflection at the bottom surface 910 of substrate 112,
the light from the light source 120 is linearly polarized with the
E vector in the light parallel to the plane of incidence, which is
the plane containing the incident ray 901 and the normal N of
substrate 112. According to Brewster's Law, when the angle of
incidence .theta. is equal to or near the Brewster's angle
.theta..sub.B, the transmittance, i.e., the ratio of transmitted
power in ray 914 to the incident power in ray 901 across bottom
surface 910 of substrate 112 should be one or near to one and the
reflected power in ray 912 from surface 910 should be zero or near
zero. Brewster's angle .theta..sub.B is given by:
.theta. B = tan - 1 ( n 2 n 1 ) = tan - 1 2 1 ##EQU00001##
where n.sub.1 and n.sub.2 are the refractive indices of the
respective media, i.e., air and substrate 112, and .epsilon..sub.1
and .epsilon..sub.2 are their respective electric permittivity
values.
[0077] In some embodiments, system 100 is configured to achieve
total internal reflection so that a significant amount of the
excitation light from light source 120 is recycled within substrate
112, as shown in FIGS. 10A-10C. Total internal reflection is a
phenomenon that light incident upon a boundary from a denser medium
to a less dense medium is completely reflected off the boundary.
Since the light ray 916 reflected from metallic film 114 has to
travel through the substrate 112 toward the boundary 1010 between
the substrate 112 and air, it is possible to achieve total
reflection such that the light ray 916 is recycled in the substrate
112.
[0078] FIG. 10A illustrates an optical arrangement for achieving
total internal reflection according to exemplary embodiments of the
present teaching. As shown in FIG. 10A, prism 108 is ideally made
of the same material as substrate 112 and is in direct or fluidic
contact with the substrate. In exemplary embodiments, prism 108 is
fused with substrate 112 at a first surface 1012 of the prism.
Prism 108 has a second surface 1014 disposed at an angle .alpha.
with respect to the first surface 1012. In some embodiments, angle
.alpha. is selected to be equal to the incident angle .theta. of
light ray 901 from light source 120. Thus, light ray 901 from light
source 120 is directed toward the second surface 1012 of prism 108
along a normal direction of the second surface 1014 of the prism
108. While a small portion of light ray 901 may be reflected by
surface 1014, the rest of light ray 901 enters substrate 112
without any change in direction because prism 108 and substrate 112
are ideally made of a same material and are fused together or
optically coupled with each other with a fluid. A large portion of
light ray 901 is reflected by metallic film 114 and comes off from
the metallic film 114 as a light ray 1112. Light ray 1112 impinges
on the boundary 1010 between substrate 112 at angle equal to the
angle .theta. with respect to the normal N of the substrate 112
from the inside of substrate 112.
[0079] In exemplary embodiments of the present teaching, .theta. is
selected to be equal or larger than a critical angle .theta., such
that light ray 1112 is totally reflected from boundary 1010 and
comes back towards metallic film 114 as light ray 1114. The above
reflection from the metallic film 114 and the total reflection at
the boundary 1010 are repeated for light ray 1114 and its
derivatives, which are the reflected portion of light ray 1114 and
reflected portion thereof and so on, as shown in
FIG. 10A. In exemplary embodiments, metallic film 114 is formed to
extend to the side surfaces 1020 and in some embodiments to the
edge portions 1030 of the bottom surface 910 of the substrate 112
so that light rays reflected from the metallic film 114 have little
chance of escaping substrate 112 at the side surfaces 1020 but are
recycled and used repeatedly as excitation light for the source
points 210, as shown in FIG. 10A. According to Snell's Law, the
critical angle .theta..sub..tau. is determined by:
.theta. .tau. = sin - 1 ( n 1 n 2 ) , ##EQU00002##
where n.sub.1 and n.sub.2 are the refractive indices of the
respective media, i.e., air and substrate 112, respectively.
[0080] In further embodiments, collection efficiency of optical
assembly can be increased by using a fluid 104 having a refractive
index between that of the air and that of the transparent material
used to construct the substrate 112. For example, when substrate
112 is made of fused silica having a refractive index of about
1.46, water can be used as the fluid 104 because it has a
refractive index of 1.33, which is between the refractive index of
air (.about.1) and that of fused silica (.about.1.46). The fluid
104 is placed between the substrate 112 and the optical assembly
130. In the embodiments employing the fluid 104, the critical angle
is determined by:
.theta. .tau. = sin - 1 ( n f n 2 ) , ##EQU00003##
where n.sub.f is the refractive index of the fluid. The critical
angle .theta..sub..tau. is therefore increased by employing the
fluid. With the increase in the critical angle .theta..sub..tau.,
the collection efficiency is increased because more emitted light
from the source points is able to escape through the bottom surface
910 of the substrate 112 without going through total internal
reflection, and can therefore be collected by the optical assembly
130. The angle .alpha. of the prism 108 and the incident angle
.theta. of the excitation light may be adjusted accordingly to
allow total internal reflection of the excitation light to still
occur in the presence of the fluid 104.
[0081] In another exemplary embodiment, as shown in FIG. 10B, total
internal reflection is facilitated by directing the excitation
light toward a side 1060 of the substrate 112. Excitation light
1070 from the light source 120 is directed to a side surface 1062
at an angle .theta..sub.1 with respect to a normal N' of the side
surface 1062. A refracted portion 1072 of the excitation light 1070
leaves the side surface 1062 at an angle .theta..sub.2 that is
dependent on the angle .theta..sub.1 according to the Snell's Law,
and impinges on the aluminum film 114 on top of the substrate 112
at an angle .theta..sub.3=.alpha.-.theta..sub.2, where .alpha. is
the angle between the side surface 1062 and the bottom surface 1010
of the substrate 112. After reflection from the aluminum film, a
reflected portion 1074 of the excitation light impinges on the
surface 1010 at an angle .theta..sub.4 that is equal to the angle
.theta..sub.3. .theta..sub.2 and .alpha. can be selected such that
.theta..sub.3 or .theta..sub.4 is equal to or larger than the
critical angle .theta..sub..tau. for total reflection at the bottom
surface 1010 of the substrate 112. For example, a in this case can
be selected to be at or near 90.degree. in order to create a large
.theta..sub.3 or .theta..sub.4 angle. Thus, most of the refracted
portion 1072 of the excitation light 1070 can be repeated used to
illuminate the source points on the sample holder 110 before
exiting the substrate 112.
[0082] In another exemplary embodiment, as shown in FIG. 10C, the
side 1060 has a beveled surface 1062 forming an angle .alpha. with
the bottom surface 910 of the substrate 112, where a is less than
90.degree. and larger or equal to the critical angle
.theta..sub..tau.. Excitation light 1070 from the light source 120
is directed to the beveled surface 1062 along a normal N' of the
beveled surface 1062. A refracted portion 1072 of the excitation
light 1070 leaves the beveled surface 1062 at an angle
.theta..sub.2 that is dependent on the angle .theta..sub.1
according to the Snell's Law, and impinges on the aluminum film 114
on top of the substrate 112 at an angle .theta..sub.3=.alpha..
After reflection from the aluminum film, a reflected portion 1074
of the excitation light impinges on the surface 1010 at an angle
.theta..sub.4 that is equal to the angle .theta..sub.3. With a
being equal or larger than the critical angle .theta..sub..tau.,
.theta..sub.4 is also equal or larger than .theta..sub..tau. and
total reflection occurs at the bottom surface 1010 of the substrate
112.
[0083] In another exemplary embodiment, as shown in FIG. 10D, the
side 1060 has a beveled surface 1062 forming an angle .alpha. with
the bottom surface 910 of the substrate 112, and excitation light
1070 from the light source 120 is directed to the beveled surface
1062 at an angle .theta..sub.1 with respect to a normal N' of the
beveled surface 1062. A refracted portion 1072 of the excitation
light 1070 leaves the beveled surface 1062 at an angle
.theta..sub.2 that is dependent on the angle .theta..sub.1
according to the Snell's Law, and impinges on the aluminum film 114
on top of the substrate 112 at an angle
.theta..sub.3=.theta..sub.2+.alpha.. After reflection from the
aluminum film, a reflected portion 1074 of the excitation light
impinges on the surface 1010 at an angle .theta..sub.4 that is
equal to the angle .theta..sub.3. .theta..sub.2 and a can be
selected such that .theta..sub.3 or .theta..sub.4 is equal to or
larger than the critical angle .theta..sub..tau. for total
reflection at the bottom surface 1010 of the substrate 112. Thus,
most of the refracted portion 1072 of the excitation light 1070 can
be repeated used to illuminate the source points on the sample
holder 110 before exiting the substrate 112.
[0084] In another exemplary embodiment, the excitation light 901
from the light source 120 is coupled into the substrate 112 through
a grism, which is a prism and grating combination, or grating 1080
formed on or attached to a portion of the bottom surface 1010 of
the substrate 112, as shown in FIG. 10E. With the use of the grism
or grating 1080, the excitation light 901 can enter the substrate
at an angle .theta. with respect to the normal N of the substrate
that is equal or larger than the critical angle .theta..sub..tau.,
and after being reflected from the aluminum film 114, would impinge
on the bottom surface 1010 of the substrate 112 at the angle
.theta. and be totally reflected from the bottom surface 1010 back
into the substrate. Thus, the excitation light is recycled within
the substrate, as shown in FIG. 10C.
[0085] The arrangements in FIGS. 9 through 10E are advantageous
over conventional systems in part because, by placing the
detector(s) and the optical assembly 130 under sample holder 110,
fluorescent signals can be collected through the bottom surface 910
of the sample holder 110 without the interference of reflected
light from metallic film 114. Furthermore, since the excitation
light and the fluorescent signals entering the optical assembly 130
do not have a common light path, there is no need of heavy
filtering to separate the excitation light entering the substrate
112 from the fluorescent signals exiting the substrate 112 through
the bottom surface 910.
[0086] In various embodiments, optical assembly 130 comprises at
least one pixilated or multi-element detector configured to sense
light signals landed thereon and a set of optical components
configured to direct light emissions from the source points toward
the multi-element detector(s). FIG. 11 illustrates a top view of a
portion of the sample holder 110 showing a plurality of source
points 210. In exemplary embodiments, as shown in FIG. 12, the
pixilated or multi-element detector comprises a plurality of
addressable light-sensing elements 1210 organized in an imaging
plane 1220, such as the x-y plane. The set of optical components is
configured to direct light emissions from different source points
toward different areas 1230 of the imaging plane 1220 so that light
emissions from different source points 210 can be separately and
substantially simultaneously detected.
[0087] Thus, as shown in FIG. 12, light emissions from each source
point 210 form an image of the source point in an area 1230 on the
imaging plane 1220. In exemplary embodiments, the set of optical
components further comprises a light-dispersing setup configured to
separate light emissions from the multiple source points 210 into
multiple spectral components so that the detected light from each
source point is spread out spectrally along the y axis and images
1230 represent spectrally resolved images of the source points 210,
as shown in FIG. 12. When the light-dispersing setup is provided,
enough separation between neighboring source points 210 on the
sample holder 110 is provided to insure that the spectrally
resolved images 1230 of the source points do not overlap with each
other. In addition, sufficient gap g along the y-direction between
an image 1230 of a source point and an image 1230 of a neighbor
source point are provided to prevent overlap of data associated
with the two source points due to cutoff filter tolerances.
[0088] The position of the images 1230 can be determined by a
spatial calibration to associate each source point on the sample
holder with an area 1230 on the image plane 1220. The calibration
can be done by using a dye solution or a light source that is not
blocked by system filters. Such calibration, however, may not be
required if there is no need to correspond the images 1230 with the
source points 210. In addition, tolerance should be allowed to
insure that there is sufficient separation d between the areas 1230
and the edges of the image plane 1220, and the separation should be
controlled to allow detection of all of the source points 210 on
the image plane 1220. As a non-limiting example, the buffer zone d
between a side 1232 of an areas 1230 facing an edge 1222 of the
image plane 1220 is no more than 8 pixels wide.
[0089] Although FIG. 12 shows that the source point images 1230
each having a width .omega. of 2 pixels, a pitch between two
neighboring images in the x-direction being 4 pixels, a gap g
between two adjacent rows of images being 4 pixels, and a buffer
distance d of the images 1230 from each edge of the image plane
1220 being 8 pixels, these numbers are shown as examples and can be
different in different applications or are adjustable to suit
different applications. Moreover, although
FIG. 12 shows only two rows of source points 210 and two rows of
images 1230 corresponding to the source points, in practice, there
may be more or less rows of source points or images. Also, the
source points 210 do not have to be arranged in rows and can be
spread out on the sample holder in any order or even randomly as
long as they are sufficiently separated so that their images 1230
do not overlap on the image plane 1220.
[0090] In exemplary embodiments, the optical assembly 130 is
similar to the one in the optical system disclosed in U.S. Pat. No.
6,690,467 B1 by Reel, which is incorporated herein by reference. As
shown in FIG. 13, as a non-limiting example, the optical assembly
comprises a collection lens assembly 1310, a reimaging lens
assembly 1320, and at least one CCD detector 1330 as the pixilated
detector. The collection lens assembly 1310 comprises at least one
collection lens configured to collect light emissions from the
source points 210. The reimaging assembly 1320 comprises at least
one reimaging lens configured to focus the collected light
emissions from different source points into different areas 1230 of
the imaging plane 1220 of the detector 1330.
[0091] The use of the collection lens assembly 1310 may also
provides a substantially collimated region between the collection
lens assembly 1310 and the reimaging lens assembly 1320, which is
suitable for insertion of a variety of optical devices such as a an
aperture 1340, a light-dispersing assembly 1350, and/or a laser
line filter 1360. In exemplary embodiments, the light-dispersing
assembly 1350 comprises at least one grating, prism, or grism
configured to spread spectrally rays of light that pass through it.
For example, a transmission grating deflects rays of light that
strike thereon at an angle roughly proportional to the wavelength
of the light. Thus, the collimated light emissions from the source
points 210, after going through the transmission gratings, are
dispersed spectrally. With the spectral dispersion, a first light
ray of a first wavelength and a second light ray of a second
wavelength coming from a same source point 210 should arrive at the
reimaging lens assembly 1320 at different angles with respect to an
optical axis of the reimaging lens assembly 1320 and thus be
focused onto different locations 1234 and 1236 of the area 1230
corresponding to the source point, as shown in FIG. 12. Locations
1234 and 1236 are spaced apart from each other along the y-axis
because of the spectral dispersion.
[0092] Instead of prism, grating, or grism in the light dispersing
assembly 1350, dichoic or bandpass filters can be used to separate
the spectral components in the fluorescent signals from each source
point. FIG. 14A illustrates other embodiments of optical assembly
130. As shown in FIG. 14A, optical assembly 130 comprises a
collection assembly 1310, an imaging assembly comprising imaging
lenses 1332-1, 1332-2, 1332-3, and 1332-4 disposed at 90.degree.
angles with respect to each other, a plurality of CCD detectors
1330, and a light dispersing assembly comprising dichroic or
bandpass filters D1, D2, and D3 placed at 90.degree. angles with
respect to each other. Each dichroic or bandpass filter is
configured to allow passage of one of the first, second, and third
bands of fluorescent signals, respectively, and to reflect all
other frequencies. The plurality of CCD detectors 1330 comprises
CCD detectors 1330-1, 1330-2, 1330-3, and 1330-4. CCD detector
1330-1 is placed behind dichroic filter D1 to collect the first
band of fluorescent signals, CCD detector 1330-2 is placed behind
dichroic filter D2 to collect the second band of fluorescent
signals, CCD detector 1330-3 is placed behind dichroic filter D3 to
collect the third band of fluorescent signals, and CCD detector
1330-4 is placed in front of dichroic filter D3 to collect the
signals reflected therefrom, which should comprise the fourth band
of fluorescent signals. Other filters (not shown) can be placed
before CCD detectors 1330, respectively, for improved frequency
selection.
[0093] Alternatively, a dichroic or bandpass filter can be
configured to reflect the first, second, third, or fourth band of
fluorescent signals, and to allow passage of all other frequencies.
It is also possible to combine bandpass, notch, lowpass and
highpass filters in any combination that permits appropriate
separation of the emission wavelengths. FIG. 14B illustrates still
other embodiments of optical assembly 130. As shown in FIG. 14B,
optical assembly 130 comprises a collection lens assembly 1310, a
reimaging assembly comprising reimaging lenses 1320-1, 1320-2,
1320-3 and 1320-4, and CCD detectors 1330-1, 1330-2, 1330-3, and
1330-4 each behind a respective one of the reimaging lenses 1320-1,
1320-2, 1320-3 and 1320-4. Optical assembly 130 further comprises a
light dispersing assembly comprising dichroic or bandpass filters
DF1, DF2, DF3 and DF4 placed in a row under collection assembly
1310 and each at an angle .gamma. to an optical axis (shown by the
dashed line) of the collection assembly 1310. Dichroic or bandpass
filters DF1, DF2, DF3, and DF4 are each placed in front of a
respective one of the reimaging lenses 1320-1, 1320-2, 1320-3 and
1320-4 and are configured to reflect the first, second, third and
fourth bands of fluorescent signals, respectively, while allowing
passage of signals of other frequencies. Other filters (not shown)
can be placed before CCD detectors 1330, respectively, for improved
frequency selection.
[0094] Imaging lenses 1320-1, 1320-2, 1320-3 and 1320-4 can be
separate lenses or sections of a single lens, CCD detectors 1330-1,
1330-2, 1330-3, and 1330-4 in FIG. 14B can be separate CCD
detectors or sections of a single CCD detector. Although FIG. 14B
shows that the dichroic or bandpass filters DF1, DF2, DF3 and DF4
are at a roughly 45.degree. angle with respect to the optical axis
of the collection assembly 1310, such placement is not necessary
and the angle .gamma. can be larger or smaller than 45.degree..
FIG. 14C illustrates an exemplary configuration of the optical
assembly 130 when the angle .gamma. is close to 90.degree. so that
the collimated light emissions from a source point would impinge on
the dichroic or bandpass filters DF1, DF2, DF3 and DF4 at a small
incident angle .beta. and be imaged by the reimaging lens assembly
1320 onto the CCD detector(s) 1330.
[0095] The CCD assembly 1330 comprises at least one charge-coupled
device (CCD) array, such as a regular CCD array, a complimentary
metal-oxide-semiconductor (CMOS) array, an electron-multiplying CCD
(EMCCD) array, an intensified CCD (ICCD) array, or an
electron-bombarded CCD (EBCCD) array. A CCD array is advantageous
over other multi-element detectors, such as an array of avalanche
photodiode (APD) based detectors or photomultiplier tube (PMT)
based detectors, because the number of elements in a CCD array is
much higher, as the size of a CCD pixel in the CCD array can be as
small as 3 .mu.m or even smaller. Therefore, signals from different
source points can be differentiated by detecting them using
different groups of elements in the CCD array, as discussed above.
A CCD array can be much less costly than an APD or PMT array.
[0096] To amplify the low light signals from fluorescing labels on
the incorporated bases above background noises in CCD arrays, a
high-sensitivity CCD-based device such as EMCCD, ICCD, or EB-CCD,
is used in exemplary embodiments. Due to fast base incorporation
rates of DNA molecules, in addition to sensitivity, the speed of
reading data out from a CCD detector is also important because it
is associated with the ability to capture event data and to readout
the data out over a short period of time to allow the next event to
be observed. Through careful design of a readout scheme, a CCD
array can be made to be fast enough to resolve fluorescent
emissions from two consecutive incorporation events associated with
a same source point. Moreover, a CCD with multiple outputs or taps
can be used to increase the CCD readout speed. For example, a CCD
with 4 taps can allow a 4-times increase in readout speed, which
allows images of more source points to be read for increased
throughput.
[0097] To further improve the readout speed, a frame transfer CCD
(FTCCD) array 1500, as illustrated in FIG. 15A, is used in detector
1330 according to exemplary embodiments of the present teaching.
The FTCCD array 1500 allows data readout operations to be performed
concurrently with data collection operations. As shown in FIG. 15A,
CCD array 1500 comprises a dark area 1510, an image area 1520, a
masked storage area 1530, and a horizontal register 1540. The image
area 1520 of CCD array 1500 is where light signals are detected and
is constructed as a two-dimensional array of light-sensitive
elements or pixels. Storage area 1530 comprises an array of storage
elements covered with an opaque mask to provide temporary storage
for an image frame transferred from the image area. The signal that
is accumulated in each pixel in image area 1520 is read out by a
process of parallel transfer in the negative y-direction shown in
the figure, whereby charges in each horizontal row within image
area 1520 and storage area 1530 are transferred to the next row and
so forth until ultimately they reach the horizontal register 1540,
which is a serial readout register that allows charges to be
transferred in the x-direction to an output node (not shown), from
which they are read out. While data from storage array 1530 is
read, image area 1520 is available to collect a next round of light
signals.
[0098] Dark area 1510 is a region of excess pixels. Because these
pixels are not illuminated, they do not have to be cleared during
each readout. Usually, the combination of dark area 1510 and image
areas 1520 maps directly onto the storage area 1530. In one
embodiment, image area 1520 occupies a small fraction (e.g. 1/10)
of the combination so that source point data can be read out at,
for example, 10 times the normal frame rate. In exemplary
embodiments, CCD array 1500 is kept cool at about 80.degree. C.
below zero so that minimal dark current charges are generated. In
certain embodiments, the dark area is eliminated when CCD 1500 is
custom built to have just the right amount of rows in the image
area 1520.
[0099] In some embodiments, an interline CCD or a combined
interline and frame transfer CCD may be employed. FIG. 15B
illustrates an interline CCD array 1550 in CCD detector 1330
according to exemplary embodiments of the present teaching. As
shown in FIG. 15B, interline CCD array 1550 comprises separate
image regions 1560 and CCD storage regions 1570. CCD storage
regions 1570 are protected with a mask structure and positioned
alongside respective ones of image regions 1560 such that CCD
storage regions 1570 and image regions 1560 together form an
alternating parallel array. Image regions 1560 may comprise circuit
elements such as photodiodes for capturing the images of the source
points while CCD storage regions 1570 shift previously acquired
images in a parallel fashion towards a horizontal register 1590.
The horizontal register 1590 then sequentially shifts image
information from each CCD storage region to an output amplifier or
other processing circuits (not shown) as a serial data stream. The
readout process is repeated until data from all of the CCD storage
regions 1570 are transferred to the output amplifier.
[0100] Where an actual two-dimensional image is desired from the
CCD, the image data in a digital format is reconstructed to yield
the final image. Where the data is to be used for non-pictorial or
non-imaging applications, the relevant pixel data may be identified
and processed according to its intended purpose. One advantage of
the interline CCDs is their ability to operate without a shutter or
synchronized strobe, allowing for an increase in device speed and
faster frame rates. An interline CCD array can be used to eliminate
blurring or image smear, which is a common problem with
frame-transfer CCDs, by effectively doing horizontal shifts
directly from the image regions to the respective ones of the
storage regions.
[0101] In exemplary embodiments, readout speed is further improved
by limiting the number of source points to be imaged on the CCD so
that the number of data rows to be read are minimized. The number
of data rows to be read may also be minimized by binning vertically
(in the y-direction), especially when the source points 210 and
thus the images of the source points 1230 are in an array so that
the positions of the images can be fairly accurately predicted, as
shown in FIG. 12. With the fluorescent signals dispersed spectrally
over a range of pixels along the y-direction, the pixels can be
binned in the y-direction to capture informative wavelength
groupings for base determination. For example, as shown in FIG. 12,
each source point image 1230 may comprise four interested
wavelength groups C1, C2, C3, and C4 interleaved with uninterested
wavelength groups U1, U2, and U3. Data in each of the interested
wavelength groups can be binned, while data in the uninterested
wavelength groups can be cleared without being read, as discussed
below. Binning can also be done in the x-direction for further
increase in readout speed because shifting is often faster than
reading. With a CCD array, binning can be done on-chip in a
conventional manner that does not introduce noise.
[0102] The CCD array in detector 1330 may also be made to allow
clearing of the horizontal register 1540 or 1590. This can speed up
readouts if desired data is separated by rows of unneeded pixels.
FIG. 16A illustrates examples of four normalized fluorescent
spectra associated with the four different fluorescent labels used
to label respective ones of four different nucleotides. The four
spectra have peak regions 1610, 1620, 1630, and 1640 corresponding
to respective ones of the first, second, third and fourth frequency
bands. Only four fluorescent frequency bands need to be collected
by detector 1330. Data can be over-determined, however, to comprise
more than the spectrum of data associated with an incorporation
event. With the light dispersing setup shown in FIG. 13, a
continuous portion of a spectrum of data is spread along the
y-direction, as shown in FIG. 16B. FIG. 16C illustrates the
over-determined data with the most informative wavelengths
corresponding to the four frequency bands in gray and the less
informative wavelength ranges shown in white. Data rows in the less
informative wavelength range between two gray bands is referred to
as a band gap bg, which may not be needed and can be cleared
without being read out for increased readout speed. The width and
position of each informative frequency band in FIG. 16C can be
optimized for best signal to noise (S/N) ratio after
multicomponenting, as explained in more detail below.
[0103] While the frequency bands in FIGS. 12, 16B, and 16C are
shown to spread along the y-direction, this is not essential and
orienting the frequency bands in a different direction on the CCD
may be advantageous in some cases.
[0104] FIG. 17A is a flow chart of a method 1700 for reading out
image data associated with a plurality of source points according
to exemplary embodiments of the present teaching. As a non-limiting
example, method 1700 is described in the context of a frame
transfer EMCCD array of 512.times.512 pixels in the image area that
allows clearing of the horizontal register. Nevertheless, the
method can be readily adapted to other types of CCD array. As shown
in FIG. 17A, method 1700 comprises step 1710 in which the pixels in
the image area are shifted down by a frame having, for example, 128
rows. So, 128 rows of pixels are shifted from the image area into
the masked storage area. These pixels are not read out for a number
of (e.g., 4) frames (512/128=4). Pixels from the dark area will be
shifted down to collect the next images, but the provision of a
dark area is not necessary because the CCD is kept cool
(-80.degree. C.) so minimal dark current charge is generated.
Method 1700 further comprises step 1720 in which a first number of
(e.g., 8) buffer rows in a frame is shifted down to the register
and cleared. The number 8 is arbitrarily chosen. This number should
correspond to the distance d shown in FIG. 12. Method 1700 further
comprises step 1730 in which the pixels associated with the first
frequency band is vertically binned and shifted. As a non-limiting
example, 4 rows of pixels are associated with a frequency band, and
these rows are binned and read out as one row. Method 1700 further
comprises step 1740 in which at least some of the rows in the band
gap bg before the next frequency band are cleared. Steps 1730 and
step 1740 are thereafter repeated for each of the other frequency
bands. Method 1700 further comprises step 1750 in which at least
some of the buffer rows in the gap g between two rows of images
1230 are shifted and cleared. Steps 1730 through 1750 are repeated
for each row of source points when the source points are arranged
in an array.
[0105] FIG. 17B is a spreadsheet for estimating the throughput for
system 100 using a frame transfer EMCCD as detector 1330 according
to exemplary embodiments. As shown in FIG. 17B, for a frame
transfer CCD having 512.times.512 pixels in the image area, a
normal read out time of 1.times.10.sup.-7 second, a vertical shift
time of 4.times.10.sup.-7 second, and a horizontal register clear
time of 5.times.10.sup.-6 second, using method 1700, the time to
read out emission data from 4 rows of source points is about
0.00096 seconds, resulting in a readout speed higher than 1000 Hz,
which is much faster than the normal readout speed of 30 Hz for the
CCD. FIG. 17C is a spreadsheet for estimating the throughput for
system 100 using a 1004.times.1002 EMCCD as detector 1330 according
to exemplary embodiments. As shown in FIG. 17C, the time for
reading out emission data from 4 rows of source points in this case
is about 0.00113 seconds, resulting in a readout speed close to
1000 Hz.
[0106] In exemplary embodiments of the present teaching, optical
data collected by detector 1330 is sent to computer system 140 and
optionally or additionally DSP or FPGA 150 for base determination.
FIG. 18 is a block diagram of computer system 140 according to
exemplary embodiments of the present teaching. As shown in FIG. 18,
computer system 140 comprises a central processing unit (CPU) 1810,
a memory unit 1820, a data input port 1830, and a user interface
1840, interconnected by one or more buses 1850. Memory unit 1820
preferably stores operating system software 1822 and other software
programs including a program 1824 for base determination. Memory
unit 1820 further comprises a data storage unit 1826 for storing
optical data collected by detector 1330 and sent to computer system
140 through data input port 1830. Program 1824 comprises coded
instructions, which, when executed by CPU 1810, cause computer
system 140 to carry out methods for detecting light emissions from
multiple source points as described above and/or methods for base
determination based on the detected data according to exemplary
embodiments of the present teaching, as explained in more detail
below.
[0107] With the illumination of excitation light, a labeled and
incorporated nucleotide should fluoresce by emitting photons from
an associated source point. The spectrum of the photons collected
at detector 130 from this single incorporation event should be a
collection of photons with different energies or frequencies. When
the number of collected photons is large, the spectrum should
resemble the normal dye spectrum corresponding to the fluorescent
dye used to label the incorporated nucleotide. The spectrum will
vary, however, due to the small number of photons that are
typically collected by detector 1330 from the single incorporation
event in each collection time period.
[0108] For example, a fluorescing dye may emit 10,000 photons over
a 10 micro second period, and about 4% of the 10,000 photons may be
detected by detector 1330 in ten time bins each corresponding to,
for example, a one micro second period. Thus, roughly 40 photons
may be collected in each time bin. Plotted spectrally over 10
spectral bins, the 40 expected photons might spread out like the
histogram shown in FIG. 19. Because of the small number of photons,
the distribution in FIG. 19 may not match the normal dye spectrum
corresponding to the incorporated nucleotide, and the mismatch may
lead to a chance of incorrect base determination.
[0109] In exemplary embodiments, to avoid a base determination
problem caused by a small number of photons from a single
incorporation event, the present teaching comprises a method 2000
for base determination illustrated by the flowchart in FIG. 20. As
shown in FIG. 20, method 2000 comprises step 2010 in which data
from all spectral bins collected in each of a plurality of
consecutive time bins are copied and combined to form composite
light data. Method 2000 further comprises step 2020 in which the
composite data from the plurality of consecutive time bins are used
to determine an incorporation time interval T.
[0110] FIG. 21 is a plot of the composite data over a plurality of
24 time bins showing the number of photons detected in each time
bin. As shown in FIG. 21, during time bins 1, 2, 3, 4, and 5, and
time bins 16, 17, 18, and 19, the composite data indicate small
numbers of photons coming from a background of diffusion events,
trial events, and substrate fluorescence. In other time bins, the
numbers of photons are significantly larger, resulting in large
rises 2110 and 2120 above the background noises. Rises 2110 and
2120 indicate incorporation events. The incorporation time T for
the incorporation event corresponding to rise 2110 can be
determined by measuring the width of rise 2110, as shown in FIG.
21.
[0111] Since most of the photons detected during the incorporation
time interval T are from a single incorporation event, for each
color bin, data associated with the same spectral bin but collected
in different time bins in the incorporation time interval can be
combined, resulting in increased data points for the spectral bin.
The increase in the number of data points leads to an improved
multicomponenting process, which is used to convert color data to
dye composition. Thus, method 2000 further comprises step 2030 in
which data associated with each spectral bin or frequency band of
interest but collected in different time bins in the incorporation
time interval T are combined, and step 2040 in which the combined
data is used in a conventional multicomponenting process to
determine a dominant dye, which is used to determine the base being
incorporated. Method 2000 further comprises step 2050 in which the
residuals of the multicomponenting process is used to determine a
confidence level.
[0112] Method 2000 for improving the signal to noise ratio by
combining data from multiple time bins may be coded as a computer
program and executed by computer system 140. Alternatively, since
the same algorithm in method 2000 is executed a large number of
times, hardware solutions such as field program gate arrays (FPGA)
or digital signal processors (DSP) 150 and the like can be used to
reduce the computation load and data stream size. The FPGAs or DSPs
150 could be integrated in detector(s) 1330, between detector 1330
and computer system 140, as shown in FIG. 1, or installed into
computer system 140.
[0113] The foregoing descriptions of specific embodiments of the
present teaching have been presented for purposes of illustration
and description. They are not intended to be exhaustive or to limit
the teaching to the precise forms disclosed, and obviously many
modifications and variations are possible in light of the above
teaching. The embodiments were chosen and described in order to
best explain the principles of the teaching and its practical
application, to thereby enable others skilled in the art to best
use the teaching and various embodiments with various modifications
as are suited to the particular use contemplated. It is intended
that the scope of the teaching be defined by the claims appended
hereto and their equivalents.
* * * * *