Note: Descriptions are shown in the official language in which they were submitted.
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METHODS AND SYSTEMS FOR ANALYZING FLUORESCENT
MATERIALS WITH REDUCED AUTOFLUORESCENCE
FIELD
[0001] The invention is in the field of reducing autofluorescence
background noise.
BACKGROUND
[0002] Typical fluorescence based optical analysis of analytical
reactions employs
reactants or other reagents in the reaction of interest that bear a
fluorescent moiety, such as a
labeling group, where the detection of that moiety is indicative of a
particular reaction result or
condition. For example, reactions may be engineered to produce a change in the
amount,
location, spectrum, or other characteristic upon occurrence of a reaction of
interest.
[0003] During analysis, an excitation light source is directed through an
optical system
or train at the reaction to excite fluorescence from the fluorescent moiety.
The emitted
fluorescence is then collected by the optical train and directed toward a
detection system, which
quantifies, records, and/or processes the signal data from the fluorescence.
Fluorescence-based
systems are generally desired for their high signal levels deriving from the
high quantum
efficiency of the available fluorescent dye moieties. Because of these high
signal levels,
relatively low levels of the materials are generally required in order to
observe a fluorescent
signal.
[0004] For example, simple multi-well plate readers have been
ubiquitously employed
in analyzing optical signals from fluid based reactions that were being
carried out in the various
wells of a multiwell plate. These readers generally monitor the fluorescence,
luminescence or
chromogenic response of the reaction solution that results from a given
reaction in each of 96,
384 or 1536 different wells of the multiwell plate. Other optical detection
systems have been
developed and widely used in the analysis of analytes in other configurations,
such as in
flowing systems, i.e., in the capillary electrophoretic separation of
molecular species.
Typically, these systems have included a fluorescence detection system that
directs an
excitation light source, e.g., a laser or laser diode, at the capillary, and
is capable of detecting
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when a fluorescent or fluorescently labeled analyte flows past the detection
region (see, e.g.,
ABI 3700 Sequencing systems, Agilent 2100 Bioanalyzer and ALP systems, etc.).
Other
detection systems direct a scanning laser at surface bound analytes to
determine where, on the
surface, the analytes have bound. Such systems are widely used in molecular
array based
systems, where the positional binding of a given fluorescently labeled
molecule on an array
indicates a characteristic of that molecule, e.g., complementarity or binding
affinity to a given
molecule (See, e.g., U.S. Patent No. 5,578,832).
[0005] Notwithstanding the great benefits of fluorescent reaction systems,
the
development of real-time, highly multiplexed, single molecule analyses and the
application of
these systems does have some drawbacks particularly when used in extremely low
signal level
reactions, e.g., low concentration or even single molecule detection systems.
In particular,
these systems often have a number of components that can potentially generate
amounts of
background signal, e.g., detected signal that does not emanate from the
fluorescent species of
interest, when illuminated with relatively high intensity radiation. This
background signal can
contribute to signal noise levels, and potentially overwhelm relatively low
reaction derived
signals or make more difficult the identification of signal events, e.g.,
increases, decreases,
pulses etc., of fluorescent signal associated with the reactions being
observed.
[0006] Background signal, or noise, can derive from a number of sources,
including, for
example, fluorescent signals from non-targeted reaction regions, fluorescence
from targeted
reaction regions but that derive from non-relevant sources, such as non-
specific reactions or
associations, such as dye or label molecules that have nonspecifically
adsorbed to surfaces,
prevalence or build up of labeled reaction products, other fluorescent
reaction components,
contaminants, and the like. Other sources of background signals in fluorescent
systems include
signal noise that derives from the use of relatively high-intensity excitation
radiation in
conjunction with sensitive light detection. Such noise sources include those
that derive from
errant light entering the detection system that may come from inappropriately
filtered or
blocked excitation radiation, and/or contaminating ambient light sources that
may impact the
overall system. Other sources of signal noise resulting from the application
of high intensity
excitation illumination derives from the auto-fluorescence of the various
components of the
system when subjected to such illumination, as well as Raman scattering of the
excitation
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illumination. The contribution of this systemic fluorescence is generally
referred to herein as
autofluorescence background noise (ABN).
[0007] It would be therefore desirable to provide methods, components and
systems in
which background signal, such as autofluorescence background noise, were
minimized. This is
particularly the case in relatively low signal level reactions, such as single
molecule
fluorescence detection methods and systems, e.g., real-time, highly
multiplexed single
molecule detection systems that are capable of detecting large numbers of
different events at
relatively high speed and that are capable of deconvolving complex, multi-
wavelength signals.
SUMMARY
[0008] This disclosure provides methods and systems that have improved
abilities to
monitor fluorescent signals from analytical reactions by virtue of having
reduced levels of
background signal noise that derives from autofluorescence created within one
or more
components of the overall system.
[0009] In a first aspect, this disclosure provides systems for monitoring
a plurality of
discrete fluorescent signals from a substrate. The systems include a substrate
onto which a
plurality of discrete fluorescent signal sources has been disposed, an
excitation illumination
source, and a detector for detecting fluorescent signals from the plurality of
fluorescent signal
sources. In addition, the systems include an optical train positioned to
simultaneously direct
excitation illumination from the excitation illumination source to each of the
plurality of
discrete fluorescent signal sources on the substrate and direct fluorescent
signals from the
plurality of fluorescent signal sources to the detector. The optical train of
the systems
comprises an objective lens focused in a first focal plane at the substrate
for simultaneously
collecting fluorescent signals from the plurality of fluorescent signal
sources on the substrate, a
first focusing lens for receiving the fluorescent signals from the objective
lens and focusing the
fluorescent signals in a second focal plane, and a confocal filter placed
within the second focal
plane to filter fluorescent signals from the substrate that are not within the
first focal plane.
[0010] Optionally, the systems for monitoring a plurality of discrete
fluorescent signals
from a substrate can include a substrate that comprises first and second
opposing surfaces that
is positioned such that the first surface of the substrate is more proximal to
the optical train than
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the second surface, and such that the first focal plane is substantially
coplanar with the second
surface. The systems can optionally include an optical train that
simultaneously directs
excitation radiation at and collects fluorescent signals from at least 100
discrete fluorescent
signal sources, at least 500 discrete fluorescent signal sources, at least
1000 discrete signal
sources, or at least 5000 discrete signal sources. The systems can optionally
include an optical
train that comprises a microlens array and/or a diffractive optical element to
simultaneously
direct excitation illumination at the plurality of discrete fluorescent signal
sources on the
substrate.
[0011] Each of the plurality of discrete signal sources in the systems
described above
can optionally comprise a reaction region, e.g., an optically confined region
on the substrate,
into which a complex comprising a nucleic acid polymerase, a template
sequence, and a primer
sequence, and at least one fluorescently labeled nucleotide has been disposed.
Optionally, the
optically confined regions can comprise zero mode waveguides.
[0012] This disclosure also provides second set of systems for monitoring
a plurality of
discrete fluorescent signals from a substrate, which includes a substrate onto
which a plurality
of discrete fluorescent signal sources has been disposed, an excitation
illumination source, and
a detector for detecting fluorescent signals from the plurality of fluorescent
signal sources. In
addition, the second set of systems of monitoring a plurality of discrete
fluorescent signals from
a substrate includes an optical train that is positioned to direct excitation
illumination from the
excitation illumination source to each of the plurality of discrete
fluorescent signal sources on
the substrate in a targeted illumination pattern. In addition, the optical
train directs fluorescent
signals from the plurality of fluorescent signal sources to the detector.
[0013] Optionally, the optical train in the second set systems for
monitoring a plurality
of discrete fluorescent signals from a substrate can comprise a microlens
array and/or a
diffractive optical element to direct excitation radiation to each of the
plurality of discrete
fluorescent signal sources in a targeted illumination pattern. The diffractive
optical element
can optionally be configured to direct excitation radiation to at least 100
discrete fluorescent
signal sources, at least 500 discrete fluorescent signal sources, at least
1000 discrete fluorescent
signal sources, or at least 5000 discrete fluorescent signal sources in a
targeted illumination
pattern.
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[0014] In the second set systems for monitoring a plurality of discrete
fluorescent
signals from a substrate, each of the plurality of discrete signal sources can
optionally comprise
a reaction region, e.g., an optically confined region on the substrate, into
which a complex
comprising a nucleic acid polymerase, a template sequence, and a primer
sequence, and at least
one fluorescently labeled nucleotide has been disposed. The optically confined
regions can
optionally comprise zero mode waveguides.
[0015] In a related aspect, this disclosure provides methods of reducing
fluorescence
background signals in detecting fluorescent signals from a substrate that
comprises a plurality
of fluorescent signal sources. The methods include directing excitation
radiation
simultaneously at a plurality of fluorescent signal sources on a substrate in
a first focal plane,
collecting fluorescent signals simultaneously from the plurality of
fluorescent signal sources,
filtering the fluorescent signals to reduce fluorescence not in the first
focal plane to provide
filtered fluorescent signals, and detecting the filtered fluorescent signals.
The filtering step in
the methods can optionally comprise confocally filtering the fluorescent
signals to provide
filtered fluorescent signals.
[0016] This disclosure also provides methods of detecting fluorescent
signals from a
plurality of discrete fluorescent signal sources on a substrate. These methods
include providing
a substrate onto which a plurality of discrete fluorescent signal sources has
disposed, directing
excitation illumination at the substrate in a targeted illumination pattern,
and detecting
fluorescent signals from each of the plurality of discrete fluorescent signal
sources. The step of
directing excitation at the substrate in a targeted illumination pattern can
optionally comprise
passing the excitation illumination through a microlens array and/or a
diffractive optical
element. The targeted illumination pattern can optionally comprise at least
100 discrete
illumination spots positioned to be incident upon at least 100 discrete
fluorescent signal
sources, at least 500 discrete illumination spots positioned to be incident
upon at least 500
discrete fluorescent signal sources, at least 1000 discrete illumination spots
positioned to be
incident upon at least 1000 discrete fluorescent signal sources, or at least
5000 discrete
illumination spots positioned to be incident upon at least 5000 discrete
fluorescent signal
sources.
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[0017] In addition, this disclosure provides three sets of methods of
monitoring
fluorescent signals from a source of fluorescent signals. In the first set,
the methods include
providing a fluorescent signal detection system that comprises a substrate
comprising a
plurality of discrete fluorescent signal sources, providing a source of
excitation illumination,
providing a fluorescent signal detector, and providing an optical train for
directing excitation
illumination from the source of excitation illumination to the substrate and
for directing
fluorescent signals from the substrate to the fluorescent signal detector. In
this set of methods,
at least one optical component in the optical train is photobleached so as to
reduce a level of
autofluorescence produced by the at least one optical component in response to
passing
excitation illumination therethrough.
[0018] The second set of methods of monitoring fluorescent signals from a
source of
fluorescent signals includes providing a substrate onto which a plurality of
discrete fluorescent
signal sources have been disposed, directing excitation illumination at the
substrate in a
targeted illumination pattern to excite fluorescent signals from the
fluorescent signal sources,
collecting the fluorescent signals from the plurality of discrete fluorescent
signal sources
illuminated with the targeted illumination pattern, confocally filtering the
fluorescent
emissions, and separately detecting the fluorescent emissions from the
discrete fluorescent
signal sources.
[0019] The third set of methods of monitoring fluorescent signals from a
source of
fluorescent signals includes providing an excitation illumination source,
providing a substrate
onto which at least a first fluorescent signal source has been disposed, and
providing an optical
train comprising optical components that is positioned to direct excitation
illumination from the
illumination source to the at least first fluorescent signal source and for
transmitting fluorescent
signals from the at least first fluorescent signal source to a detector. The
third set of methods
includes photobleaching at least one of the optical components to reduce an
amount of
autofluorescence produced by the at least one optical component in response to
the excitation
illumination, directing excitation illumination through the at least one
optical component and at
the at least first fluorescent signal source, and detecting fluorescent
signals from the at least
first fluorescent signal source. In the third set of methods, the fluorescent
signals can
optionally be confocally filtered prior to being detected.
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[0020] Relatedly, this disclosure provides systems for detecting
fluorescent signals
from a plurality of signal sources on a substrate. These systems include a
source of excitation
illumination, a detection system, and an optical train positioned to direct
excitation illumination
from the source of excitation illumination to the plurality of signal sources
on the substrate and
transmit emitted fluorescence from the plurality of fluorescent signal sources
to the detector.
The optical train in these systems includes an objective lens that has a ratio
of excitation
illumination to autofluorescence of greater than 1 X 1010
.
[0021] This disclosure is generally directed to highly multiplexed
optical interrogation
systems, and particularly to highly multiplexed fluorescence-based detection
systems. In one
aspect, the present disclosure is directed at systems and methods for high
resolution, highly
multiplexed analysis of optical signals from large numbers of discrete signal
sources, and
particularly signal sources that are of very small dimensions and which are
arrayed on or within
substrates at regularly spaced intervals.
[0022] In a first aspect, this disclosure includes multiplex fluorescence
detection
systems that comprise an excitation illumination source, and an optical train
that comprises an
illumination path and a fluorescence path. In the context of certain aspects,
the illumination
path comprises an optical train that comprises multiplex optics that convert a
single originating
illumination beam from the excitation illumination source into at least 10
discrete illumination
beams, and an objective lens that focuses the at least 10 discrete
illumination beams onto at
least 10 discrete locations on a substrate. The fluorescence path comprises
collection and
transmission optics that receive fluorescent signals from the at least 10
discrete locations, and
separately direct the fluorescent signals from each of the at least 10
discrete locations through a
confocal filter and focus the fluorescent signals onto a different location on
a detector.
[0023] In a related aspect, this disclosure provides a system for
detecting fluorescence
from a plurality of discrete locations on a substrate, which system comprises
a substrate, an
excitation illumination source a detector, and an optical train positioned to
receive an
originating illumination beam from the excitation illumination source. In the
context of certain
aspects, the optical train is configured to convert the originating
illumination beam into a
plurality of discrete illumination beams, and focus the plurality of discrete
illumination beams
onto a plurality of discrete locations on the substrate, wherein the plurality
of discrete locations
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are at a density of greater than 1000 discrete illumination spots per mm2,
preferably greater
than 10,000 discrete spots per mm2, more preferably greater than 100,000
discrete illumination
spots per mm2, in many cases greater than 250,000 discrete illumination spots
per mm2, and in
some cases up to and greater than 1 spot per gm2. In terms of inter-spot
spacing upon the
substrate, the illumination patterns of the invention will typically provide
spacing between
adjacent spots (in the closest dimension), of less than 100 gm, center to
center, preferably, less
than 20 gm, more preferably, less than 10 gm, and in many preferred cases,
spacing between
spots of 1 um or less, center to center. As will be appreciated, such spacing
generally refers to
inter-spot spacing in the closes dimension, and does not necessarily reflect
inter-row spacing
that may be substantially greater, due to the allowed spacing for spectral
separation of adjacent
rows, as discussed elsewhere herein. The optical train is further configured
to receive a
plurality of discrete fluorescent signals from the plurality of discrete
locations, and focus the
plurality of discrete fluorescent signals through a confocal filter, onto the
detector.
[0024] In other aspects, this disclosure provides systems for collecting
fluorescent
signals from a plurality of locations on a substrate, which comprise
excitation illumination
optics configured to simultaneously provide excitation radiation to an area of
a substrate that
includes the plurality of locations, and fluorescence collection and
transmission optics that
receive fluorescent signals from the plurality of locations on the substrate,
and separately direct
the fluorescent signals from each of the plurality of locations through a
separate confocal
aperture in a confocal filter and image the fluorescent signals onto a
detector.
[0025] Relatedly, this disclosure also provides systems for detecting
fluorescent signals
from a plurality of discrete locations on a substrate, that comprise an
excitation illumination
source, a diffractive optical element or holographic phase mask, positioned to
convert a single
originating illumination beam from the excitation illumination source into at
least 10 discrete
beams each propagating at a unique angle relative to the originating beam, an
objective for
focusing the at least ten discrete beams onto at least 10 discrete locations
on a substrate,
fluorescence collection and transmission optics, and a detector. In the
context of certain
aspects, the fluorescence collection and transmission optics are positioned to
receive
fluorescent signals from the plurality of discrete locations and transmit the
fluorescent signals
to the detector.
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[0026] In other aspects, this disclosure provides methods of detecting a
plurality of
discrete fluorescent signals from a plurality of discrete locations on a
substrate. The methods
comprise simultaneously and separately illuminating each of the plurality of
discrete locations
on the substrate with excitation illumination. Fluorescent signals from each
of the plurality of
locations are simultaneously and separately collected and each of the
fluorescent signals from
the plurality of discrete locations is separately directed through a confocal
filter, and separately
imaged onto a discrete location on a detector.
[0027] Those of skill in the art will appreciate that the methods
provided herein, e.g.,
for detecting a plurality of discrete fluorescent signals from a plurality of
discrete locations on a
substrate, for reducing fluorescence background signals in detecting
fluorescent signals from a
substrate that comprises a plurality of fluorescent signal sources, and/or for
monitoring
fluorescent signals from a source of fluorescent signals, can be used alone or
in combination
and can be used in combination with any one or more of the systems described
herein.
Likewise, the systems provided herein, e.g., multiplex fluorescence detection
systems, systems
for monitoring a plurality of discrete fluorescent signals from a substrate,
and/or systems for
detecting fluorescence from a plurality of discrete locations on a substrate,
can be used alone or
in combination. In addition to the foregoing, this disclosure is also directed
to the use of any of
the foregoing systems and/or methods in a variety of analytical operations.
[0028] Various embodiments of the claimed invention relate to a method of
monitoring
fluorescent signals from a source of fluorescent signals, comprising:
providing a fluorescent
signal detection system that comprises: a substrate comprising a plurality of
discrete
fluorescent signal sources; a source of excitation illumination; a fluorescent
signal detector; an
optical train for directing excitation illumination from the source of
excitation illumination to
the substrate, and for directing fluorescent signals from the substrate to the
fluorescent signal
detector; and, photobleaching at least one optical component in the optical
train, so as to reduce
a level of autofluorescence produced by the at least one optical component in
response to
passing excitation illumination therethrough.
[0029] Various embodiments of the claimed invention relate to a method of
monitoring
fluorescent signals from a source of fluorescent signals, comprising:
providing an excitation
illumination source, a substrate having at least a first fluorescent signal
source disposed
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thereon, and an optical train comprising optical components and positioned to
direct excitation
illumination from the illumination source to the at least first fluorescent
signal source, and for
transmitting fluorescent signals from the at least first fluorescent signal
source to a detector;
photobleaching at least one optical component of the optical train to reduce
an amount of
autofluorescence produced by the at least one optical component in response to
the excitation
illumination; directing excitation illumination through the at least one
optical component and at
the at least first fluorescent signal source; and, detecting fluorescent
signals from the at least
first fluorescent signal source.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] Figure 1 provides a schematic overview of a fluorescence
detection system.
[0031] Figure 2 shows a plot of fluorescent signals as a function of the
number of
illumination lines applied to a given fluorescently spotted substrate, showing
increasing
background fluorescence levels with increasing illumination.
[0032] Figure 3 schematically illustrates a targeted illumination
pattern generated from
an originating beam passed through differently oriented diffraction gratings.
[0033] Figure 4 provides an example of a microlens array for use in the
present
invention.
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[0034] Figure 5 shows an image of diffractive optical element ("DOE") and
the
illumination pattern generated when light is passed through the DOE.
[0035] Figure 6 shows an illumination pattern from a DOE designed to
yield very
high illumination multiplex.
[0036] Figure 7 schematically illustrates a targeted illumination pattern
generated
from overlaying illumination patterns from two DOEs but offsetting them by a
half period.
[0037] Figure 8 schematically illustrates an illumination path including
a polarizing
beam splitting element.
[0038] Figure 9 shows a comparison plot of autofluorescence of a
fluorescent
detection system in the absence and presence of a confocal mask in the system,
to filter out
of focus autofluorescence components.
[0039] Figure 10 schematically illustrates a portion of a confocal mask.
[0040] Figure 11 provides a schematic of an optical train incorporating a
confocal
mask.
[0041] Figure 12 is a comparative plot of autofluorescence imaged at a
discrete
detector location in the absence of a confocal mask, and in the presence of
confocal slits of
decreasing cross sectional dimensions.
[0042] Figure 13 provides a schematic illustration of a fluorescence
detection
system that can be used with the methods and systems of the present invention.
[0043] Figure 14 schematically illustrates the illumination and
fluorescence paths of
one exemplary system according to the invention.
DETAILED DESCRIPTION
I. General Discussion of Invention
[0044] The present invention generally provides methods, processes and
systems for
monitoring fluorescent signals associated with reactions of interest, but in
which
background signal levels and particularly autofluorescence background noise of
system
components, is reduced.
[0045] The methods, processes and systems of the invention are
particularly suited
to the detection of fluorescent signals from signal sources, e.g., reaction
regions, on
substantially planar substrates, and particularly for detection of relatively
low levels of
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fluorescent signals from such reaction regions, where signal background has a
greater
potential for negative impact.
[0046] Increasing throughput of chemical, biochemical and/or biological
analyses
has generally relied, at least in part, on the ability to multiplex the
analysis. Accordingly, in
a preferred embodiment, the methods, processes and systerrls of the invention
can be used
with multiplexed optical systems for high-throughput analysis of fluorescent
signal sources,
e.g., fluorescent signal sources associated with chemical, biochemical, or
biological
reactions. Such multiplex generally utilizes the simultaneous analysis of
multiple different
samples that are either physically discrete or otherwise separately
identifiable within the
analyzed material. Examples of such multiplex analysis include, e.g., the use
of multi-well
plates and corresponding plate readers, to optically interrogate multiple
different fluorescent
reactions simultaneously. Such plate systems have been configured to include
16 wells, 32
wells, 96 wells, 384 wells and even 1536 wells in a single plate that can be
interrogated
simultaneously.
[0047] Multiplexed systems in which autofluorescence background noise is
beneficially minimized, e.g., by the methods and systems of the invention,
include array
based technologies in which solid substrates bearing discrete patches of
different molecules
are reacted with a certain set of reagents and analyzed for reactivity, e.g.,
an ability to
generate a fluorescent signal. Such arrays are simultaneously interrogated
with the reagents
and then analyzed to identify the reactivity of such reagents with the
different reagents
immobilized upon different regions of the substrate.
[0048] In the context of the present invention, the optical signal
sources that are
analyzed using the methods and systems typically can comprise any of a variety
of
materials, and particularly those in which optical analysis may provide useful
information.
Of particular relevance to the present invention are optical signal sources
that comprise
chemical, biochemical or biological materials that can be optically analyzed
to identify one
or more chemical, biochemical and/or biological properties. Such materials
include
chemical or biochemical reaction mixtures that may be analyzed to determine
reactivity
under varying conditions, varying reagent concentrations, exposure to
different reagents, or
the like. Examples of materials of particular interest include proteins such
as enzymes, their
substrates, antibodies and/or antigens, biochemical pathway components, such
as receptors
and ligands, nucleic acids, including complementary nucleic acid associations,
nucleic acid
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processing systems, e.g., ligases, nucleases, polymerases, and the like. These
materials may
also include higher order biological materials, such as prokaryotic or
eukaryotic cells,
mammalian tissue samples, viral materials, or the like.
[0049] Optical interrogation or analysis of these materials can generally
involve
known optical analysis concepts, such as analysis of light absorbance,
transmittance and/or
reflectance of the materials being analyzed. In other aspects, such analysis
may determine a
level of optical energy emanating from the system. In some cases, material
systems may
produce optical energy, or light, as a natural product of the process being
monitored, as is
the case in systems that use chemiluminescent reporter systems, such as
pyrosequencing
processes (See, e.g., U.S. Patent No. 6,210,891). In particularly preferred
aspects, the
optical analysis of materials in accordance with the present invention
comprises analysis of
the materials' fluorescent characteristics, e.g., the level of fluorescent
emissions emanating
from the material in response to illumination with an appropriate excitation
radiation. Such
fluorescent characteristics may be inherent in the material being analyzed, or
they may be
engineered or exogenously introduced into the system being analyzed. By way of
example,
the use of fluorescently labeled reagent analogs in a given system can be
useful in providing
a fluorescent signal event associated with the reaction or process being
monitored.
[0050] In certain aspects, the optical signal sources analyzed using
methods,
processes, and systems provided by the invention are referred to as being
provided on a
substrate. Such substrates may comprise any of a wide variety of supporting
substrates
upon which such signal sources may be deposited or otherwise provided,
depending upon
the nature of the material and the analysis to be performed. For example, in
the case of
fluid reagents, such substrates may comprise a plate or substrate bearing one
or more
reaction wells, where each fluorescent signal source may comprise a discrete
reaction well
on the plate, or even a discrete region within a given reaction well. In terms
of multi-well
plates, as noted above, such plates may comprise a number of discrete and
fluidically
isolated reaction wells. In fact, such plates are generally commercially
available in a variety
of formats ranging from 8 wells, to 96 wells, to 384 wells to 1536 wells, and
greater. In
certain aspects, each discrete well on a multi-well plate may be considered a
discrete, e.g.,
fluorescent, signal source. However, in some aspects, a single well may
include a number
of discrete fluorescent signal sources. As used herein, a discrete fluorescent
signal source
typically denotes a fluorescent signal source that is optically resolvable and
separately
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,
- identifiable from another adjacent fluorescent signal source. Such
separate identification
,. may be a result of different chemical or biochemical characteristics
of each fluorescent
_
signal source or merely result from spatial differentiation between
fluorescent signal
sources.
[0051] Other substrates that can be used with the methods and
systems of the
invention, particularly in the field of biochemical analysis, include planar
substrates upon
which are provided arrays of varied molecules, e.g., proteins or nucleic
acids. In such cases,
different features on the array, e.g., spots or patches of a given molecule
type, may comprise
a discrete signal source.
[0052] The methods and systems of the invention are generally
applicable to a wide
variety of multiplexed analysis of a number of discrete optical signal sources
on a substrate.
Of particular benefit in the present invention is its applicability to
extremely high-density
arrays of such optical signal sources and/or arrays of such signal sources
where each signal
source is of extremely small area and/or signal generating capability.
Examples of such
arrayed signal sources include, for example, high density arrays of molecules,
e.g., nucleic
acids, high density multi-well reaction plates, arrays of optical
confinements, and the like.
[0053] For ease of discussion, the present invention is
described in terms of its
application to multiplexed arrays of single molecule reaction regions on
planar substrates
from which fluorescent signals emanate, which signals are indicative of a
particular reaction
occurring within such reaction regions. Though described in terms of such
single molecule
arrays, it will be appreciated that the invention, as a whole, or in part,
will have broader
applicability and may be employed in a number of different applications, such
as in
detection of fluorescent signals from other array formats, e.g., spotted
arrays, arrays of
fluidic channels, conduits or the like, or detection of fluorescent signals
from multi-well
plate formats, fluorescent bar-coding techniques, and the like.
[0054] One exemplary analytical system or process in which the
invention is applied
is in a single molecule DNA sequencing operation in which an immobilized
complex of
DNA polymerase, DNA template and primer are monitored to detect incorporation
of
nucleotides or nucleotide analogs that bear fluorescent detectable groups.
See, e.g., U.S.
Patent Nos. 7,033,764, 7,052,847, 7,056,661, and 7,056,676. In brief, these
arrays
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typically comprise a transparent substrate e.g., glass, quartz, fused silica,
or the like, having
an opaque, e.g., typically a metal, layer disposed over its surface. A number
of apertures
are provided in the metal layer through to the transparent substrate. In
waveguide
nomenclature, the apertures are typically referred to as cores, while the
metal layer
functions as the cladding layer. Typically, large numbers of cores are
provided immobilized
upon the substrates, and positioned such that individual biological or
biochemical
complexes are optically resolvable when associated with a fluorescent labeling
group or
molecule, such as a labeled nucleotide or nucleotide analog.
[0055] In preferred aspects, e.g., that maximize throughput of the
sequencing
process, the individual complexes may be provided within an optically confined
space, such
as a zero mode waveguide, where the substrate comprises an array of zero mode
waveguides housing individual complexes. In this aspect, an excitation light
source is
directed through a transparent substrate at an immobilized complex within a
zero mode
waveguide core. Due to the cross-sectional dimension of the waveguide core in
the
nanometer range, e.g., from about 20 to about 200 nm, the excitation light is
unable to
propagate through the core, and evanescent decay of the excitation light
results in an
illumination volume that only extends a very short distance into the core. As
such, an
illumination volume that contains one or a few complexes results. Thus,
multiple different
reactions represented in multiple waveguide cores in individual arrays can be
illuminated
and interrogated simultaneously. Zero mode waveguides and their application in
sequencing and other analyses are described in, e.g., U.S. Patent Nos.
6,917,726, 7,013,054,
7,181,122, 7,292,742; 7,302,146; 7,315,019 and Levene et al., Science
2003:299:682-686.
[0056] Other approaches to optical confinement can also be used with
the methods
and systems provided by the invention. For example, total internal reflectance
fluorescence
microscopy may be used to confine the illumination to near the surface of a
substrate. This
provides a similar confining effect as the zero mode waveguide, but does so
without
providing a structural confinement as well. Still other optical confinement
techniques may
generally be applied, such as those described in U.S. Patent Nos. 7,033,764,
7,052,847,
7,056,661, and 7,056,676.
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[0057] Because of the dimensions and density of features, e.g., waveguide
cores
and/or other optical confinements, on such substrates, highly multiplexed
illumination and
collection/detection systems that maximize the signal-to-noise ratio, e.g., by
minimizing the
production of and/or detection of background signal levels and
autofluorescence, can be of
beneficial use in analyzing fluorescent signals.
[0058] The multiplexed ZMW arrays described above are typically
interrogated
using a fluorescence detection system that directs excitation radiation at the
various reaction
regions in the array and collects and records the fluorescent signals emitted
from those
regions. A simplified schematic illustration of these systems is shown in
Figure 1. As
shown, the system 100 includes substrate 102 that includes a plurality of
discrete sources of
fluorescent signals, e.g., array of zero mode waveguides 104. An excitation
illumination
source, e.g., laser 106, is provided in the system and is positioned to direct
excitation
radiation at the various fluorescent signal sources. This is typically done by
directing
excitation radiation at or through appropriate optical components, e.g.,
dichroic 108 and
objective lens 110 that direct the excitation radiation at substrate 102, and
particularly signal
sources 104. Emitted fluorescent signals from sources 104 are then collected
by the optical
components, e.g., objective 110, and passed through additional optical
elements, e.g.,
dichroic 108, prism 112 and lens 114, until they are directed to and impinge
upon an optical
detection system, e.g., detector array 116. The signals are then detected by
detector array
116, and the data from that detection is transmitted to an appropriate data
processing unit,
e.g., computer 118, where the data is subjected to interpretation, analysis,
and ultimately
presented in a user ready format, e.g., on display 120, or printout 122, from
printer 124.
[0059] While the ability to multiplex is theoretically only limited by
the amount of
area in which you can place your multiple samples and then analyze them,
realistic
analytical systems face constraints of laboratory space and cost. As such, the
amount of
multiplex that can be derived in the analysis of discrete fluorescent signal
sources or sample
regions using a realistic instrumentation system, e.g., an array of ZMWs, is
somewhat
limited by the ability to obtain useful signal information from increasingly
small amounts of
materials or small areas of substrates, plates or other analysis regions. In
particular, as such
signal sources are reduced in size, area or number of molecules to be
analyzed, the amount
of detectable signal likewise decreases, as does the signal to noise ratio of
the system, e.g.,
due to autofluorescence background noise.
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[0060] With respect to the exemplary sequencing systems described above,
sources
of autofluorescence background noise can typically include the components of
the optical
train through which the excitation radiation is directed, including the
objective lens 110 or
lenses, the dichroic filter(s) 108, and any other optical components, i.e.,
filters, lenses, etc.,
through which the excitation radiation passes. Also contributing to this
autofluorescence
background noise are components of the substrate upon which the monitored
sequencing
reactions are occurring, which, in the case of zero mode waveguide arrays for
example,
include the underlying transparent substrate that is typically comprised of
glass, quartz or
fused silica, as well as the cladding layer that is disposed upon the
substrate, typically a
metal layer such as aluminum.
[0061] In general, the present invention provides both preventive and
remedial
approaches to reducing impacts of autofluorescence background noise, in the
context of
analyses that employ illuminated reactions, e.g., multiplexed illuminated
reactions.
Restated, in a first general preventive aspect, the invention is directed to
processes and
systems that have a reduced level of autofluorescence background noise that is
created and
that might be ultimately detected by the system. In the additional or
alternative remedial
aspects, the invention provides methods and systems in which any
autofluorescence
background noise that is created, is filtered, blocked or masked substantially
or in part from
detection by the system. As will be appreciated, in many cases, both
preventative and
remedial approaches may be used in combination to reduce autofluorescence
background
noise.
II Preventive Measures
[0062] In a first aspect, the present invention reduces the level of
autofluorescence
background noise generation by preventing or reducing the production of that
background
noise in the first instance. In particular, this aspect of the invention is
directed to providing
illumination of the optical signal source or sources in a way that reduces or
minimizes the
generation of such autofluorescence background noise.
[0063] In accordance with one aspect of the invention, the reduction in
autofluorescence creation is accomplished by reducing the amount of
illumination input into
the system and/or directed at the substrate, e.g., by providing highly
targeted illumination of
only the locations that are desired to be illuminated, and preventing
illumination elsewhere
in the array or system. By using highly targeted illumination, one
simultaneously reduces
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the area of the substrate that might give rise to autofluorescence, and
reduces the overall
amount of input illumination radiation required to be input into the system,
as such input
illumination is more efficiently applied.
[0064] In particular, the amount of illumination power required to be
applied to the
system increases with the number of signal sources that are required to be
illuminated. For
example, in a zero mode waveguide array that is configured in a gridded format
of rows
and/or columns of waveguides, multiple waveguides are generally illuminated
using a linear
illumination format (See, e.g., International Patent Application Nos.
US2007/003570 and
US2007/003804). Multiple rows and/or columns are then illuminated with
multiple
illumination lines. While linear beam spot illumination can be effective for
illuminating
multiple discrete regions on a substrate, e.g., multiple signal sources that
are disposed in a
line, there are certain deficiencies associated with this method, including
excessive
illumination, inefficient illumination power usage, and excessive
autofluorescence
background noise.
[0065] As shown in Figure 2, as the number of illumination lines
increases, it
results in a linear increase in the amount of autofluorescence emanating from
the system. In
particular, Figure 2 shows a plot of fluorescent signals emanating from a
spotted array of
A1exa488 fluorescent dye spots on a fused silica slide. As can be seen, as
more illumination
lines are applied to the array, the baseline fluorescence level attributable
to autofluorescence
background noise increases linearly with the number of illumination lines.
Further, it has
been demonstrated that this autofluorescence background noise derives not only
from the
substrate, but also from the other optical components of the system, such as
the objective
lens and dichroic filter(s).
[0066] Accordingly, in a first aspect, the invention reduces the amount
of
autofluorescence background noise by reducing the amount of excitation
illumination put
into the system, while still producing the desired fluorescent signals. In
general, providing
the same or similar levels of excitation illumination at desired locations,
e.g., on the
substrate, while reducing overall applied excitation illumination in the
system, is
accomplished through more efficient use of applied illumination by targeting
that
illumination only to the desired locations. In particular, by targeting
illumination only at the
relevant locations, e.g., primarily at only the waveguides on an array, one
can reduce the
amount of power required to be directed into the system to accomplish the
desired level of
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illumination and at the substrate, yielding a consequent reduction in the
amount of
autofluorescence background noise that is generated at either of the substrate
or those
optical components through which such illumination power is directed.
Additionally,
because less of the substrate is being illuminated by virtue of the targeted
nature of the
illumination, less of the substrate will be capable of contributing to the
autofluorescence
background noise.
[0067] By targeted illumination or targeted illumination pattern, in
accordance with
the foregoing, is meant that the illumination directed at the substrate is
primarily incident
upon the desired locations, rather than other portions, e.g., of a substrate.
For example, as
alluded to above, where one desires to interrogate a number of discrete
locations on a
substrate for fluorescent signals, using targeted illumination would include
directing
discrete illumination spots at each of a plurality of the different discrete
locations. Such
targeted illumination is in contrast to illumination patterns that illuminate
multiple locations
with a single illumination spot or line, in flood or linear illumination
profiles. Again, as
noted above, targeting illumination provides the cumulative benefits of
reducing the
required amount of illumination input into the system, and illuminating less
area of the
substrate, both of which contribute to the problem of autofluorescence
background noise.
[0068] In particular, targeted illumination, as used herein, can be
defined from a
number of approaches. For example, in a first aspect, a targeted illumination
pattern refers
to a pattern of illuminating a plurality of discrete signal sources, reaction
regions or the like,
with a plurality of discrete illumination spots. While such targeted
illumination may
include ratios of illumination spots to discrete signal sources that are less
than 1, i.e., 0.1,
0.25, or 0.5 (corresponding to one illumination spot for 10 signal sources, 4
signal sources
and 2 signal sources, respectively) in particularly preferred aspects, the
ratio will be 1 (e.g.,
one spot for one signal source, i.e., a waveguide).
[0069] In accordance with preferred aspects of the present invention,
optical systems
that can be used with the methods, processes and systems of analyzing
fluorescent materials
with reduced autofluorescence can separately illuminate large numbers of
discrete regions
on a substrate or discrete signal sources. As used herein, separate
illumination of discrete
regions or locations refers to multiple individual illumination spots that are
separate from
each other at at least the resolution of optical microscopy. In particular
embodiments, the
optical systems, e.g., that can be used with the methods and systems of
analyzing
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fluorescent materials with reduced autofluorescence described herein, provide
the further
advantage of providing such separate illumination of densely arrayed or
arranged discrete
regions. Such illumination patterns can provide discrete illumination spots at
a density of
on the order of at least 1000 discrete illumination spots per mm2, preferably
at least 10,000
discrete illumination spots per mm2, and in some cases, greater than 100,000
discrete
illumination spots per mm2, or even 250,000 discrete illumination spots per
mm2 or more.
As will be appreciated, the foregoing illumination pattern densities will
typically result in
intra-spot spacing upon an illuminated substrate (in the closest dimension),
of less than 100
pm, center to center, preferably, less than 20 p.m, more preferably, less than
10 m, and in
many preferred cases, spacing between spots of 1 pm or less, center to center.
As noted
previously, such spacing generally refers to inter-spot spacing in the closes
dimension, and
does not necessarily reflect inter-row spacing that may be substantially
greater, due to the
allowed spacing for spectral separation of adjacent rows, as discussed
elsewhere herein.
[0070] The optical systems that can be used with the methods and systems
of the of
analyzing fluorescent materials with reduced autofluorescence are generally
capable of
separately illuminating 100 or more discrete regions on a substrate,
preferably greater than
500 discrete regions, more preferably greater than 1000 discrete regions, and
still more
preferably, greater than 5000 or more discrete regions. Further, such high
number multiplex
optics will preferably operate at the densities described above, e.g., from
densities of about
1000 to about 1,000,000 discrete illumination spots per mm2.
[0071] The optical systems that can be used with the methods, processes,
and
systems for analyzing fluorescent materials with reduced autofluorescence can
provide
illumination targets on the substrate that are regularly arranged over the
substrate to be
analyzed, e.g., provided in one or more columns and/or rows in a gridded
array. Such
regularly oriented target regions provide simplicity in production of the
optical elements
used in the optical systems. Notwithstanding the foregoing, in many cases, the
optical
systems that can be used in methods and systems to analyze fluorescent
materials with
reduced autofluorescence may be configured to direct excitation illumination
in any of a
variety of regular or irregular illumination patterns on the substrate. For
example, in some
cases, it may be desirable to target illumination at a plurality of regions
that are arranged
over the substrate in a non-repeating irregular spatial orientation.
Accordingly, having
identified such arrangement one could provide multiplex optics that direct
excitation light
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accordingly. Likewise, such optics can readily provide for targeted
illumination of rows or
columns of signal sources that are disposed at irregular intra or inter-row
(or column)
spacings or pitches, e.g., where spots within a row are more closely spaced
than spots in
adjacent rows.
[0072] In the context of the multiplex optical systems that can be used
with the
methods and systems for analyzing fluorescent materials with reduced
autofluorescence,
such targeted illumination also typically refers to the direction of
illumination to multiple
discrete regions on the substrate, which regions preferably do not overlap to
any substantial
level. As will be appreciated, such targeted illumination preferably directs a
large number
of discrete illumination beams to a large number of substantially discrete
locations on a
substrate, in order to separately interrogate such discrete regions. As will
also be
appreciated the systems of the invention do not necessarily require a complete
absence of
overlap between adjacent illumination regions, but may include only a
substantial lack of
overlap, e.g., less than 20%, preferably less than 10% overlap and more
preferably less than
5% of the illumination in one spot will overlap with an adjacent spot (when
plotted as spot
illumination intensity, e.g., from an imaging detector such as a CCD or
EMCCD).
[0073] In still other aspects, the multiplex optics that can be used with
the methods
and systems of analyzing fluorescent materials with reduced autofluorescence
can
optionally direct in-focus illumination in a three dimensional space, thus
allowing the
systems of the invention to illuminate and detect signals from three
dimensional substrates.
Such substrates may include solid tissue samples, encased samples, bundles,
layers or stacks
of substrates, e.g., capillaries, planar arrays, or multilayer microfluidic
devices, and the like.
[0074] A variety of components may be used to provide large numbers of
illumination spots from a few, or a single illumination beam. As discussed in
greater detail
below, the multiplex optical element can comprise one, two, three, four or
more discrete
optical elements that work in conjunction to provide the desired level of
multiplex as well as
provide controllability of the direction of the multiplexed beams. For example
and as
discussed in greater detail below, one may use two or more diffraction
gratings to first split
a beam into a plurality of beams that will provide a plurality of collinear
spots arrayed in a
first dimension. Each of these beams may then be subjected to additional
manipulation to
provide a desired targeted illumination pattern. For example, each resulting
beam may be
passed through appropriate linearization optics, such as a cylindrical lens,
to expand each
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collinear spot into an illumination line oriented orthogonal to the axis of
the original series
of spots. The result is the generation of a series of parallel illumination
lines that may be
directed at the substrate. Alternatively and preferably in some cases, the
series of beams
resulting from the first diffraction grating can be passed through a second
diffraction grating
that is rotated at a 90 degree angle (or other appropriate angle) to the first
diffraction grating
to provide a two dimensional array of illumination beams/spots, i.e.,
splitting each of the
collinear spots into an orthogonally oriented series of collinear spots. In
particular, if one
provides a diffraction grating that provides equal amplitude to the different
orders, and
illuminates it with a laser beam, it will result in a row of illuminated
spots, corresponding to
discrete beams each traveling at a unique angle after they impinge on the
grating. If a
second similar grating is placed adjacent to the first but rotated by 90
degrees, it will
provide a 2 dimensional grid of beamlets, each traveling with a unique angle.
If the 2
gratings are identical, a square grid will result, but if the 2 gratings have
different period, a
rectangular grid will result. By selecting each of the diffraction gratings
and the angle of
rotation of the two gratings relative to each other, one can adjust spacing
between and/or
positioning of the columns or rows of illumination spots in the array, as
desired.
[0075] Figure 3 provides a schematic illustration of the illumination
pattern
generated from a first diffraction grating, and for a first and second
diffraction grating
oriented 90 relative to each other. As shown, passing a single laser beam
through an
appropriate diffraction grating will give rise to multiple discrete beams (or
"beamlets") that
are oriented in a collinear array and are represented in Panel A of Figure 3
as a linear array
of unfilled spots. By subsequently passing the linear array of beamlets
through a second
diffraction grating rotated orthogonally to the first, e.g., 90 , around the
optical axis, one
will convert each of the first set of beamlets (unfilled spots), into its own,
orthogonally
arrayed collinear array of beamlets (illustrated as hatched spots in Panel B
of Figure 3).
The resulting set of beamlets results in a gridded array of spots, as shown in
Panel B of
Figure 3.
[0076] Targeting illumination to each of an array of point targets such
as zero mode
waveguides, can be also accomplished by a number of other methods. For
example, in a
first aspect, excitation radiation may be directed through a microlens array
in conjunction
with the objective lens, in order to generate spot illumination for each of a
number of array
locations. In particular, a lens array can be used that would generate a
gridded array of
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illumination spots that would be focused upon a gridded array of signal
sources, such as
zero mode waveguides, on a substrate. An example of a microlens array is shown
in Figure
4, Panel A. In particular, shown is an SEM image of the array. Panel B of
Figure 4
illustrates the illumination pattern from the microlens array used in
conjunction with the
objective lens of the system. As will be appreciated, the lens array is
fabricated so as to be
able to focus illumination spots on the same pitch and position as the
locations on the array
that are desired to be illuminated.
[0077] In alternative and/or additional aspects, a plurality of
illumination spots for
targeted illumination of signal sources may be generated by passing excitation
illumination
through one or more diffractive optical elements ("DOE") upstream of the
objective lens.
In particular, DOEs can be fabricated to provide complex illumination
patterns, including
arrays of large numbers of illumination spots that can, in turn, be focused
upon large
numbers of discrete targets.
[0078] For example, as shown in Figure 5, a DOE Phase mask, as shown in
Panel
A, can generate a highly targeted illumination pattern, such as that shown in
Panel B, which
provides targeted illumination of relatively large numbers of discrete
locations on a
substrate, simultaneously. In particular, the DOE equipped optical system can
generally
separately illuminate at least 100 discrete signal sources, e.g., zero mode
waveguides,
simultaneously and in a targeted illumination pattern. In preferred aspects,
the DOE may be
used to simultaneously illuminate at least 500 discrete signal sources, and in
more preferred
aspects, illuminate at least 1000, at least 5000, or at least 10,000 or more
discrete signal
sources simultaneously, and in a targeted illumination pattern, e.g., without
substantially
illuminating other portions of a substrate such as the space between adjacent
signal sources
or preferably between adjacent illumination spots.
[0079] Several approaches can be used to design and fabricate a DOE for
use in the
present invention. The purpose here is to evenly divide the single laser beam
into a large
number of discrete new beams, e.g., up to 5000 or more new beams, each with
1/5000 of the
energy of the original beam, and each of the 5000 "beamlets" traveling in a
different
direction. By way of example, the DOE design requirement is to evenly space
the beamlets
in angles (the 2 angles are referred to herein as Ox and Or).
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[0080] As will be appreciated, the DOE (and/or a Microlens Array) will
divide the
light into numerous beams that are propagating at unique angles. In a
preferred illumination
scheme the DOE is combined with the objective lens in a planned way, such that
the
objective lens will perform a Fourier transform on all of the beamlets. In
this Fourier
transform, angle information is converted into spatial information at the
image plane of the
objective. After the beamlets pass through the objective, each unique 0, and
Oy will
correspond to a unique x,y location in the image plane of the objective. The
objective
properties must be known in order to correctly design the DOE or microlens.
The formula
for the Fourier transform is given by:
[0081] (x,y) = EFL x Tangent (0, , 007
[0082] where EFL is the Effective Focal Length of the objective.
[0083] There are several different approaches to producing a DOE that
will meet the
needs of a fluorescence detection system that can be used with the methods and
systems for
reducing autofluorescence background noise. For example, one approach is
through the use
of a phase mask that is pixilated such that each pixel will retard the
incident photons by a
programmed amount. This phase retardation can again be achieved in different
ways. For
example, one preferred approach uses thickness of the glass element. For
example, the
phase mask might include a 1/2 inch square piece of SiO2. Material is etched
away from the
top surface of the SiO2 plate to, e.g., 64 different etch depths. This is
referred to as a 64-
level gray scale pattern. The final phase mask then is comprised of a
pixilated grid where
each pixel is etched to a particular depth. The range of etch depths
corresponds to a full 27t
of phase difference. Restated, a photon which impinges on a pixel with the
minimum etch
depth (no etching) will experience exactly 2ic additional phase evolution
compared to a
photon which strikes a maximum etch depth (thinnest part of the Si02). The
pixilated
pattern etched into the DOE is repeated periodically, with the result that the
lateral position
of the laser beam impinging on the mask is unimportant.
[0084] Figure 6 shows an illumination pattern generated from a DOE that
provides
an array of 5112 discrete illumination spots. The DOE is configured such that
the
illumination spots are on a period that, when focused upon the substrate
appropriately, will
correspond to a discrete signal source in an arrayed substrate, e.g., a zero
mode waveguide
array.
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[0085] In some cases, it may be desirable to provide illumination
patterns that have
a higher density of illumination spots than may be provided using a single
DOE. In
particular, the period size or spacing between adjacent illumination spots
resulting from a
DOE is a function of the minimum spot size of the originating illumination
beam. As such,
in order to obtain a higher density or smaller period size, for the
illumination pattern, one
may be required to employ an originating beam spot size that is smaller than
desired,
resulting in incomplete illumination of a desired target or enhanced
difficulty in targeting a
small spot to a small target. For example, in many cases, the originating beam
size typically
must be at least twice the period size between two adjacent resulting
illumination spots from
a DOE. However, where one desires an illumination spot of a larger size, the
period is
consequently increased.
[0086] In addressing this issue, one particularly preferred approach is
to utilize
multiple multiplex optical elements in parallel (rather than in series). In
particular, one may
use two or more similar or identical DOEs in an illumination path where each
DOE results
in illumination spots at a period size that is twice that desired in one or
more dimensions,
but where each of which provides an illumination spot size that is desired. By
way of
example, an originating beam is first split into two identical beams using,
e.g., a 50% beam
splitter. Each beam is then directed through its own copy of the DOE, and the
resulting
multiplexed beams are imaged one half a period off from each other. As a
result, the period
size of the illumination spots is half that obtained with a single DOE. Figure
7 provides a
schematic illustration of the resulting illumination pattern when the
illumination pattern
(unfilled spots) from a first DOE having a first period P1 (shown in panel A)
and a second
DOE having the same illumination pattern period P1 (hatched spots) are
overlaid as a single
projection (shown in Panel B) having a new effective period P2. As alluded to
above, two,
three, four or more DOEs may be used in parallel and their resulting spots
overlaid, to
provide different spot spacing regardless of the originating illumination spot
size, providing
spacing is maintained sufficient to avoid undesirable levels of spot overlap
at the target
locations. In addition, and as apparent in Figure 7, by overlaying multiple
illumination
patterns, one can provide different spacing of illumination spots in one
dimension while
preserving the larger spacing. In particular, one can provide more densely
arrayed
illumination spots in rows while preserving a larger intra-row spacing. Such
spacing is
particularly useful where one wishes to preserve at least one dimension of
larger spacing to
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CA 02687062 2009-11-10
account for spectral separation of signals emanating from each illuminated
region. Such
spacing is discussed in detail in, e.g., U.S. Patent Application Nos.
11/704,689, filed
February 9, 2007, 11/483,413, filed July 7, 2006, and 11/704,733, filed
February 9, 2007.
[00871 In addition to the foregoing considerations, and as will
be appreciated, the
actual phase evolution for the DOE is a function of the optical wavelength of
the light being
transmitted through it, so DOE devices will generally be provided for a
specific wavelength
of excitation illumination. As such, for applications in which broad spectrum
or
multispectral illumination is desired, the optical systems used in the methods
and processes
for analyzing fluorescent materials with reduced autofluorescence will
typically include
multiple multiplex elements, e.g., DOEs. For example, in the case of
multispectral
fluorescent analysis, different fluorescent dyes are typically excited at
different
wavelengths. As such, multiple different excitation light sources, e.g.,
lasers are used, e.g.,
one for each peak excitation spectrum of a dye. In such cases, a different
multiplex element
would preferably be provided for each illumination source. In the case of
systems
employing DOEs as the multiplex component for example, the optical path
leading from
each different laser would be equipped with its own DOE tailored or selected
for that laser's
spectrum. Accordingly, the optical systems that can be used with the methods
and systems
to analyze fluorescent materials with reduced autofluorescence will typically
include at least
one multiplex component, preferably, two, three or in many cases four or more
different
multiplex components to correspond to the at least one, preferably two, three,
four or more
different excitation light sources of varying illumination spectra.
[0088] In addition to accounting for variation in the excitation
wavelength in the
selection of the DOEs, the need for high-density discrete illumination may
also impact the
DOE specifications. In particular, as will be appreciated, because adjacent
beamlets or
spots may be either perfectly in or out of phase with each other, any overlap
between
adjacent spots on a surface may be constructive, i.e., additive, or
destructive, i.e.,
subtractive. As such, in particularly preferred aspects where it is desired
that optical
systems used with the methods and systems for reducing autofluorescence
provide uniform
illumination of spots across the field of illumination spots, spots must be
substantially
separated with little or no overlap within the desired illumination region.
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[0089] In alternative embodiments, optical systems used with the methods
and
systems for reducing autofluorescence, in conjunction with the multiple DOE
approach
described above, can employ a polarization splitter to divide the originating
beam into two
or more separate beams of differing polarization. Each different beam may then
be split
into multiple beamlets that may be overlaid in closer proximity or with
greater overlap
without concern for destructive interference in the overlapping regions. While
a
conventional polarizing beam splitter may be used to divide the originating
beam, in
preferred aspects, a Wollaston prism may be employed. Wollaston prisms provide
for a
slightly different deviation angle for s and p polarizations, resulting in the
generation of two
closely spaced beamlets that may be directed through the same or multiple DOEs
without
concern for interference from overlapping beamlets. In addition to avoiding an
interference
issue, the use of the Wollaston prism provides additional control of the intra-
illumination
spot spacing. In particular, by rotating the prism, one can adjust the spacing
between grids
of beamlets generated from passing the two or more different polar beam
components
through the DOE(s). An example of an illumination optical path including this
configuration is illustrated in Figure 8. For ease of discussion, the
fluorescence path is
omitted from Figure 8. As shown, the illumination path 800 includes excitation
light
source 802. The excitation light is directed through polarizing splitter such
as Wollaston
prism 804, which splits the originating beam into its polar p and s
components. Each polar
beam is then passed through a multiplex component, such as one or more DOEs
804. These
doubled multiplexed beams are then passed through lens 806, dichroic 810 and
objective
812, to be focused as an array of illumination spots on substrate 814. As with
Figure 7, the
array of illumination spots comprise overlaid patterns separated by the
separation imparted
by the Wollaston prism 804. Further, by rotating the prism 804, one can
modulate the
separation between the overlaid polar illumination patterns to adjust intra-
spot spacing.
[0090] As noted above, in some cases, it may be desirable to direct
excitation
illumination at targets that exist in three-dimensional space, as opposed to
merely on a
planar substrate. In such cases, DOEs may be readily designed to convert an
originating
beam into an array of beamlets with different focal planes, so as to provide
for three
dimensional illumination and interrogation of three dimensional substrates,
such as layered
fluidic structures (See, U.S. Patent No. 6,857,449) capillary bundles, or
other solid
structures that would be subjected to illuminated analysis.
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[0091] For many applications the desired intensity of the different
beamlets
provided by optical systems used with the methods and systems to analyze
fluorescent
materials with reduced autofluorescence could be variable. For example, it may
be
advantageous to prescribe a varying pattern of intensities to provide a
variable range of
intensities that can be sampled by a grid of sample regions. Or, the desired
intensity could
be selected in real time by moving the sample to a beamlet of the desired
intensity. Or, the
grid of variable intensities could be in a repeating pattern such that a grid
of sample regions
with the periodicity of the repeating pattern, and the intensity of the entire
grid can be
selected by moving to the desired location. More importantly, variations in
optical
throughput can be compensated by programming the beamlet intensity. In most
optical
systems light near the edges of the field-of-view is vignetted such that the
optical
transmission is maximum at the center and falls off slowly as the observation
point moves
away from the center. In a typical system based on an objective lens, the
vignetting may
cause up to or even more than 10% lower throughput at the edge of the optical
field, as
compared to the center of the field. In this case, the DOE beamlet intensity
pattern can be
pre-programmed to accommodate such variations, e.g., to be 10% higher at the
edge of the
field than the center, and to vary smoothly according to the vignetting. More
complicated
variations in throughput can also exist in particular optical systems, and can
be pre-
compensated in the DOE design. For a discussion on the design of DOE phase
masks, see,
e.g., "Digital Diffractive optics" by Bernard Kress and Patrick Meyrueis,
Wiley 2000.
[0092] Accordingly, one may provide DOEs that present multiplexed
beamlets that
have ranges of different powers or intensities depending upon the desired
application and/or
system used. In particular, the DOE may be designed and configured to present
beamlets
that differ in their respective power levels. As such, at least two beamlets
presented will
typically have different power levels, and in some cases larger subsets (e.g.,
10 or more
beamlets), or all of the presented beamlets may be at different power levels
as a result of
configuration of the DOE. Restated, a DOE can generate beamlets having power
profiles to
fit a given application, e.g., correcting for optical aberrations such as
vignetting, providing a
range of illumination intensities across a substrate, and the like. The
resulting beamlets may
fall within two, 5, 10, 20 or more different power profiles.
[0093] When the DOE beamlet pattern is used in combination with a
microscope
objective lens, the size of the individual beamlets can be modified as desired
by 1) adjusting
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the diameter of the beam into the DOE and 2) defocusing the pattern slightly.
In the case of
1) the size of the beamlet is a function of the size of the input beam, and
increasing the input
beam size will increase the beamlet size. In any case the final beamlet size
at the ZMW
plane obeys the diffraction limit, which is affected by the aperture size, and
changing the
input beam diameter is equivalent to changing the aperture related to the
optical diffraction
limit. In the case of defocusing the entire pattern, the diffraction limit is
no longer obeyed
but the beamlets can be made to have larger size than the diffraction limit.
Further, the
beamlets need not be circular ¨ they could be elliptical by either starting
with an elliptical
beam input into the DOE or by defocusing the pattern in one or both
dimensions. See, e.g.,
"Principles of Optics" by Born and Wolf, Wiley, 2006 edition.
[0094] Alternative multiplex optics systems for converting a single
illumination
source into multiple targeted illumination beams, e.g., to reduce
autofluorescence
background noise, includes, for example, fiber optic approaches, where
excitation light is
directed through multiple discrete optical fibers that are, in turn directed
at the substrate,
e.g., through the remainder of the optical train, e.g., the objective. In such
context, the fiber
bundles are positioned to deliver excitation illumination in accordance with a
desired
pattern, such as a gridded array of illumination spots.
[0095] In addition to multiplex optics that convert a single illumination
beam into
multiple discrete beams, as described above, certain aspects of the optical
systems that can
be used with the present invention may employ multiplexed illumination sources
in place of
a single illumination source with a separate multiplex optic component to
split the
illumination into multiple beamlets. Such optical systems are particularly
useful in
combination with the spatial filters described in greater detail below, and
include, for
example, arrayed solid state illumination sources, such as LEDs, diode lasers,
and the like.
[0096] Alternatively, as a goal of targeted illumination in the context
of the present
invention is to reduce autofluorescence from excessive illumination, targeted
illumination
denotes illumination where a substantial percentage of the illumination that
is incident upon
the substrate is incident upon the desired signal source(s) as opposed to
being incident on
other portions of the substrate. Accounting for the often small size of signal
sources, e.g., in
the case of nanoscale zero mode waveguides, as well as the tolerance in
direction of
illumination by optical systems, such targeted illumination will typically
result in at least
5% of the illumination incident upon the overall substrate being incident upon
the discrete
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signal sources themselves. This corresponds to 95% or less wasted illumination
that is
incident elsewhere. In preferred aspects, that percentage is improved such at
least 10%,
20% or in highly targeted illumination patterns, at least 50% of the
illumination incident
upon the substrate is incident upon the discrete signal sources. Conversely,
the amount of
illumination incident upon other portions of the substrate is less than 90%,
less than 80% or
in highly targeted aspects, less than 50%. Determination of this percentage is
typically a
routine matter of dividing the area of a substrate that is occupied by the
relevant signal
discrete source divided by the area of total illumination, multiplied by 100,
where a region
is deemed "illuminated" for purposes of this determination if it exceeds a
threshold level of
detectable illumination from the illumination source, e.g., 5% of that at the
maximum point
of a given illumination spot on the same substrate.
[0097] In still a further aspect, targeted illumination may be identified
through the
amount of laser power required to illuminate discrete signal sources vs.
illuminating such
signal sources using a single flooding illumination profile, e.g., that
simultaneously
illuminates an entire area in which the plurality of discrete sources is
located, as well as the
space between such sources. Preferably, the efficiency in targeted
illumination over such
flood illumination will result in the use of 20% less laser power, preferably
30% less laser
power, more preferably more than 50% less laser power, and in some cases more
than 75%,
90% or even 99% less laser power to achieve the same illumination intensity at
the desired
locations, e.g., the signal sources. As will be appreciated, the smaller the
discrete
illumination spot size, e.g., the more targeted the illumination, the greater
the susceptibility
of the system to alignment and drift issues, and calibration efforts will need
to be increased.
[0098] In addition to the advantages of reduced autofluorescence, as set
forth above,
targeted illumination also provides benefits in terms of reduced laser power
input into the
system which consequently reduces the level of laser induced heating of
reaction regions.
[0099] In another preventive approach, an overall optical system or one
or more
components through which the excitation illumination passes, may be treated to
reduce the
amount of autofluorescence background noise generated by the system
components. By
way of example, in an overall optical system, e.g., as schematically
illustrated in Figure 1,
illumination may be applied to the system that results in a photobleaching of
some or all of
the elements of the various components that are fluorescing under normal
illumination
conditions. Typically, this will require an elevated illumination level
relative to the normal
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analytical illumination conditions of the system. Photobleaching of the
optical components
may be carried out by exposing the optical train to illumination that is
greater in one or both
of intensity or power and duration. Either or both of these parameters may be
from 2X, 5X
10X or even greater than that employed under conventional analysis conditions.
For
example, exposure of the optical train to the excitation illumination for a
prolonged period,
e.g., greater than 10 minutes, preferably greater than 20 minutes, more
preferably greater
than 50 minutes, and in some cases greater than 200 or even 500 minutes, can
yield
substantial decreases in autofluorescence background noise emanating from the
system
components. In one particular exemplary application, a 20 mW, 488 nm laser can
be used
to illuminate the overall system for upwards of 20 hours in order to
significantly reduce
autofluorescence from the components of such system. Figure 9 shows a plot of
autofluorescence counts in a system illuminated with a 20 mW 488nm laser,
following
exposure of the optical train to 'burn in' illumination from a 7.5mW laser at
488 nm from 0
to 1000 minutes, followed by illumination from a 162mW laser at 488 nm from
1000 to
4600 minutes. Alternatively or additionally, other illumination sources may be
employed to
photobleach the optical components, including, e.g., lasers of differing
wavelengths,
mercury lamps, or the like. As will be appreciated, the photobleaching of the
optical
components may be carried out at a targeted illumination profile, e.g., a
relatively narrow
wavelength range such as 488 nm laser illumination, or it may be carried out
under a
broader spectrum illumination, depending upon the nature of the components to
be
photobleached and the underlying cause of the autofluorescence.
[0100] In addition to providing large numbers of discrete beams to be
directed at
arrayed regions on substrates, the fluorescence detection systems that can be
used with the
systems and methods to reduce autofluorescence optionally include additional
components
that provide controlled beam-shaping functionalities, in order to present
optimal
illumination for a given application.
[0101] For example, in the case of systems employing lens arrays, as
described
previously, such lens arrays may comprise a rectangular shape that results in
illumination
spots that are asymmetrically shaped, e.g., elliptical. Accordingly, one may
include within
the illumination path, one or more relatively shallow cylindrical lenses to
correct the beam
shape and provide a more symmetrical spot.
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[0102] In addition to the various optical components described above, a
number of
additional cooperating optical elements may be employed with lens arrays in
order to
provide finer tuning of the resulting illumination pattern emanating from the
multiplex
component or components of the optical systems that can be used with the
methods and
systems for analyzing fluorescent materials with reduced autofluorescence.
[0103] In a number of cases, it will be desirable to control, and
preferably
independently control the direction of individual beams or subsets of beams
that have been
multiplexed using the systems described herein. In particular, preferred
applications of the
optical systems will direct multiple beams at arrays of targets that are on a
pre-selected
spacing, orientation and/or pitch. However in some cases, the spacing,
orientation and/or
pitch of target regions may not be precisely known at the time of designing
the optical path,
and/or may be subject to change over time. Accordingly, in some cases it will
be desired to
provide for independent adjustment of the direction of individual beams, or
more routinely,
subsets of beams multiplexed from a single originating beam.
[0104] By way of example, in the case of arrays of discrete reaction
regions,
typically such reaction regions will be provided at substantially known
relative locations,
pitch and/or orientation. In particular, such arrays may generally be
presented in a gridded
format of regularly spaced columns and/or rows. However, variations in the
processes used
to create such arrays may result in variations in such relative location,
within prescribed
tolerances. This is particularly an issue where the features of such arrays
are on the scale of
nanometers, e.g., from 10 to 500 nm in cross section.
[0105] For example, in the case of linear illumination patterns, one may
wish to
adjust the inter-line spacing of the illumination pattern, e.g., to adjust for
variations in the
inter-row or inter-column spacing of signal sources. One particular approach
involves the
case where a series of parallel illumination lines is created from the
linearization of a row of
co-linear beamlets or spots. In particular, a collinear arrangement of
illumination spots
generated by passing a single illumination beam through, e.g., a diffraction
grating or DOE,
may be converted to a series of parallel illumination lines by directing the
beams through
one or more cylindrical lenses. Accordingly, by simply rotating the
diffraction grating or
DOE around its optical axis, one can adjust the spacing of the illumination
lines emanating
from the cylindrical lens(es). Such adjustment both optimizes the illumination
of discrete
signal sources and reduces the production of autofluorescence background
noise.
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[0106] Additionally or alternatively, in some cases, it may be desirable
to provide
tunable lens or lenses between the multiplex component(s) and the objective of
the system,
in order to compensate for potential focal length variation or distortion in
the objective.
Such lenses may include, for example, a zoomable tube lens having a variable
focal length
that may be adjusted as needed. Alternatively, additional pairs of field
lenses may be
employed that are adjustable relative to each other, in order to provide the
variable focal
length. In addition to the foregoing advantages, the use of such field lenses
also provide
for: transformation of diverging beamlets from DOEs or other multiplex
elements, into
converging beamlets into the objective (as shown in Figure 14, Panel B);
provide the
ability to finely adjust the angular separation of the beamlets; and provide
an intermediate
focusing plane so that additional elements can be incorporated, such as
additional spatial
filters. For example either in conjunction with field lenses as set forth
above, or in some
cases, in their absence, spatial filters may be applied in the illumination
path. The
flexibility of such an optical system can advantageously reduce the production
of
autofluorescence background noise while optimizing the illumination of signal
sources on a
substrate.
[0107] A schematic illustration of a system employing such pairs of field
lenses is
shown in Figure 14. As shown, the excitation illumination source 1402 directs
the
originating beam through the multiplex component(s) such as DOE 1404 to create
multiple
beamlets. The beamlets are then passed through a pair of lenses or lens
doublets, such as
doublets 1406 and 1408. As noted above, the lens pair or doublet pairs 1406
and 1408,
provide a number of control options over the illumination beams. For example,
as shown in
Panel B to Figure 14, these doublet pairs can convert diverging beamlets into
converging
beamlets in advance of passing into objective 1416. Likewise, such doublet
pairs may be
configured to adjust the angular separation of the beamlets emanating from DOE
1404. In
particular, by adjusting spacing between lenses in each doublet, one can
magnify the angle
of separation between beamlets. One example of this is shown in the table,
below, that
provides the calculated angular magnification from adjustment of spacing
between lenses in
each doublet of a pair of exemplary doublet lenses, e.g., corresponding to the
lenses in
doublets 1406 and 1408 of Figure 14.
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Spacing in First Spacing in Incoming Outgoing Angular
Doublet Second Doublet Beamlet Angle Beamlet Angle
Magnification
(mm) (mm) (0) (0)
2 0 2.5505 -2.4234 -0.95
1 0 2.5505 -2.48858 -0.97
=
0 0 2.5505 -2.5505 -1.00
0 1 2.5505 -2.6161 -1.03
0 2 2.5505 -2.6816 -1.05
[0108] Additionally, an optional spatial filter (as shown Figure 13 as
spatial filter
1310) may be provided between the doublets 1406 and 1408, to provide
modulation of the
beamlets as described elsewhere herein.
[0109] The beamlets are then directed through dichroic 1414, e.g., by
reflecting off
optional directional mirror 1410, and through objective 1416, which focuses
the
illumination pattern of the beamlets onto substrate 1418. Fluorescent
emissions from each
discrete location that is illuminated by the discrete beamlets are then
collected by the
objective 1416 and reflected off dichroic 1414 to pass into the separate
portion of the
fluorescence path of the system. The fluorescent signals are then focused by
focusing or
field lens, e.g., shown as a doublet lens 1420, through a spatial filter such
as confocal mask
1422, that is positioned in the focal plane of lens doublet 1420, so that only
in focus
fluorescence is passed. Doublet 1420 is preferably paired with objective 1416
to provide
optimal image quality (both at the confocal plane and the detector image
plane). The
confocally filtered fluorescence is then refocused using field lens 1424 and
is focused onto
detector 928 using another focusing lens or lens doublet, such as doublet
1426. By
providing a doublet-focusing lens, one again yields advantages of
controllability as applied
to the fluorescent signals, which can reduce both power usage and the
generation of
autofluorescence background noise.
[0110] In addition to independent adjustment of subsets of beams
multiplexed from
a single originating beam, it may also be desirable to independently adjust
subsets of signals
emanating from a substrate in response to illumination. In particular, in some
cases, it may
be desirable to selectively adjust certain subsets of signals in order to
direct them through
selected regions of the optical train, e.g., aligning with confocal masks, or
to direct such
signals to desired detector regions. In general, adjusting the direction of
the multiple
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discrete fluorescent signals may be accomplished using substantially the same
methods and
components as those described for use in the adjustment of the excitation
beams.
[0111] The use of spatial filters in the illumination path can provide a
number of
control advantages for the system, including dynamic and uniform control over
the
multiplex illumination pattern and reduction in autofluorescence background
noise. In
particular, one can employ a simple aperture or iris shaped or shapeable to
narrow the array
of beamlets that reaches the objective, and consequently the substrate. As a
result, one can
narrowly tailor the illumination pattern to avoid extraneous illumination of
the substrate, or
to target a sub-set of illumination regions or sub-region of an overall
substrate. More
complex spatial filters may also be employed to target different and diverse
patterns of
regions on the substrate by providing a mask element that permits those
beamlets that
correspond to the desired illumination pattern on the substrate. For example,
one could
target different rows and /or columns of reaction regions on an arrayed
substrate, to monitor
different reactions and/or different time points of similar reactions, and the
like. As will be
appreciated, through the use of controllable apertures, e.g., apertures that
may be adjusted
in situ to permit more, fewer, or different beamlets pass to the objective and
ultimately the
substrate, one could vary the illumination patterns dynamically to achieve a
variety of
desired goals.
[0112] Other types of optical elements also may be included within the
illumination
path. For example, in some cases, it may be desirable to include filters that
modulate laser
power intensity that reaches the objective. Such filters may include uniform
field filters,
e.g., modulating substantially all beamlets to the same extent, or they may
include filters
that are pixilated to different levels of a gray scale to apply adjusted
modulation to different
beamlets in an array. Such differential modulation may be employed to provide
a gradient
of illumination over a given substrate or portion of a substrate, or it may be
used to correct
for power variations in beamlets as a result of aberrations in the multiplex
optics., or other
components of the optical train, or it may be used to actively screen off or
actively adjust
the modulation of illumination at individual or subsets of illumination
targets. As will be
appreciated, LCD based filters can be employed that would provide active
control on a
pixel-by-pixel basis.
[0113] Any of a variety of other optical elements may similarly be
included in the
illumination path depending upon the desired application, including, for
example,
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polarization filters to adjust the polarization of the illumination light
reaching the substrate,
scanning elements, such as galvanometers, rotating mirrors or prisms or other
rastering
optics such as oscillating mirrors or prisms, that may provide for highly
multiplexed
scanning systems, compensation optics to correct for optical aberrations of
the system, e.g.,
vignetting, patterned spectral filters that can direct illumination light of
different spectral
characteristics to different portions of a given substrate, and the like. Use
of such optical
elements in targeting and/or polarizing the illumination light can reduce
power usage and
decrease autofluorescence background noise.
[0114] In particular, such spatial filter may be configured to block
extraneous
beamlets resulting from the diffractive orders of the multiplex components,
which
extraneous beamlets may contribute to noise issues. By way of example, a
simple square or
rectangular aperture may be provided in the illumination path after the
multiplex component
to permit only a limited array of beamlets to pass ultimately to the objective
and substrate.
Further, additional and potentially more complex spatial filters may be used
to selectively
illuminate portions of the substrate, which filters may be switched out in
operation to alter
the illumination profile and minimize the production of autofluorescence
background noise.
As noted above, the use of such fine-tuning optical components may be included
not only in
the illumination path of the system, but also in the fluorescence transmission
path of the
system
[0115] Although described as including various components of both an
illumination
path and a fluorescence path, it will be appreciated that certain aspects of
the invention do
not require all elements of both paths as described above. For example, in
certain aspects,
spectral separation of fluorescent signals may not be desirable or needed, and
as such may
be omitted from the systems of the invention. Likewise, in other aspects,
optical signals
from a substrate may not be based upon fluorescence, but may instead be based
upon
reflected light from the illumination source or transmitted light from the
illumination
source. In either case, the optical train may be configured to collect and
detect such light
based upon known techniques. For example, in the case of the detection of
transmitted
light, a light collection path may be set up that effectively duplicates the
fluorescence path
shown in the Figures hereto, but which is set up at a position relative to the
sample opposite
to that of the illumination path. Such path would typically include the
objective, focusing
optics, and optionally spectral filters and or confocal filters to modify the
detected
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transmitted light, e.g., reduce scattering and autofluorescence. In such
cases, dichroic filters
may again, not be desired or needed.
[0116] In other preventive approaches to autofluorescence mitigation, the
present
invention also utilizes optical elements in the optical train or the overall
system that are less
susceptible to generating autofluorescence background noise. In particular, it
has been
determined that a substantial amount of autofluorescence from more complex
optical
systems derives from coatings applied to the optical components of the system,
such as the
coatings applied to dichroic filters and objective lenses. As a result, it
will be appreciated
that additional gains in the reduction of autofluorescence can be obtained
through the
selection of appropriate optical components, e.g., that have reduced
autofluorescence. For
example, in selecting an objective lens, it will typically be desirable to
utilize an objective
that provides a reasonably low ratio of autofluorescence to illumination, as
determined on a
photon count ratio. For example, in the case of a variety of objective lenses,
this ratio has
been determined at, e.g., 1.5X10-1 and 3.2X10-1 for Olympus model objective
lenses
U1S2Fluorite 60X Air objective and 40X Air Objective, respectively.
Conversely,
objectives that have been selected or treated to have reduced autofluorescence
will typically
have a ratio that is greater than this, e.g., greater than 1X10-10. By way of
example, an
Olympus model UIS1 APO 60X Air Objective provided a ratio of 6X10-11 following
a
photobleaching exposure as described above.
[0117] As noted above, selection of components to fall within the desired
levels of
autofluorescence will in many cases select for components that have fewer or
no applied
coating layers, or that have coating layers that are selected to have lower
autofluorescence
characteristics under the particular applied illumination conditions. Of
particular relevance
to the instant aspect is the selection of dichroic filters that have been
selected to have lower
autofluorescence deriving from their coatings, either through selection of
coating materials
or use of thinner coating layers.
Prevention of Detection of Autofluorescence
[0118] In an alternative or additional aspect, the invention is directed
to a remedial
approach to background signal levels, e.g., that reduce the amount of
background signal or
autofluorescence that is detected or detectable by the system. Typically, this
aspect of the
invention is directed to filtering signals that are derived from the signal
sources or arrays in
such a way that highly relevant signals, e.g., those from the signal sources
and not from
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irrelevant regions, are detected by the system. As will be appreciated, this
aspect of the
invention may be applied alone, or in combination with the preventive measures
set forth
above, in order to maximize the reduction of the impact of background signal
levels.
[0119] In the context of one aspect of the invention, it has been
determined that a
large amount of the autofluorescence background noise constitutes "out of
focus"
fluorescence, or fluorescence that is not within the focal plane of the system
when analyzing
a given reaction region or regions. For example, autofluorescence that derives
from the
substrate portion of the overall systems of the invention, e.g., substrate 102
in Figure 1,
derives from locations in the substrate that are outside of the focal plane of
the optical
system. In particular, where the optical system is focused upon the back
surface of the
substrate, the autofluorescence that derives from the entirety of the
thickness of the
substrate, from the cladding layer above the back surface of the substrate, or
from other
points not within the focal plane of the system, will generally be out of
focus. Likewise,
autofluorescence from optical components of the system that are subjected to
excitation
illumination also are typically not within the focal plane of the instrument.
Such
components include, for example and with reference to Figure 1, objective lens
110, and
dichroic 108. Because these components transmit the full excitation
illumination, they are
more prone to emitting autofluorescence.
[0120] In order to mitigate the contribution of the out of focus
components in the
systems of the invention, the collected signals from each of these signal
sources is subjected
to a spatial filtering process whereby light noise contributions that are not
within the focal
plane of the optical system are minimized or eliminated.
[0121] Accordingly, in at least one aspect, the invention employs a
spatial filter
component to filter out autofluorescence that is out of the focal plane of the
objective lens.
One example of such a spatial filter includes a confocal mask or filter placed
in the optical
train. In particular, the fluorescent signals from the discrete regions on the
substrate that are
collected by the objective and transmitted through the optical train, are
passed through a
focusing or field lens and a confocal filter placed in the image plane of that
lens. The light
passed through the confocal filter is subsequently refocused and imaged onto a
detector.
Fluorescence that is not in the focal plane of the objective will be blocked
by the confocal
aperture, and as a result, will not reach the detector, and consequently will
not contribute to
the fluorescent noise. This typically includes scattered or reflected
fluorescence,
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autofluorescence of substrates and other system components and the like. In
the context of
the present invention, the spatial filtering process is applied to the
fluorescent signals from a
large number of discrete signal sources, simultaneously, e.g., without the use
of scanning,
galvo or other rastering systems. In particular, the confocal filters applied
in the systems of
the invention typically include a large number of confocal apertures that
correspond to the
number of regions on the substrate from which signals are desired to be
obtained. In
accordance with array sizes as set forth elsewhere herein, for example, the
confocal masks
used in this context can typically include an array of at least about 100 or
more discrete
confocal apertures, preferably greater than 500 discrete confocal apertures,
more preferably
greater than 1000 discrete confocal apertures, and still more preferably,
between about 1000
and about 5000 apertures, and in some cases greater than 5000 or more discrete
confocal
apertures. Such confocal masks will also typically be arrayed in a concordant
pitch and/or
alignment with the signal source arrays, so that signal from each discrete
source that is
desired to be observed will pass through a separate confocal aperture in the
confocal mask.
The actual size and spacing of the confocal pinholes will typically vary
depending upon the
desired illumination pattern, e.g., number and spacing of illumination
beamlets, as well as
the characteristics of the optical system.
[0122] While individual pinhole apertures corresponding to individual
signal
sources are generally preferred, it will be appreciated that other spatial
filters may also be
employed that provide for simpler alignment, such as using narrow slits to
reduce out of
focus signal components in at least one dimension. Individual slits could be
employed in
filtering signals from a plurality of signal sources in a given row, column or
other defined
region, e.g., adjacent signal sources on the diagonal. Figure 10 shows a
schematic of a
partial confocal mask showing apertures that are provided on the same pitch
and
arrangement as the signals being focused therethrough, e.g., corresponding to
fluorescent
signals imaged from an array of zero mode waveguides. As noted previously,
where
confocal slits, or other filters applied to multiple signal sources are used,
they may number
less than the total number of individual signal sources and may conform to the
number of
columns and or rows of signal sources, e.g., greater than 10, 20, 50 or even
100 or more
confocal apertures.
[0123] An example of an optical train including such a confocal filter is
schematically illustrated in Figure 11. As shown, an objective lens 1102 is
positioned
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adjacent to a substrate, such as zero mode waveguide array 1104 having the
reaction regions
of interest disposed upon it, so as to collect signals emanating from the
substrate, as well as
any autofluorescence that emanates from the substrate. The collected
fluorescence is then
focused through a first focusing lens 1106. A confocal mask 1108 is placed in
the focal
plane of the first focusing lens 1106. Spatially filtered fluorescence that is
passed by the
confocal mask is then refocused through a second focusing lens 1110 and passed
through
the remainder of the optical train. As shown, this includes a wedge prism 1112
to spatially
separate spectral components of the fluorescence, and third focusing lens
1114, that focuses
the image of the fluorescence derived from the focal plane of the objective
1102, onto a
detector, such as EMCCD 1116. By placing the confocal mask in the focal plane
of the first
focusing lens 1106, autofluorescence components that are out of the focal
plane of the
objective lens (and thus not focused by the focusing lens at the confocal mask
1108) will be
blocked or filtered, and only fluorescence that is in the focal plane, e.g.,
fluorescent signals
and any autofluorescence that exists in the focal plane, will be passed and
imaged upon the
detector 1116, and detected. In comparative experiments, autofluorescence
background
signals were reduced approximately 3 fold through the incorporation of a
confocal mask, in
both two and three laser systems.
[0124] Figure 9 provides an illustration of the effects of out of focus
autofluorescence as well as the benefits of a confocal mask in reducing such
autofluorescence. In particular, Figure 9 shows a plot of autofluorescence
levels as a
function of the location of the image of the autofluorescence on an EMCCD
detector, from
a substrate that was illuminated with four illumination lines at 488nm. As
shown, the upper
plot 902 corresponds to autofluorescence image from 4 illumination lines, but
in the
absence of a confocal mask filtering the out of focus components. The 4 peaks
(904-910)
correspond to the elevated autofluorescence at the illumination lines on the
substrate while
the baseline corresponds to the overall global autofluorescence across the
remainder of the
substrate. By contrast, inclusion of a confocal mask provides a substantial
reduction in the
amount of the out of focus autofluorescence from the system. In particular,
the lower plot
912, reflects the confocally filtered traces through a number of different
slit sizes, where
each aggregate peak (914-948) corresponds to the position of the slits in the
confocal masks
used. As can be seen, peaks 928-934 correspond to the location of the
illumination lines,
and as such have a higher amount of in focus autofluorescence. The remaining
peaks also
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represent autofluorescence that is in the focal plane and thus not filtered by
the confocal
mask. Figure 12 shows an expanded view of the various plots with illumination
at 633 nm,
with the upper plot reflecting an unfiltered level of autofluorescence imaged
at a given
position on the detector, while the lower plots reflect the autofluorescence
at the same
position but filtered using confocal masks having slit sizes of 60 nm, 55 nm,
50 nm, 45 nm,
40 nm, and 30 nm. The decreasing size of the autofluorescence peak is
correlated to the
reduction in the dimensions of the slit in the confocal mask used.
[0125] Notwithstanding this in focus component, it can be easily seen that
the
provision of the confocal mask provides a significant reduction in the overall
autofluorescence that is detected (as indicated by the area under each of the
two plots). As
noted, the confocal mask used in the example shown in Figure 9 employed
confocal slits
for a linear illumination profile. It will be appreciated that alternative
mask configurations
may be employed as well, such as the use of arrayed pinholes in the confocal
mask, in order
to provide arrayed spot or targeted illumination as discussed elsewhere
herein.
[0126] Other additional approaches to reduction of generated
autofluorescence
include spectral filtering of autofluorescence noise, through the
incorporation of appropriate
filters within the optical train, and particularly the collection aspects of
the optical train. It
has been observed that a substantial amount of autofluorescence signal in a
typical
illumination profile, e.g., in a wavelength range of from about 720 nm to
about 1000 nm,
falls within spectral ranges that do not overlap with desired detection
ranges, e.g., from
about 500 nm to about 720 nm. As such, elimination of at least a portion of
autofluorescence noise may be accomplished by incorporating optical filters
that block light
outside of the desired range, e.g., long or short pass filters that block
light of a wavelength
greater than about 720 nm or less than about 500 nm. Such filters are
generally made to
order from optical component suppliers, including, e.g., Semrock, Inc.,
Rochester NY, Barr
Associates, Inc., Westford, MA, Chroma Technology Corp., Rockingham VT.
[0127] Figure 13 provides a general schematic for an embodiment of a
fluorescence
detection system comprising optical elements that can reduce both the
production and
detection of autofluorescence background noise. As shown, the overall system
1300
generally includes an excitation illumination source 1302. Typically, such
illumination
sources will comprise high intensity light sources such as lasers or other
high intensity
sources such as LEDs, high intensity lamps (mercury, sodium or xenon lamps),
laser diodes,
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and the like. In preferred aspects, the sources will have a relatively narrow
spectral range
and will include a focused and/or collimated or coherent beam. For the
foregoing reasons,
particularly preferred light sources include lasers, solid-state laser diodes,
and the like.
[0128] The excitation illumination source 1302 is positioned to direct
light of an
appropriate excitation wavelength or wavelength range, at a desired
fluorescent signal
source, e.g., substrate 1304, through an optical train. In accordance with the
present
invention, the optical train typically includes a number of elements, e.g.,
one or more
microlens and/or one or more DOE, to appropriately direct excitation
illumination at the
substrate 1304, and receive and transmit emitted signals, e.g., with reduced
autofluorescence background noise, from the substrate to an appropriate
detection system
such as detector 1328. In accordance with the present invention, the
excitation illumination
from illumination source 1302 is directed first through optical multiplex
element (or
elements 1306), e.g., one or more microlens and/or one or more DOE, to
multiply the
number of illumination beams or spots from an individual beam or spot from the
illumination source 1302. The multiplexed beam(s) is then directed via
focusing lens 108
through optional first spatial filter 1310, and focusing lens 1312. As
discussed in greater
detail above, spatial filter 1310 optionally provides control over the extent
of multiplex
beams continuing through the optical train reduces the amount of any scattered
excitation
light from reaching the substrate. The spatially filtered excitation light is
then passed
through dichroic 1314 into objective lens 1316, whereupon the excitation light
is focused
upon the substrate 1304. Dichroic 1314 is configured to pass light of the
spectrum of the
excitation illumination while reflecting light having the spectrum of the
emitted signals
from the substrate 1304. Because the excitation illumination is multiplexed
into multiple
beams, multiple discrete regions of the substrate are separately illuminated.
[0129] Fluorescent signals that are emitted from those portions of the
substrate that
are illuminated, are then collected through the objective lens 1316, and,
because of their
differing spectral characteristics, they are reflected by dichroic 1314,
through focusing lens
1318, and second spatial filter, such as confocal mask 1320, and focusing lens
1322.
Confocal mask 1320 is typically positioned in the focal plane of lens 1318, so
that only in-
focus light is passed through the confocal mask, and out-of focus light
components are
blocked. This results in a substantial reduction in noise levels from the
system, e.g., that
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derive from out of focus contributors, such as autofluorescence of the
substrate and other
system components.
[0130] As with the excitation illumination, the signals from the
multiple discrete
illuminated regions on the substrate are separately passed through the optical
train. The
fluorescent signals that have been subjected to spatial filtering are then
passed through a
dispersive optical element, such as prism assembly 1324, to separately direct
spectrally
different fluorescent signal components, e.g., color separation, which
separately directed
signals are then passed through focusing lens 1326 and focused upon detector
1328, e.g., an
imaging detector such as a CCD, ICCD, EMCCD or CMOS based detection element.
Again, the spectrally separated components of each individual signal are
separately imaged
upon the detector, so that each signal from the substrate will be imaged as
separate spectral
components corresponding to that signal from the substrate. For a discussion
of the spectral
separation of discrete optical signals, see, e.g., Published U.S. Patent
Application No. 2007-
0036511.
[0131] As will be appreciated, a more conventional configuration
that employs
reflected excitation light and transmitted fluorescence may also be employed
by altering the
configuration of and around dichroic 1314. In particular, dichroic 1314 could
be selected to
be reflective of the excitation light from illumination source 1302, and
transmissive to
fluorescence from the substrate 1304. The various portions of the optical
train are then
arranged accordingly around dichroic 1314. Notwithstanding the foregoing,
fluorescence
reflective optical trains are particularly preferred in the applications of
the systems of the
invention. For a discussion on the advantages of such systems, see, e.g., U.S.
Patent
Application Nos. 11/704,689, filed February 9, 2007, 11/483,413, filed July 7,
2006, and
11/704,733, filed February 9, 2007.
[0132] As noted with reference to Figure 13, the fluorescence path
of the system
typically includes optics for focusing the signals from the various regions
onto discrete
locations on a detector. As with the direction of excitation illumination onto
a plurality of
discrete regions on a relatively small substrate area, likewise, each of the
plurality of
discrete fluorescent signals is separately imaged onto discrete locations on a
relatively small
detector area. This is generally accomplished through focusing optics in the
fluorescence
path positioned between the confocal filter and the detector (optionally in
combination with
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optical components provided with the confocal filter (see discussion below).
As with the
illumination path, the fluorescence path can typically direct at least 10,
preferably at least
100, more preferably at least 500, or 1000 or in some cases at least 5000
discrete fluorescent
signals to discrete locations on the detector. Because these detectors, e.g.,
EMCCDs have
relatively small areas, these signals will typically be imaged at relatively
high densities at
the EMCCD plane. Such densities typically reflect the illumination spot
density at the
substrate plane divided by the relative size of image of the substrate as
compared to the
actual substrate size, due to magnification of the system, e.g., imaging
signal sources on an
area that is 3600X larger than the illumination pattern (e.g., 250,000
illumination
density/3600). Although in preferred aspects, the images of the fluorescent
signal
components will be oriented in an array of two or more rows and/or columns of
imaged
signals, in order to provide the densities set forth herein, it will be
appreciated that density
may be determined from images arrayed in other formats, such as linear arrays,
random
arrays, and the like. Further, while the imaged signals of the invention will
preferably
number greater than 10, 100, 500, 1000 or even greater than 5000, density may
be readily
determined and applicable to as few as two discrete images, provided such
images are
sufficiently proximal to each other to fit within the density described.
[0133] The systems of fluorescence detection that can be used with the
methods and
systems provided herein, e.g., for reducing autofluorescence, also typically
include spectral
separation optics to separately direct different spectral components of the
fluorescent signals
emanating from each of the discrete regions or locations on the substrate, and
image such
spectral components onto the detector. In some cases, the image of the
spectral components
of a given discrete fluorescent signal will be completely separate from each
other. In such
cases, it will be appreciated that the density of the discrete images on the
detector may be
increased by the number of discrete fluorescent components. For example, where
a
fluorescent signal is separated into four spectral components, each of which
is discretely
imaged upon the detector, such density could be up to 4 times that set forth
above. In
preferred aspects, however, the separate direction of spectral components from
a given
fluorescent signal will not impinge upon completely discrete regions of the
detector, e.g.,
image of one spectral component would impinge on overlapping portions of the
detector as
another spectrally distinct component (See, e.g., Published U.S. Patent
Application No.
2007-0036511, U.S. Patent Application Nos. 11/704,689, filed February 9,2007,
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11/483,413, filed July 7, 2006, and 11/704,733, filed February 9, 2007.
[0134] While the separation optics may include multiple elements
such as
filter/mirror combinations to separately direct spectrally distinct components
of each
fluorescent signal, in preferred aspects, a dispersive optical element is used
to separately
direct the different spectral components of the fluorescent signals to
different locations on
the substrate.
IV. Other Applications for the Optical Systems and Fluorescence
Detection
Systems Described Herein
[0135] As noted previously, the optical systems, fluorescence
detection systems and
the methods of their use that are described herein are broadly applicable to a
wide variety of
applications where it is desirable to illuminate multiple discrete regions of
a substrate and
obtain responsive optical signals from such regions, e.g., with reduced
autofluorescence
background noise. Such applications include analysis of fluorescent or other
optically
monitored reactions or other processes, optical interrogation of, e.g.,
digital optical media,
spatial characterization, e.g., holography, laser driven rapid prototyping
techniques,
multipoint spatial analysis, e.g., for mobility/motility analysis, as well as
a large number of
other general uses.
[0136] In one particularly preferred example, the methods and
systems of the
invention are applied in the analysis of nucleic acid sequencing reactions
being carried out
in arrays of optically confined reaction regions, such as zero mode
waveguides. In
particular, the methods and systems are useful for analyzing fluorescent
signals that are
indicative of incorporation of nucleotides during a template dependent
polymerase mediated
primer extension reaction, where the fluorescent signals are not just
indicative of the
incorporation event but also can be indicative of the type of nucleotide
incorporated, and as
a result, the underlying sequence of the template nucleic acid. Such nucleic
acid sequencing
processes are generally referred to as "sequencing by incorporation" or
sequencing by
synthesis" methods, in that sequence information is determined from the
incorporation of
nucleotides during nascent strand synthesis. Although the systems and methods
of the
invention are much more broadly applicable than this preferred application,
the advantages
and benefits of these systems and methods are exploited to a great degree in
such
applications. As such, for ease of discussion, the systems and methods of the
invention are
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described in greater detail with respect to such applications, although they
will be
appreciated as having much broader applicability.
[0137] Typically, in sequencing by synthesis processes, a complex of a
polymerase
enzyme, a target template nucleic acid sequence and a primer sequence is
provided. The
complex is generally immobilized via the template, the primer, the polymerase
or
combinations of these. When the complex comes into contact with a nucleotide
that is
complementary to the base in the template sequence immediately adjacent to
where the
primer sequence is hybridized to that template, the polymerase will typically
incorporate
that nucleotide into the extended primer. By associating a fluorescent label
with the
nucleotide, one can identify the incorporation event by virtue of the presence
of the label
within the complex. In most SBI applications, the incorporation event
terminates primer
extension by virtue of a blocked 3' group on the newly incorporated
nucleotide. This
generally allows the immobilized complex to be washed to remove any non-
incorporated
label, and observed, to identify the presence of the label. Subsequent to
identifying
incorporation, the complex is typically treated to remove any terminating
blocking group
and/or label group from the complex so that subsequent base incorporations can
be
observed. In some processes, a single type of base is added to the complex at
a time and
whether or not it is incorporated is determined. This typically requires
iterative cycling
through the four bases to identify extended sequence stretches. In alternative
aspects, the
four different bases are differentially labeled with four different
fluorescent dyes that are
spectrally distinguishable, e.g., by virtue of detectably different emission
spectra. This
allows simultaneous interrogation of the complex with all four bases to
provide for an
incorporation even in each cycle, and also provide for the identification of
the base that was
incorporated, by virtue of its unique spectral signature from its own label.
In general, such
systems still typically require addition of a terminated nucleotide followed
by a washing
step in order to identify the incorporated nucleotide.
[0138] In another approach, nucleotide incorporation is monitored in real
time by
optically confining the complex such that a single molecular complex may be
observed.
Upon incorporation, a characteristics signal associated with incorporation of
a labeled
nucleotide, is observed. Further, such systems typically employ a label that
is removed
during the incorporation process, e.g., a label coupled to the polyphosphate
chain of a
nucleotide or nucleotide analog, such that additive labeling effects do not
occur. In
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particular, such optical confinements typically provide illumination of very
small volumes
at or near a surface to thereby restrict the amount of reagent that is subject
to illumination to
at or near the complex. As a result, labeled nucleotides that are associated
with the
complex, e.g., during incorporation, can yield a distinct signal indicative of
that association.
Examples of optical confinement techniques include, for example, total
internal reflection
fluorescence (TIRF) microscopy, where illumination light is directed at the
substrate in a
manner that causes substantially all of the light to be internally reflected
within the substrate
except for an evanescent wave very near to the surface.
[0139] Other preferred optical confinement techniques include the use of
zero mode
waveguide structures as the location for the reaction of interest. Briefly,
such zero mode
waveguides comprise a cladding layer disposed over a transparent substrate
layer with core
regions disposed through the cladding layer to the transparent substrate.
Because the cores
have a cross-sectional dimension in the nanometer range, e.g., from about 10
to about 200
nm, they prevent propagation of certain light through the core, e.g., light
that is greater than
the cut-off frequency for the given cross-sectional dimension for such core.
As a result, and
as with the TIRF confinement, light entering the waveguide core through one or
the other
end, is subject to evanescent decay, that results in only a very small
illumination volume at
the end of the core from which the light enters.
[0140] In the context of SBI applications, immobilizing the complex at
one end of
the core, e.g., on the transparent substrate, allows for illumination of the
very small volume
that includes the immobilized complex, and thus the ability to monitor few or
individual
complexes. Because these systems focus upon individual molecular interactions,
they
typically rely upon very low levels of available signal. This in turn
necessitates more
sensitive detection components. Further, in interrogating large numbers of
different
reactions, one must apply a relatively large amount of illumination radiation
to the substrate,
e.g., to provide adequate illumination to multiple reaction regions. As a
result, there is the
potentiality for very low signal levels coming from individual molecules
coupled with very
high noise levels coming from highly illuminated substrates and systems and
sensitive
optical detection systems.
[0141] Although described primarily in terms of single molecule analysis,
and
particularly for sequence determination applications, the optical systems and
fluorescence
detection systems described herein, with their highly multiplexed confocal
optics, are useful
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in almost any application in which one wishes to interrogate multiple samples
for a
fluorescent signal or signals and detect the signals with reduced
autofluorescence
background noise. For example, in related research fields, the systems of the
invention are
directly applicable to the optical interrogation of arrays of biological
reactions and/or
reactants. These may range from the simple embodiment of a highly multiplexed
multi-well
reaction plates, e.g., 96, 386 or 1536 well plates, or higher multiplexed
"nanoplates", such
as the Openarray0 plates from Biotrove, Inc., to the more complex systems of
spotted or in-
situ synthesized high density molecular or biological arrays. In particular,
biological arrays
typically comprise relatively high density spots or patches of molecules of
interest that are
interrogated with and analyzed for the ability to interact with other
molecules, e.g., probes,
which bear fluorescent labeling groups. Such arrays typically employ any of a
variety of
molecule types for which one may desire to interrogate another molecule for
its interaction
therewith. These may include oligonucleotide arrays, such as the Genechipe
systems
available from Affymetrix, Inc (Santa Clara, CA), protein arrays that include
antibodies,
antibody fragments, receptor proteins, enzymes, or the like, or any of a
variety of other
biologically relevant molecule systems.
[0142] In its most prolific application, array technology employs arrays
of different
oligonucleotide molecules that are arrayed on a surface such that different
locations, spots
or features have sequences that are known based upon their position on the
array. The array
is then interrogated with a target sequence, e.g., an unknown sample sequence
that bears a
fluorescent label. The identity of at least a portion of that target sequence
is then
determinable from the probe with which it hybridizes, which is, in turn, known
or
determinable from the position on the array from which the fluorescent signal
emanates.
[0143] As feature sizes in arrays are reduced in order to provide greater
numbers of
molecules, the needs for highly multiplexed optical systems and fluorescence
detection
systems described herein are increased. Likewise, as array sizes increase, the
demands on
conventional scanning systems are further increased. As such, the systems of
the invention,
either as static array illumination, or as scanning or otherwise translatable
systems, as
described above, are particularly useful in this regard.
[0144] In commercially available systems, interrogation of large arrays
of molecules
has been carried out through either the use of image capture systems, or
through the
iterative scanning of the various spots or features of the array using, e.g.,
confocal scanning
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microscopes. The optical systems and fluorescence detection systems described
herein, in
contrast, provide a simultaneous, confocal examination of highly multiplexed
arrays of
different molecules through their discrete illumination and signal collection,
e.g., signal
collection with reduced autofluorescence background noise. Further, the
spectroscopic
aspects of the invention further enhance this functionality in the context of
multi-label
applications, e.g., where different targets/probes are labeled with spectrally
distinguishable
fluorescent labels.
[0145] The optical systems and fluorescence detection systems described
herein are
similarly useful in a variety of other multiplexed spectroscopic analyses. For
example, in
the field of microfluidic systems, large numbers of microfluidic conduits may
be arrayed
and analyzed using the systems of the invention. Such microfluidic systems
typically
comprise fluidic conduits disposed within a glass or plastic substrate,
through which
reagents are moved, either electrokinetically or under pressure. As reagents
flow past a
detection point, they are interrogated with an excitation source, e.g., a
laser spot, and the
fluorescence is monitored, e.g., with an increased signal-to-noise ratio.
Examples of
microfluidic systems include, for example, capillary array electrophoresis
systems, e.g., as
sold by Applied Biosystems Division of Applera, Inc., as well as monolithic
systems, such
as the LabChip microfluidic systems available from Caliper Life Sciences,
Inc.
(Hopkinton, MA), and the BiomarkTm and Topaz systems available from Fluidigm
, Inc.
(So. San Francisco, CA). While the fluidic conduits of these systems are
predominantly
arrayed in two dimensions, e.g., in a planar format, the systems of the
invention may be
configured to provide confocal illumination and detection from a three
dimensional array of
signal sources. In particular, diffractive optical elements used in certain
aspects of the
multiplex optics of the invention may be configured to provide illumination
spots that are
all in focus in a three dimensional array. Such three dimensional arrays may
include
multilayer microfluidic systems, bundled capillary systems, stacked multi-well
reaction
plates, or the like.
[0146] In addition to the foregoing, these optical systems and
fluorescence detection
systems described herein are similarly applicable to any of a variety of other
biological
analyses, including, for example, multiplexed flow cytometry systems,
multiplexed in-vivo
imaging, e.g., imaging large numbers of different locations on a given
organism, or multiple
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organisms (using, e.g., infrared illumination sources, e.g., as provided in
the Ivis0 series of
imaging products from Caliper Life Sciences, Inc.
101471 While the primary applications for the systems of the
invention are geared
toward multiplexed analysis of chemical, biochemical and biological
applications, it will be
appreciated that the highly multiplexed systems of the invention, with their
high signal to
noise capability, also find use, in whole or in part, in a variety of other
optical interrogation
techniques. For example, the highly multiplexed confocal optics and detection
methods of
the invention may be readily employed in high bandwidth reading and/or writing
of digital
data to or from optical media. Likewise, the highly multiplexed illumination
systems of the
invention may be employed in optically driven tools, such as laser based rapid
prototyping
techniques, parallel lithography techniques, and the like, where highly
multiplexed laser
beams can be applied in the fabrication and/or design processes.
[0148] Although described in some detail for purposes of
illustration, it will be
readily appreciated that a number of variations known or appreciated by those
of skill in the
art may be practiced within the scope of present invention.
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