U.S. patent application number 14/500682 was filed with the patent office on 2015-04-23 for luminescence reference standards.
The applicant listed for this patent is APPLIED BIOSYSTEMS, LLC. Invention is credited to Kevin S. Bodner, Steven J. Boege, Aldrich N. K. Lau, Mark F. Oldham, J. Michael PHILLIPS, Donald R. Sandell, David H. Tracy.
Application Number | 20150111200 14/500682 |
Document ID | / |
Family ID | 40254674 |
Filed Date | 2015-04-23 |
United States Patent
Application |
20150111200 |
Kind Code |
A1 |
PHILLIPS; J. Michael ; et
al. |
April 23, 2015 |
Luminescence Reference Standards
Abstract
The present teachings provide for systems, and components
thereof, for detecting and/or analyzing light. These systems can
include, among others, optical reference standards utilizing
luminophores, such as nanocrystals, for calibrating, validating,
and/or monitoring light-detection systems, before, during, and/or
after sample analysis.
Inventors: |
PHILLIPS; J. Michael; (San
Carlos, CA) ; Bodner; Kevin S.; (Belmont, CA)
; Lau; Aldrich N. K.; (Palo Alto, CA) ; Oldham;
Mark F.; (Emerald Hills, CA) ; Sandell; Donald
R.; (San Jose, CA) ; Tracy; David H.;
(Champaign, IL) ; Boege; Steven J.; (San Mateo,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
APPLIED BIOSYSTEMS, LLC |
Carlsbad |
CA |
US |
|
|
Family ID: |
40254674 |
Appl. No.: |
14/500682 |
Filed: |
September 29, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14307353 |
Jun 17, 2014 |
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14500682 |
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14189040 |
Feb 25, 2014 |
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14307353 |
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13765568 |
Feb 12, 2013 |
8659755 |
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14189040 |
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12813389 |
Jun 10, 2010 |
8373854 |
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13765568 |
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12338778 |
Dec 18, 2008 |
7742164 |
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12813389 |
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11173609 |
Jun 30, 2005 |
7480042 |
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12338778 |
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60584890 |
Jun 30, 2004 |
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Current U.S.
Class: |
435/5 ;
422/82.08; 435/287.2; 435/288.3; 435/29; 435/6.1; 435/6.11;
435/6.12; 436/163; 436/165; 436/501 |
Current CPC
Class: |
G01J 1/10 20130101; G01N
21/6452 20130101; G01N 2201/0612 20130101; G01N 21/4785 20130101;
B82Y 15/00 20130101; G01N 2201/0697 20130101; Y10S 977/943
20130101; G01N 2201/0691 20130101; G01N 2201/062 20130101; G01N
21/01 20130101; Y10S 977/949 20130101; G01N 21/278 20130101; G01N
2201/06113 20130101; G01N 21/76 20130101 |
Class at
Publication: |
435/5 ;
422/82.08; 435/287.2; 435/288.3; 436/165; 435/6.12; 435/6.1;
435/6.11; 436/163; 435/29; 436/501 |
International
Class: |
G01N 21/27 20060101
G01N021/27; G01N 21/64 20060101 G01N021/64; G01N 21/76 20060101
G01N021/76 |
Claims
1-5. (canceled)
6. A system for biological reactions, comprising: a frame
configured to support a sample support configured to support one or
more biological samples; a reference standard disposed to provide
optical access to the one or more biological samples, the reference
standard includes a plurality of layers, comprising: a
light-producing element layer; and a second layer comprising at
least one of a spectral filter layer, an intensify filter layer, a
subtractive element layer, or a protective coating layer positioned
adjacent the light-producing element; a light source configured to
illuminate (1) the one or more biological samples and (2) the
light-producing element through the second layer; a detector
configured to receive light from the one or more biological samples
and to receive light that is emitted, reflected, or scattered from
the light-producing element; an optical system configured to direct
light from the biological samples and the light-producing element
to the detector.
7. The system of claim 6, further comprising a support layer
configured to provide structural support for the reference
standard.
8. The system of claim 7, wherein the light-producing element
comprises a layer positioned on the support layer.
9. The system of claim 7, further comprising a mask disposed above
the light-producing element layer, wherein the light-producing
element layer and the mask are separated by the second layer.
10. The system of claim 6, further comprising a light-blocking
element defining a calibration mark on the light-producing element
layer.
11. The system of claim 10, wherein the light-blocking element
forms a mask.
12. The system of claim 6, further comprising a thermal cycler and
an insulator configured to thermally insulate the reference
standard from the thermal cycler.
13. The system of claim 12, wherein the insulator is coupled to the
light-blocking element.
14. The system of claim 6, further comprising the sample
support.
15. The system of claim 6, wherein the sample support comprises a
microtiter plate or a cell culture plate.
16. The system of claim 6, wherein the sample support comprises a
cell culture plate.
17. The system of claim 6, wherein the light-producing element
layer comprises at least one nanocrystalline material.
18. The system of claim 6, wherein the light-producing element
layer comprises at least one of Ultem,
photoluminescent-nanocrystal-containing material, quantum dots, or
luminescent-glass-containing material.
19. The system of claim 6, wherein the light-producing element
layer is configured to produce light by one or more of emission,
reflection, transmission, refraction, diffraction, or
scattering.
20. The system of claim 6, wherein the light-producing element
layer is configured to produce light by one or more of
luminescence, photoluminescence, fluorescence, phosphorescence, or
chemiluminescence.
21. A method for biological reactions, comprising: providing a
sample support configured to support one or more biological
samples; providing a reference disposed to provide optical access
to the one or more biological samples, comprising: a
light-producing element; and a second layer comprising at least one
of a spectral filter layer, an intensify filter layer, a
subtractive element, or a protective coating layer; illuminating
the one or more biological samples with a light source;
illuminating the light-producing element with the light source
through the second layer; receiving at a detector at least one of
(1) light from the one or more biological samples or (2) light that
is emitted, reflected, or scattered from the light-producing
element; using an optical system, directing light from at least one
of the one or more biological samples or the light-producing
element to the detector.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of patent application Ser.
No. 12/338,778 filed Dec. 18, 2008; which is a continuation of
patent application Ser. No. 11/173,609 filed Jun. 30, 2005, now
U.S. Pat. No. 7,480,042; which claims a priority benefit under 35
U.S.C. .sctn.119(e) from U.S. Patent Application No. 60/584,890
filed Jun. 30, 2004; all of which are incorporated herein by
reference.
INTRODUCTION
[0002] The identity, properties, and interactions of samples such
as biomolecules can be analyzed by detecting light emitted by or
otherwise originating from the sample. The emitted light can arise
naturally in the sample and/or be induced by illumination or other
stimulus. In either case, the emitted light can be detected, and
the illumination optionally can be provided, by a suitable
light-detection system. The ability of the light-detection system
to collect and quantify emitted light and thus to characterize
samples is determined in part by the extent to which the
light-detection system is and can be calibrated.
SUMMARY
[0003] The present teachings provide systems, and components
thereof, for detecting and/or analyzing light. These systems can
include, among others, optical reference standards for calibrating,
validating, and/or monitoring light-detection systems, before,
during, and/or after sample analysis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0004] FIG. 1 is a schematic view of an exemplary light-detection
system, showing selected optical components and an optical
reference standard, in accordance with aspects of the present
teachings.
[0005] FIG. 2 is a top view of an exemplary optical reference
standard, in accordance with aspects of the present teachings.
[0006] FIG. 3 is an exploded isometric view of two exemplary
embodiments of an optical reference standard, with an upper
light-blocking portion and a lower light-producing portion, in
accordance with aspects of the present teachings. The embodiment
shown at left is configured generally for use in an optical plane
intermediate between a light source and the object plane. The
embodiment shown at right is configured generally for use in the
object plane.
[0007] FIG. 4 is an exploded isometric view of two alternative
exemplary embodiments of an optical reference standard, with an
upper light-producing portion and a lower light-blocking portion,
in accordance with aspects of the present teachings. The embodiment
shown at left is configured generally for use in an optical plane
intermediate between a light source and the object plane. The
embodiment shown at right is configured generally for use in the
object plane.
[0008] FIG. 5 is a schematic sectional view of an exemplary
subtraction-based optical reference standard, showing
light-producing and light-blocking elements, in accordance with
aspects of the present teachings.
[0009] FIG. 6 is an exemplary emission spectrum for Ultem.RTM. 1000
plastic, showing relative emission intensity as a function of
emission wavelength, following excitation at a fixed excitation
wavelength, in accordance with aspects of the present teachings.
The shaded envelope is constructed from the lowest and highest
intensities measured at each emission wavelength, based on analysis
of 384 identically prepared samples.
[0010] FIG. 7 is a plot of transmission versus wavelength showing
individual and composite transmission spectra of subtractive
elements of the subtraction-based,
Ultem.RTM.-1000-plastic-containing reference standard of FIG. 6.
The dashed, dot-dashed, and thin solid lines represent transmission
spectra of individual layers. The thick solid line represents the
composite transmission spectrum.
[0011] FIG. 8 is a plot of an exemplary calculated emission
spectrum for the subtraction-based,
Ultem.RTM.-1000-plastic-containing reference standard of FIG. 6.
The calculated emission spectrum (solid line) is a product of the
emission spectrum of a broadband photoluminescent component (thick
dashed line) and the composite transmissivity of various spectral
filter components (thin dashed line).
[0012] FIG. 9 is an exemplary emission spectrum for a
subtraction-based, Ultem.RTM.-1000-plastic-containing reference
standard, showing relative emission intensity as a function of
emission wavelength, following excitation at a fixed excitation
wavelength, in accordance with aspects of the present teachings.
The shaded envelope is constructed from the lowest and highest
intensities measured at each emission wavelength, based on analysis
of 384 identically prepared samples.
[0013] FIG. 10 is an exemplary emission spectrum for an
addition-based, photoluminescent-nanocrystal-containing reference
standard, showing relative emission intensity as a function of
emission wavelength, following excitation at a fixed excitation
wavelength, in accordance with aspects of the present teachings.
The shaded envelope is constructed from the lowest and highest
intensities measured at each emission wavelength, based on analysis
of 20 identically prepared samples.
[0014] FIG. 11 is an exemplary emission spectrum for an
addition-based, luminescent-glass-containing reference standard,
showing relative intensity as a function of emission wavelength,
following excitation at a fixed excitation wavelength, in
accordance with aspects of the present teachings. The shaded
envelope is constructed from the lowest and highest intensities
measured at each emission wavelength, based on analysis of a
plurality of identically prepared samples.
[0015] FIG. 12 is a schematic sectional view of another exemplary
light-detection system, showing selected optical components and an
optical reference standard, in accordance with aspects of the
present teachings.
DESCRIPTION OF VARIOUS EMBODIMENTS
[0016] The present teachings provide systems, including components
thereof and aids thereto, for detecting and/or analyzing light.
These systems can include, among others, optical reference
standards for calibrating, validating, and/or monitoring
light-detection systems, including components and/or aspects
thereof, before, during, and/or after sample analysis. Suitable
uses can include aligning portions of a light detection system,
relative to one another and/or one or more samples. Suitable uses
also can include validating system performance, including
attainment and/or maintenance of threshold conditions. Suitable
uses also can include monitoring and/or correcting for underages,
overages, and/or variations in illumination intensity, among
others. These underages, overages, and/or variations can occur with
respect to space and/or time, and can be determined and/or
corrected for before, during, and/or after sample analysis. Thus,
the optical reference standards can be used in methods of
calibrating, validating, and/or monitoring a light-detection system
and/or methods of performing an assay (e.g., involving measuring
properties of the reference standard and sample, and performing a
correlation and/or correction based on such measurements), among
others.
[0017] Exemplary usages of the optical reference standards can
include (1) aligning optical components, (2) validating system
performance, (3) confirming performance and settings for excitation
and/or emission filters (before or after their installation in the
same or a different instrument), (4) verifying threshold
illumination (e.g., intensity and/or uniformity) and detection
(e.g., sensitivity) parameters, (5) compensating for spatial and/or
temporal variations in illumination, (6) tracking drift in
illumination intensity and/or looking for signs of (and acting to
prevent) impending component (e.g., light source) failure, (7)
locating and/or aligning sample holders, or portions thereof (e.g.,
in high-density array grids), and/or (8) calibrating intensity,
wavelength, and/or other parameters (e.g., by comparing values
measured in a sample against known values measured from the
standard), among others. Additional usages and applications are
described below, particularly in Section III and Example 4, among
others.
[0018] Exemplary users can include manufacturers, service and
maintenance personnel, and/or end users, among others.
[0019] FIG. 1 shows an exemplary light-detection system 100,
including selected optical components 102-108 and an optical
reference standard 110, in accordance with aspects of the present
teachings.
[0020] The light-detection system generally comprises any mechanism
for detecting (and optionally analyzing) light from a sample.
Suitable systems can include (1) a light source 102 for producing
light for illuminating, and/or inducing a suitable or desired
response from, a sample, (2) a sample-support device 104 for
supporting one or more samples 112 at an examination area 114, (3)
a detector 106 for detecting light transmitted or otherwise
originating from a sample and optionally converting the detected
light into a representative signal, and/or (4) an optical relay
structure 108 for directing light between the light source and
examination area, and the examination area and detector, among
others. These and other aspects of the light-detection system are
described below; see particularly Section II and Example 3.
Exemplary light-detection systems, including components and uses
thereof, also are described in the following patent application,
which is incorporated herein by reference: U.S. Provisional Patent
Application Ser. No. 60/584,525, filed Jun. 30, 2004, titled
Distributed Light Detector of inventors Charles S. Vann and Steven
Boege.
[0021] The optical reference standard generally comprises any
mechanism for monitoring and/or calibrating a light-detection
system, including components and/or aspects thereof. The reference
standard can include a light-producing (e.g., luminescent) portion
116 and/or a light-blocking (e.g., mask) portion 118, among others.
The reference standard can be disposed in the light-detection
system, transiently or permanently, at any suitable position(s).
Typical positions lie in or along the optical path 120, such as in
the object plane 122 (i.e., the plane, typically at least partially
coextensive with the examination area, that is occupied by the
sample and imaged by the detector during sample analysis) and/or in
an intermediate position 124 (e.g., an intermediate image plane)
operatively disposed along the incident illumination path between
the light source and object plane, among others. The reference
standard can be positioned manually and/or automatically, as
desired. The reference standard can be placed into and/or taken out
of the light path in connection with each use and/or related series
of uses, and/or the reference standard can be positioned
permanently in the light path. The reference standard (or a
suitable set of reference standards) can be housed within the
detection system, where it can be moved more easily into or out of
position or, alternatively, maintained in a fixed position. These
and other aspects of the optical reference standard are described
below; see particularly Section I and Examples 1 and 2.
[0022] The following sections describe further aspects of the
present teachings, including (I) reference standards, (II)
light-detection systems, (Ill) applications, and (IV) examples,
among others.
I. OPTICAL REFERENCE STANDARDS
[0023] The optical reference standard, as noted above, generally
comprises any mechanism for monitoring and/or calibrating a
light-detection system, including components and/or aspects
thereof.
[0024] FIG. 2 shows an exemplary optical reference standard 200, in
accordance with aspects of the present teachings. This reference
standard can include a light-producing (e.g., luminescent) element
202, for producing light, and/or a light-blocking (e.g., mask)
element 204, for selectively blocking light, among others. These
elements can be present and/or employed singly and/or in
combination. Thus, in a given light-detection system, and/or for a
given application, a light-producing element can be used, a
light-blocking element can be used, and/or both a light-producing
and a light-blocking element can be used. Moreover, if used
together, the light-producing and light-blocking elements can be at
least substantially separate or at least substantially unitary,
depending on embodiment. Thus, in some cases (e.g., FIG. 3), the
light-blocking element can be supported by or ancillary to the
light-producing element, whereas, in other cases (e.g., FIG. 4),
the light-producing element can be supported by or ancillary to the
light-blocking element. In both cases, the light-producing and
light-blocking elements simply can be placed in contact or
proximity, or the light-producing and light-blocking elements can
be joined and/or rendered at least partially coextensive using any
suitable mechanism (including adhesives, fasteners, fusion, and/or
so on).
[0025] The light-producing and light-blocking elements (as well as
other components of the reference standard) can have any suitable
shape or size. Commonly, these elements, or portions thereof, will
be at least substantially planar. Such planar portions can be
configured so that, in use, the plane is at least substantially
perpendicular to the optical axis of the light-detection system.
More generally, the reference standard (or components thereof) can
be shaped and/or sized to match a standard sample holder, or
portion thereof, such as a microplate, a biochip, and so on.
[0026] The properties of the optical reference standard can be
correlated with properties of the intended sample(s). The
correlation can include matching properties of the light produced
by the standard with properties of the light produced by the
sample(s), including wavelength, intensity, polarization, and/or
the like. The correlation also can include matching the
concentration, size, and/or position of luminophore(s) in the
reference standard and sample(s).
[0027] The reference standard can include one or more ancillary
elements, helpful to function, but not involved directly in light
production. These ancillary elements can include an identification
element 206, among others. The identification element can include
any mechanism or feature for identifying the reference standard
and/or a characteristic or use thereof. Suitable mechanisms or
features can include a barcode and/or a shape, pattern, marking,
aperture, or the like associated with the reference standard.
Suitable information can include a unique identifier (e.g., a
serial number), general properties of the reference standard (e.g.,
emission wavelength profile, the identities of luminophores
suitable for use with the standard, assay conditions or
identifiers, operator information, date, etc.), and/or so on. The
information provided by the identification element can be used for
any suitable purpose, including setting detection-system parameters
(e.g., filter settings), inputting data to be stored electronically
with the assay results, and/or so on.
[0028] The reference standard can be configured for use in any
desired optical configuration, including trans (excitation light
directed to and emission light collected from opposite sides of the
sample), epi (excitation light directed to and emission light
collected from same side of the sample), and oblique (excitation
light directed and emission light detected at oblique angles),
among others. For example, for trans illumination, associated
support components typically would be transparent to at least one
of the excitation and emission light, whereas for epi illumination
such supports can be transparent or opaque.
[0029] The reference standard can be placed in and/or removed from
desired positions in the light-detection system by any suitable
mechanism. For example, the reference standard can be placed in the
examination area by hand, and/or carried to and/or from the
examination area (or other position) by an automated conveyor
mechanism, among others. The conveyor mechanism can be the same as
or different than any mechanism (such as a stage) used to convey
sample holders to and/or from the examination area. Where the same
conveyor is used for both standards and samples, the conveyor can
be adapted to hold standards and samples simultaneously and/or
sequentially. Where different conveyors are used for standards and
samples, the two conveyors can be configured and controlled
independently and/or coordinately. In either case, the standard
optionally can be configured (e.g., shaped and/or sized) like the
sample holder, simplifying handling, and helping to ensure that the
standard successfully emulates the sample, sample conditions,
and/or aspects thereof.
[0030] These and other aspects of optical reference standards are
described below, including (A) relationships between
light-producing and light-blocking elements, (B) light-producing
elements, and (C) light-blocking elements, among others. Attributes
of these elements, and components thereof, can be mixed and matched
as appropriate or desired.
[0031] I.A Relationships Between Light-Producing and Light-Blocking
Elements
[0032] This section describes exemplary relationship between the
light-producing and light-blocking elements, in the context of
several examples; see FIGS. 3 and 4.
[0033] I.A(i) Exemplary Relationships 1
[0034] FIG. 3 shows two exemplary embodiments of a first optical
reference standard 300, with a first set of relationships between
light-producing and light-blocking elements, in accordance with
aspects of the present teachings. These reference standards are
particularly suitable for use in epi illumination.
[0035] Reference standard 300 can include a light-producing element
302a,b (corresponding to embodiments "a" and "b") and a
light-blocking element 304, among others. Light-producing element
302a in embodiment "a" can include a luminescent portion 306a and a
set of transparent portions 308. Light-producing portion 302b in
embodiment "b" can include a luminescent portion 306b but does not
(necessarily) include any transparent portions. Light-blocking
element 304 (in both embodiments) can include an opaque portion 310
and first and second sets 312, 314 of transparent portions,
respectively. The first set of transparent portions is configured
for sample excitation; specifically, in the pictured embodiment,
there are 96 apertures configured to allow light to travel to and
from 96 samples in a 96-well microplate. The second set of
transparent portions is configured for calibration; specifically,
in the pictured embodiment, there are 8 inter-well apertures
configured to allow light to travel to and from an associated
light-producing element. Here, transparent portions can transmit at
least a substantial portion of incident light, and opaque portions
can block at least substantially all incident light.
[0036] Embodiment "a" (i.e., the left side of the reference
standard (302a+304)) is configured for placement in an intermediate
optical plane, between the light source and sample, facilitating
use before, during, and/or after sample analysis. In use, the first
set of transparent portions (312) in the light-blocking element can
be aligned with the transparent portions (308) in the
light-producing element, allowing excitation light, at first
preselected positions, to impinge on samples and induce production
of emission light that passes back through the aligned sets of
transparent portions and onto the detector, where it can be used
for sample analysis. Moreover, the second set of transparent
portions (314) in the light-blocking element can be aligned with
emissive portions (306a) of the light-producing element, allowing
excitation light, at second preselected positions, to impinge on an
adjacent light-producing element and induce production of emission
light that passes back through the first set of transparent
portions and onto the detector, where it can be used for
calibration. These concepts are further illustrated in FIG. 1.
[0037] Embodiment "b" (i.e., the right side of the reference
standard (302b+304)) is configured for placement in an object
plane, facilitating use before and/or after sample analysis. Here,
in use, both the first (312) and second (314) sets of transparent
portions in the light-blocking portion can be aligned with emissive
portions (306b) of the light-producing element, allowing excitation
light to impinge on and induce production of emission light from
both regions, where it can be used for calibration.
[0038] I.A(i) Exemplary Relationships 2
[0039] FIG. 4 shows two exemplary embodiments of a second optical
reference standard 400, with a second set of relationships between
light-producing and light-blocking elements, in accordance with
aspects of the invention. These reference standards, like those in
FIG. 3, are particularly suitable for use in epi illumination.
[0040] Reference standard 400 can include a light-producing element
402a,b (corresponding to embodiments "a" and "b") and a
light-blocking element 404a,b, among others. Light-producing
element 402a in embodiment "a" can include a set of light-emissive
portions 406, and light-producing element 402b in embodiment "b"
can include first and second sets 408, 410 of light-emissive
portions. Light-blocking element 404a can include an opaque portion
412a and a set 414 of transparent portions. Light-blocking element
404b can include an opaque portion 412b but does not (necessarily)
include any transparent portions. The pictured embodiment is
configured for use with a 96-well microplate, in parallel with FIG.
3, although, like in FIG. 3, the general principles can be applied
in any suitable manner. Here, the light-emissive portions (406,
408, and 410) are supported by (i.e., contact, rest upon, and/or
are partially or completely embedded in) the light-blocking element
(404a or 404b, respectively). The light-producing portions (in this
and/or other embodiments) can independently have the same and/or
different spectral outputs (i.e., can independently produce light
with the same and/or different color(s)).
[0041] Embodiment "a" (i.e., the left side of the reference
standard (402a+404a)) is configured for placement in an
intermediate optical plane, between the light source and sample. In
use, light can travel to and from the sample through transparent
portions 414, for use in sample analysis, and light emission can be
induced from light-emissive portions 406, for use in calibration.
This embodiment most closely resembles embodiment "a" in FIG.
3.
[0042] Embodiment "b" (i.e., the right side of the reference
standard (402b+404b)) is configured for placement in an object
plane. Here, in use, light emission can be induced from both the
first (408) and second (410) sets of light-emissive portions, for
use in calibration. This embodiment most closely resembles
embodiment "b" in FIG. 3.
[0043] I.B Light-Producing Elements
[0044] The light-producing element generally comprises any
mechanism for producing (and/or redirecting) light as part of an
optical reference standard for monitoring and/or calibrating a
light-detection system. This element can be distinguished by the
nature and/or arrangement of the mechanism(s) used to produce the
light and/or the disposition of the mechanism(s) relative to an
associated light-blocking element and/or a light-detection system,
among others. Thus, in some embodiments, and unless otherwise
noted, the light-producing element can be configured to produce
light by any suitable mechanism, including emission, reflection,
transmission, refraction, diffraction, and/or scattering, among
others. Here, emission can include luminescence, including
photoluminescence (e.g., fluorescence, phosphorescence, etc.)
and/or chemiluminescence, among others. Conversely, in other
embodiments, the light-producing element can be configured to
produce light only via particular mechanisms, for example, via
luminescence mechanisms, as described below for subtraction-based
and addition-based reference standards. In either case, the light
produced by the light-producing element can be used "as is" and/or
filtered to alter its wavelength, intensity, polarization, and/or
coherence, among others.
[0045] The optical reference standard can be configured to produce
light primarily or exclusively at preselected wavelengths and/or
over preselected ranges of wavelengths. Toward this end, in some
"subtraction-based" embodiments, the light produced by the
reference standard can be obtained by "subtracting" undesired
spectral components from a relatively broadband emission; see
Section I.D below. In other "addition-based" embodiments, the light
produced by the reference standard can be obtained by "adding"
desired spectral components from two or more materials; see Section
I.E below. In yet other embodiments, the light produced by the
reference standard can be obtained by a combination of these and/or
other mechanisms.
[0046] Luminescence-based light-producing elements can be based on
photoluminescence, in which the light-producing element is induced
to emit emission light by illumination with suitable excitation
light. The emission light corresponding to excitation with
particular excitation light can be characterized by an emission
spectrum, and the excitation light corresponding to particular
emission light can be characterized by an excitation spectrum, as
described below. Typically, the excitation light will lie at least
primarily to one side of some cutoff wavelength, and the emission
light will lie at least primarily to the other side of the cutoff
wavelength. For example, with single-photon excitation, the
wavelength(s) of the excitation light typically would be shorter
than some cutoff wavelength (corresponding to relatively higher
energies), and the wavelength(s) of the emission light typically
would be longer than the cutoff wavelength (corresponding to
relatively lower energies). Conversely, with multi-photon
excitation, these wavelength differences typically would be
reversed. The differences between the wavelengths of the excitation
and emission light can facilitate the separation and
distinguishable detection of emission light.
[0047] I.C Light-Blocking Elements
[0048] The light-blocking element generally comprises any mechanism
for blocking (and/or redirecting) light as part of an optical
reference standard for monitoring and/or calibrating a
light-detection system. This element can be distinguished by the
nature and/or arrangement of the mechanism(s) used to block the
light and/or the disposition of the mechanism(s) relative to an
associated light-producing element and/or a light-detection system,
among others. The light-blocking (or optically opaque) element can
be configured to block light by any suitable mechanism, including
absorption and/or reflection, refraction, diffraction, and/or
scattering out of the optical path, among others. The blocked light
can represent any amount between at least about half and at least
about all (or entirely all) of the incident light, among
others.
[0049] The light-blocking element can be formed of any suitable
material, including but not limited to plastic and/or metal such as
aluminum or steel (e.g., sheet metal) having a non-fluorescing
coating or paint, such as black anodizing.
[0050] The light-blocking element can include both light-blocking
(opaque) portions, as described above, and light-transmitting
(transparent) portions that allow passage of excitation and/or
emission light, as appropriate or desired. The opacity and
transparency of these portions can be absolute or relative.
Collectively, the opaque and transparent portions can create a mask
or pattern that allows selective illumination of the
light-producing portion of the reference standard and/or samples in
a light-detection system, for example, as described above for FIGS.
1, 3, and 4.
[0051] The transparent portions (and thus the opaque portions) can
have any suitable shape and size. For example, the transparent
portions can be sized and spaced to correspond to and/or allow
selective illumination of a light-producing element and/or sample
holder, among others. Thus, in specific embodiments, the reference
standard can include 96, 384, or 1536 transparent portions, among
others, corresponding to wells in a 96, 384, or 1536-well
microplate, among others. Alternatively, or in addition, the
reference standard might include another number of transparent
portions corresponding to inter-well calibration marks.
[0052] The transparent portions can have any form capable of
transmitting excitation and/or emission light, or portions thereof.
Thus, the transparent portions can include apertures and/or
optically transparent (or partially transparent) materials (at
least in the wavelength range(s) of interest). The optically
transparent materials can include glass, quartz, plastic and/or the
like, among others.
[0053] I.D Subtraction-Based Reference Standards
[0054] Subtraction-based reference standards generally comprise any
reference standards, or portions thereof, in which output light
with the desired spectral components is obtained by producing light
with both desired and undesired spectral components and then
"subtracting" the light with the undesired spectral components. The
subtraction-based reference standards can include, among others,
(1) a relatively broadband emissive element, and (2) a relatively
narrowband subtractive element. The emissive element generally is
capable of outputting light (e.g., emitting luminescence) over a
greater number or range of wavelengths than ultimately desired as
output light from the reference standard (i.e., outputting light
with both "desired" and "undesired" spectral components). The
subtractive element generally is configured to reduce or eliminate
the undesired spectral components, thereby increasing the relative
proportion of desired spectral components.
[0055] FIG. 5 shows an exemplary subtraction-based reference
standard 500. This exemplary standard can include a support element
502, a relatively broadband emissive element 504, a relatively
narrowband subtractive element 506, an intensity filter element
508, a protective coating 510, and/or a mask (light-blocking
element) 512, among others. These elements can be discrete (e.g.,
separate, defined layers) and/or interdigitated (e.g., interwoven
and/or blended layers (with shared or overlapping functions)).
Typically, the subtractive and filter elements will be disposed so
that they will lie in use between the emissive element and the
detector, and the coating element will be disposed near an outer
surface of the reference standard. More generally, these and other
elements of the subtraction-based (or other type of) reference
standard can be arranged in any suitable order.
[0056] Support element 502 generally comprises any material, or
property thereof, configured to provide structural support for the
reference standard. Exemplary support materials include glass
and/or plastic, among others. The support element can take any
suitable form, with any suitable dimensions, such as a planar slab
(e.g., sized like a microscope slide, a gel, and/or a microplate,
among others) and/or a standard sample holder (e.g., configured and
sized like a microplate, a biochip, or the like).
[0057] Emissive element 504 generally comprises any mechanism for
generating or producing light over a greater number or range of
wavelengths than the number or range(s) output by the reference
standard (i.e., with desired and undesired spectral components).
The emissive element can produce such light by any suitable
mechanism, including transmission, reflection, absorption
(generally selective absorption and/or absorption and re-emission),
and/or emission, among others. Transmission generally includes
passing light through the element, unaltered, or altered via
absorption, refraction, diffraction, scattering, and/or the like.
Emission generally includes any mechanism for producing light
within the emissive element, including photoluminescence and/or
chemiluminescence, among others. Photoluminescent sources produce
photoluminescence light in response to illumination with suitable
excitation light. Photoluminescence can include fluorescence (i.e.,
light produced by a singlet-to-singlet electronic transition)
and/or phosphorescence (i.e., light produced by a triplet-to-single
electronic transition), among others. Chemiluminescent sources
produce chemiluminescence light associated with a chemical reaction
(e.g., as part of a reaction that produces an intermediary or
product in an excited electronic state that subsequently decays by
production of light) and/or electrical stimulation (e.g.,
electrochemiluminescence). Chemiluminescence can include
bioluminescence (i.e., light produced by a biological reaction),
among others. Suitable emissive elements can include luminophores,
fluorophores, dyes, pigments, and/or the like. These elements can
be wet or dry, liquid or solid, dissolved or suspended, homogeneous
or heterogeneous, and so on.
[0058] Subtractive element 506 generally comprises any mechanism
for altering the spectrum of the excitation and/or emission light,
or portions thereof. The spectral filter element generally acts to
reduce or eliminate emission at selected wavelengths and/or over
selected ranges of wavelengths (i.e., to reduce or remove undesired
spectral components). These functions can be accomplished via any
suitable mechanism, using a single subtractive element or a
combination of subtractive elements. Suitable subtractive elements
include filter elements such as (1) short-pass (cut-off) filters,
which pass short-wavelength light and reject long-wavelength light,
(2) long-pass (cut-on) filters, which pass long-wavelength light
and reject short-wavelength light, (3) bandpass filters, which pass
light with a particular wavelength (or range of wavelengths) and
reject light with lower and higher wavelengths, and/or (4) band
reject (or notch) filters, which reject light with a particular
wavelength (or range of wavelengths) and pass light with lower and
higher wavelengths, among others. Short-pass and long-pass filters
(also know as edge filters) can be characterized by a cut-on or
cut-off wavelength, among others, and bandpass and band reject
filters can be characterized by a center wavelength and a
bandwidth, among others. Suitable subtractive elements can include
thin-film (e.g., metallic and/or interference) coatings, colored
filter glass, holographic filters, liquid-crystal tunable filters,
and/or acousto-optical tunable filters, among others. These
subtractive elements can work by absorbing, reflecting, and/or
bending (refracting or diffracting) light, among others. In some
embodiments, the subtractive element can work by filtering portions
of the excitation light whose absorption gives rise to undesired
spectral components of the emission.
[0059] Intensity filter element 508 generally comprises any
mechanism for altering the intensity of the excitation and/or
emission light, or portions thereof. In some cases, the intensity
filter element can reduce the overall intensity of the excitation
light and/or emission light, substantially independent of
wavelength. For example, the filter could reduce overall excitation
intensity to reduce photobleaching of the standard, and it could
reduce overall emission intensity to better match corresponding
sample intensities, among others. In other cases, the intensity
filter element can reduce the intensity of the excitation and/or
emission light, according to wavelength. For example, the filter
could adjust the relative intensities of different spectral
components of the emission light (e.g., making the height of
multiple emission peaks more (or less) similar by using a filter
element that blocks selectively a portion of the brighter (or
dimmer) peaks). In any case, the strength of the intensity filter
can be substantially uniform with position, so that intensities
from all portions of the reference standard are reduced
proportionally, or the strength of the intensity filter can vary
with position, so that intensities from some portions of the
reference standard are reduced more than the intensities from
others (e.g., to create a pattern). Suitable intensity filter
elements include neutral density filters, among others.
[0060] Protective coating 510 generally comprises any mechanism for
protecting the reference standard from unnecessary or unwanted
exposure to environmental conditions. These conditions can include
high humidity, harsh or corrosive chemicals, rough handling, and so
on. Thus, the protective coating can be relatively impervious to
water and/or other commonly encountered chemicals, and/or resistant
to scratching, bending, and/or breaking, among others. Consistent
with these functions, the coating can be disposed about an outward
or exterior surface of the reference standard, particularly on
portions of the standard likely to encounter the unwanted
condition(s). Suitable coating materials include plastics, among
others, particularly plastics that are at least partially
transparent to excitation and/or emission light employed during use
of the reference standard.
[0061] Mask 512 generally comprises any mechanism for selectively
rejecting most or all of the emission from selected portions of the
reference standard. Suitable masks were discussed above; see
particularly Section I.C. The mask can be positioned to block
excitation light and/or emission light, depending in part on the
embodiment and intended application.
[0062] Specific exemplary subtraction-based reference standards are
described below, in Example 1.
[0063] I.E Addition-Based Reference Standards
[0064] Addition-based reference standards generally comprise any
reference standards, or portions thereof, in which output light
with the desired spectral components is obtained by "adding"
together or combining light from different components that
individually produce light having only a subset of the desired
characteristics. Light from addition-based reference standards can
be used "as is" or spectrally filtered (as with the
subtraction-based reference standards) to remove any undesired
components present after the addition. Such filtering can be
accomplished, among other ways, using a spectral filter that is
integral to or otherwise associated with the standard and/or part
of a light-detection system. Addition-based standards optionally
can include one or more of the various components described above
for subtraction-based standards, including a support, a filter
element, a coating, and/or a mask, among others. Luminescent (and
other) components of the addition-based reference standard can take
any suitable form, including wet or dry, continuous or discrete,
separate or blended, planar or formed, planar or sample-holder
shaped, and/or so on. In some embodiments, the luminescent
components can be selected and/or adapted such that they can be
excited using light with one wavelength (or range of wavelengths),
while emitting at two or more distinguishable wavelengths (or
ranges of wavelengths). Exemplary addition-based reference
standards can include photoluminescent nanocrystals (e.g., "quantum
dots"), luminescent glass, and so on. Specific exemplary
addition-based reference standards are described below, in Example
2.
II. LIGHT DETECTION SYSTEMS
[0065] The light-detection system, as noted above, generally
comprises any mechanism for detecting (and optionally analyzing)
light from a sample. This system can include (1) a light source for
producing light for illuminating, and/or inducing a suitable or
desired response from, a sample, (2) a sample-support device for
supporting one or more samples at an examination area, (3) a
detector for detecting light transmitted or otherwise originating
from a sample and optionally converting the detected light into a
representative signal, and/or (4) an optical relay structure for
directing and/or processing light between the light source and
examination area, and/or between the examination area and the
detector, among others.
[0066] The components of the light-detection system can be selected
and/or configured according to the intended application, desired
level of automation, and so on. Typically, the light source
produces light that is directed by the optical relay structure
along an incident optical path so that it impinges on or
illuminates one or more samples disposed at one or more locations
in the examination area. (This portion of the light-detection
system can be omitted or simply left unused in chemiluminescence
applications.) Output light emitted, transmitted, reflected,
scattered, and/or otherwise originating from the sample(s) then is
directed by the optical relay structure along an output optical
path onto the detector. The light-detection system can include an
optional controller adapted to control one or more system
components, including, among others, the light source(s), the
detector(s), and/or a registration device configured to bring one
or more samples and the examination area into registration for
analysis of the sample(s). The controller can be configured to
control or otherwise coordinate the illumination of samples and/or
the detection of outputted radiation with the relative position(s)
of sample(s) and examination area. The light-detection system also
can include additional components, such as (1) a fluidics mechanism
to add, remove, and/or mix sample components, (2) a sample-handling
mechanism to convey samples and/or sample holders to and/or from
the examination area and/or registration device, (3) an analysis
mechanism to analyze or interpret assay results, and/or (4) a
sample-identification mechanism such as a barcode reader to
identify samples and/or sample holders, and optionally to configure
or operate system components accordingly, among others.
[0067] These and other aspects of light-detection systems provided
by the present teachings are described below, including (A) light
sources, (B) optical relay structures, (C) sample-support devices,
(D) registration devices, (E) color separators, (F) detectors, (G)
miscellaneous optical elements, and (H) controllers, among others,
as well as relationships and interactions there between.
[0068] II.A. Light Sources
[0069] The light source (102) generally comprises any mechanism for
producing light capable of illuminating, and/or inducing a suitable
or desired response from, a sample. For example, when used in an
optical assay, light from the light source can, as a result of
illuminating a sample, produce emitted (e.g., photoluminescence)
light, transmitted light, reflected light, and/or scattered light,
among others. These different forms of light can be present
exclusively, or in various combinations, and can include
ultraviolet, visible, and/or infrared light, among others. The
light source optionally can induce a similar or related response
from a suitably positioned optical reference standard.
[0070] Exemplary light sources can include continuous wave and
pulsed lasers, arc (e.g., xenon) lamps, incandescent (e.g.,
tungsten halogen) lamps, fluorescent lamps, electroluminescent
devices, laser diodes, and/or light-emitting diodes (LEDs), among
others. Such light sources can be capable of use in one or more
illumination modes, including continuous and/or time-varying (e.g.,
pulsed or sinusoidally varying) modes, among others, depending on
system configuration and/or intended application. For example, an
arc lamp or continuous wave laser can be used to provide continuous
illumination, and a pulsed laser can be used to provide
intermittent illumination. Such light sources also can produce
coherent, incoherent, monochromatic, polychromatic, polarized,
and/or unpolarized light, among others. For example, an arc lamp
can be used to provide (at least initially) incoherent,
polychromatic, unpolarized light, and a laser can be used to
provide (at least initially) coherent, monochromatic, polarized
light, among other possibilities.
[0071] The light source can be used alone but would be used more
commonly in combination with various optics and/or other mechanisms
(such as the optical relay structures described below). These
optics and/or other mechanisms can be used to alter the nature of
the light output by the light source (e.g., its color (spectrum or
chromaticity), intensity, polarization, and/or coherence, among
others). Alternatively, or in addition, these optics and/or other
mechanisms can be used to direct and/or alter the size, shape,
and/or numerosity of the light beam(s) (e.g., to illuminate
selected locations in an examination area with one or more light
beams). The light that ultimately is incident on the sample(s) can
be produced by one or more light sources, and can be directed
and/or modified by one or more optical devices operatively disposed
between the light source(s) and the examination area. The resultant
light beam or beams can be one or more of various forms, including
but not limited to diverging, collimated, and converging, among
others. This beam or beams can be directed onto an examination area
in a manner inducing light production from one or more samples
located in a plurality of locations within the examination
area.
[0072] II.B. Optical Relay Structures
[0073] The optical relay structure (108) generally comprises any
mechanism(s) for directing, transmitting, and/or conducting light
between two points, such as from a light source toward a sample (or
examination site) and/or from a sample (or examination site) toward
a detector. These structures can stand alone and/or be portions of
or integral to other system components, such as the light source,
color separator, and/or detector, among others. The optical relay
structures can be configured to direct one or more light beams, in
the same or different directions, along the same, multiple, or
different optical paths.
[0074] The optical relay structures can include any suitable
combination of optical elements. These elements independently can
be part of a single (e.g., excitation or emission) relay structure,
or can be shared between two or more relay structures. Exemplary
optical elements can include (1) reflective elements, such as
concave, planar, and/or convex mirrors, among others, (2)
refractive elements, such as converging, diverging, concave,
convex, and/or planoconvex lenses, including circular and/or
cylindrical lenses, among others, and/or (3) transmissive or
conductive elements, such as glass or quartz fiber optics and/or
liquid light guides, among others.
[0075] The optical relay structure(s) can be selected, in
conjunction with the light source(s) and/or detector(s), to allow
any suitable or desired combinations of illumination and/or
detection. For example, these components can be arranged to allow
same-side, (locally) anti-parallel or straight-on ("epi")
illumination and detection, such as top illumination and top
detection, or bottom illumination and bottom detection,
respectively. Alternatively, or in addition, these components can
be arranged to allow opposite side, (locally) parallel or
straight-through ("trans") illumination and detection, such as top
illumination and bottom detection, or bottom illumination and top
detection, respectively. Alternatively, or in addition, these
components can be arranged to allow illumination and/or detection
at oblique angles. For example, illumination light can impinge on
the bottom of a sample holder at an acute angle (e.g., about 45
degrees) relative to detection. Such oblique illumination and
detection can reduce the amount of excitation light reaching the
detector, relative to straight-on epi systems (light source and
detector directed at about 90 degrees to sample holder) or
straight-through trans systems (light source directed through a
sample holder directly at a detector). Epi systems are especially
suitable for photoluminescence assays, trans systems are especially
suitable for absorbance assays, and oblique systems (with the
incidence angle set above the critical angel) are especially
suitable for total internal reflection assays, among others.
[0076] II.C. Sample-Support Devices
[0077] The sample-support device (104) generally comprises any
mechanism for supporting one or more samples at a sample site in an
examination area. In typical embodiments, the sample-support device
allows for the receipt and/or transmission of light relative to one
or more samples supported by the device. General examples of
suitable sample-support devices can include trays, wells, tubes,
containers, channels, chambers, frames, carriages, holders, slides,
shelves, stages, housings, and/or the like. Specific examples of
suitable sample-support devices can include microplates, PCR
plates, microtiter plates, cell culture plates, biochips,
hybridization chambers, chromatography plates, and/or microscope
slides, among others. Specific locations in the sample-support
device, such as wells in microplates, PCR plates, microtiter
plates, and cell culture plates and array sites on biochips, can
comprise assay sites. For example, microplates (and/or PCR plates,
microtiter plates, and/or cell culture plates) can include arrays
of 6, 12, 24, 48, 96, 384, 864, 1536, 3456, and/or 9600 such assay
sites, among others. The sample-support devices can be configured
to allow top detection (e.g., by having an open or at least
partially transparent top), bottom detection (e.g., by having an at
least partially transparent bottom), and/or side detection (e.g.,
by having an at least partially transparent side), among others. In
some embodiments, the sample-support device can be configured,
additionally and/or alternatively, to support an optical reference
standard, for use before, during, and/or after sample analysis.
[0078] II.D. Registration Devices
[0079] The registration device generally comprises any mechanism
for bringing a sample(s) and an examination area into registration,
for analysis of the sample(s).
[0080] The registration device can move the sample (or associated
sample-support device), the examination area, and/or both. For
example, to effect relative movement of a sample and examination
area, the registration device can include a driver, such as a
stepper motor, that moves a carriage, tray, conveyor, stage, frame,
and/or other structure or structures adapted to support the sample,
an associated sample-support device, and/or a detector, among
others. The registration device can move the sample and/or
examination area in any suitable form or combination of motion(s),
including, among others, (1) continuous or intermittent, (2)
unidirectional, bidirectional, or multi-directional, and/or (3)
rectilinear or curvilinear. Such movement can occur in the x, y,
and/or z directions, and can occur parallel, perpendicular, and/or
skewed to the excitation and/or emission axes.
[0081] The registration device can be under manual and/or automated
control. Automated control can be particularly useful for analysis
of plural samples, allowing successive registration of multiple
samples and the examination site. Such control can be effected
using any suitable mechanism, for example, moving a sample-support
device and/or examination area in preselected increments and/or
until preselected criteria are fulfilled. Such preselected
increments can correspond to predefined separations between
samples, or sample sites, as found in a microplate, PCR plate,
biochip array, and/or the like. Such preselected criteria also can
correspond to indicia (such as increased intensity) indicative of
the presence of a sample, as found in bands on a separatory gel or
column, among others. Such preselected criteria also can correspond
to the positions of reference markings on an optical reference
standard (e.g., portions 314 in FIG. 3 and/or portions 406 and 410
in FIG. 4, among others).
[0082] II.E. Color Separators
[0083] The color separator generally comprises any mechanism for
spatially separating, or distributing, light according to its
wavelength composition (or spectrum).
[0084] The color separator can use any suitable mechanism(s) and/or
component(s) for separating light. Exemplary mechanisms can include
diffraction, interference, and/or refraction, among others.
Exemplary components can include (diffraction) gratings,
interferometers, and/or prisms, among others. In some embodiments,
the color separator can include two or more sequentially acting
components, employing the same or different mechanisms, with the
first component achieving a coarse color separation, and the second
component achieving a finer or final color separation.
[0085] The color separator can separate multi-wavelength light by
directing light with different wavelengths along different paths
(e.g., in different directions, at different angles, etc.) The
separation can be partial or complete, and can create bands or
beamlets of light, which can be continuous or discrete, and which
can be partially overlapping or completely distinct. The character
of the separation typically will be determined at least in part by
the character of the light being separated. Thus, input light with
several well-spaced wavelength components can give rise to
separated output light at several discrete (well-spaced) positions,
while input light with closely or continuously spaced wavelength
components can give rise to separated output light over a
continuous set of positions.
[0086] The separated light generally can form any distinguishable
pattern. Thus, the separated light can form a linear array, in
which the wavelength of light varies with position along the line.
Alternatively, the separated light can form a circular array, in
which the wavelength of the light varies with distance from the
center of the circle. In some embodiments, the color separator can
produce light that is spectrally separated along a first axis, and
spatially differentiated along a second axis at least substantially
transverse to the first axis. In these embodiments, position along
the first axis provides information about the spectral composition
of light emitted from the sample, and position along the second
axis provides information about the position from which the
spectrally separated light arose in the sample.
[0087] The separated light can be directed onto a common detector,
onto separate detectors, or onto a combination of detectors. The
relationship between wavelength and position on the detector(s) can
be determined empirically, for example, using input or calibration
light of known wavelength(s). Alternatively, or in addition, the
relationship between wavelength and position on the detector(s) can
be determined theoretically, for example, by calculating the
optical paths for light of different wavelengths. The position(s)
of light on the detector can be determined by a variety of factors,
including (1) the mechanism used to separate the light, (2) the
angle(s) at which the light leaves the color separator, the
distance between the color separator and the detector(s)
(generally, greater distances between the color separator and
detector(s) will give rise to greater separations between light of
different wavelengths on the detector(s)), and/or (3) the type of
pattern (e.g., linear versus circular) formed by the separated
light, among others.
[0088] The spatial distribution or pattern of detected light can be
converted into information about the distribution and identity(ies)
of components of the sample, using any suitable method. These
methods can include simply looking up a result in a look-up table
(e.g., position (x,y) on the detector corresponds to light of
wavelength .lamda. (or wavelengths within some extended range
(e.g., .lamda..sub.1 to .lamda..sub.2)) emitted from position (X,Y)
(or positions within some extended range) in the sample, evaluating
a function expressing the relationship between these parameters,
and/or the like. The desired result can be obtainable simply by
noting qualitatively the presence or absence of light at a
particular position on the detector (subject, in some cases, to
some threshold amount), or it can be obtainable by determining
quantitatively the amount of light (intensity, number of photons,
amount of energy, etc.) detected at the position, among others.
[0089] The color separator can be disposed, in some embodiments, so
that it acts only on light directed from a sample toward a
detector, without acting on (or, in most cases, even contacting)
light directed from a light source toward a sample.
[0090] The color separator can be included, in some embodiments, as
part of a spectroscope or other instrument for producing and
observing spectra from light or other electromagnetic radiation
emitted from a sample.
[0091] Exemplary color separators are described further in U.S.
Provisional Patent Application Ser. No. 60/584,525, filed Jun. 30,
2004, titled Distributed Light Detector of inventors Charles S.
Vann and Steven Boege, which is incorporated herein by
reference.
[0092] II.F. Detectors
[0093] The detector (106) generally comprises any mechanism for
detecting light transmitted or otherwise originating from a sample
and optionally converting the detected light into a representative
signal.
[0094] Exemplary detectors can include film, charge-coupled devices
(CCDs), intensified charge-coupled devices (ICCDs), charge
injection device (CID) arrays, videcon tubes, photomultiplier tubes
(PMTs), photomultiplier tube (PMT) arrays, position sensitive
photomultiplier tubes, photodiodes, and/or avalanche photodiodes,
among others. Such detectors can be capable of use in one or more
detection modes, including (1) imaging and point-reading modes, (2)
discrete (e.g., photon-counting) and analog (e.g.,
current-integration) modes, and/or (3) steady-state and
time-resolved modes, among others. Particularly if used with a
color separator, the detectors can be configured to receive a
two-dimensional array of light, which may be separated along a
first dimension according to position in a sample or sample array,
and along a second dimension according to spectral composition.
Toward this end, the detector can include bins for detecting light
of different colors, for example, corresponding to light from
different luminophores. These bins can be the same or different
sizes, and can be formed of one or more sub-bins, or pixels,
depending in part on the average separation between spectral peaks
output by the color separator.
[0095] The detector can be used alone or in combination with
various optics and/or other mechanisms (such as the optical relay
structure described above). These optics and/or other mechanisms
can be used to alter properties of the light (e.g., color,
intensity, polarization, coherence, and/or size, shape, and/or
numerosity of the light beam(s), as described elsewhere herein),
prior to its detection. In some embodiments, the detector can be
part of or coupled to a spectrograph or spectroscope for analyzing
the spectral composition of the detected light.
[0096] II.G. Miscellaneous Optical Elements
[0097] The light-detection system, and/or components thereof, also
can include miscellaneous optical elements capable of performing
additional and/or duplicative optical functions. These optical
elements can include (1) intensity filters (such as neutral density
filters) for reducing the intensity of light, (2) spectral filters
(such as interference filters, diffraction gratings, and/or prisms)
for altering or selecting the wavelength(s) of light (e.g., for
separating longer-wavelength emission light from shorter-wavelength
excitation light, in single-photon photoluminescence, and/or for
separating shorter-wavelength emission light from longer-wavelength
excitation light, in multi-photon photoluminescence), (3)
polarization filters (such as "polarizers") for altering or
selecting the polarization of light, (4) "confocal optics elements"
(such as an aperture or slit positioned in an intermediate image
plane) for reducing or eliminating out-of-focus light, (5) beam
collimators for converting input light (particularly diverging
input light) into an at least substantially collimated light beam,
(6) beam expanders for increasing the cross-sectional area of a
beam of light, (7) beam homogenizers (such as a fiber optic cable
or liquid light guide) for enhancing the spatial uniformity of
light, and/or (8) reference monitors for correcting for variations
(e.g., fluctuations and/or inhomogeneities) in light produced by a
light source and/or other optical elements. These elements can be
functional in one or more of the space, time, and/or frequency
domains, as necessary or desired.
[0098] The relative positions of any intensity, spectral,
polarization, and/or other optical elements generally can be varied
without affecting the operation of the light-detection system. In
addition, if there is more than one optical path, for example, to
permit top and bottom or oblique illumination and/or detection,
optical elements can be shared and/or used independently in each
path. The particular order, positions, and combinations of optical
elements for a particular experiment can depend on the apparatus,
the assay mode, and the sample (target material), among other
factors. In some cases, optical elements can be associated with an
exchange mechanism, such as a wheel or slider, that allows
convenient and automatable placement and exchange of optical
elements by rotating, sliding, or otherwise bringing preselected
optical elements into or out of the optical path.
[0099] II.H. Controllers
[0100] The controller generally comprises any mechanism for
controlling components and/or other aspects (including calibration
and/or monitoring) of the light-detection system. These components
and/or other aspects can include the light source, optical relay
structures, registration device, detector, and/or optical reference
standard, among others. For example, the controller can determine
and/or change (1) the wavelength, intensity, and/or (spatial and/or
temporal) uniformity of light produced by the light source, (2) the
order and timing of sample delivery by the registration device and
image acquisition by the detector, and/or (3) the wavelength and/or
intensity of light detected by the detector, among others. The
controller can include hardware, software, firmware, and/or a
combination thereof, and can be any device, or combination of
devices, adapted to store and execute instructions to control
associated detection system components. The controller can include
one or more of various devices, such as a computer, computer
server, microprocessor, memory, logic unit, and/or processor-based
system capable of performing a sequence of logic operations. In
addition, processing can be centralized (with two or more
components sharing a common controller) and/or distributed (with
one or more components having their own dedicated controllers,
acting alone, or connected to one another and/or a central
controller).
III. APPLICATIONS
[0101] Light-detection systems calibrated and/or monitored using
optical reference standards in accordance with the present
teachings can be used for any suitable purposes, such as detecting
and/or monitoring the occurrence of, and/or changes in, light or
other forms of radiation received from one or more suitable
samples.
[0102] The detection or monitoring of light can be performed
qualitatively and/or quantitatively. Qualitative detection can
include measurement of the presence or absence of a signal, and/or
a change in a signal from present to absent, or absent to present,
among others. Here, presence or absence can be in reference to a
whole signal (such as any light) and/or a component of the signal
(such as light of a particular wavelength, polarization, and/or the
like). The signal, and/or components thereof, can arise from the
sample itself and/or from labels (or other reporters) attached to
or otherwise associated with the sample. Quantitative detection can
include measurement of the magnitude of a signal, such as an
intensity, wavelength, polarization, and/or lifetime, among others.
The quantified signal can be used alone and/or compared or combined
with other quantified signals and/or calibration standards. The
standard can take the form of a calibration curve, a calculation of
an expected response, and/or a control sample measured before,
during, and/or after measurement of the sample of interest.
[0103] The detected or monitored light can be used for any suitable
purpose, for example, to determine the presence, absence, amount,
concentration, activity, and/or physical properties (including
interactions) of an analyte (such as a photoactive analyte) in a
sample. Here, the analyte can be the actual moiety of interest
and/or a reporter moiety (such as a luminophore) that reports on
the actual moiety of interest.
[0104] The moiety of interest can be a reaction component.
Exemplary reaction components can include an enzyme, enzyme
substrate, enzyme product, and/or enzyme modulator (e.g., agonist
and/or antagonist). Suitable reactions can occur in vivo and/or in
vitro, for example, as part of a cell-lysis experiment and/or a
polymerase chain reaction (PCR) preparation. Exemplary reaction
components also can include precursors and/or products of a
synthetic pathway, such as an amino acid, peptide, protein,
nucleotide, oligonucleotide, nucleic acid polymer, carbohydrate,
fatty acid, lipid, and/or the like.
[0105] The moiety of interest also can be the subject and/or
product of a separatory process, such as on a chromatograph, gel,
column, and/or the like. Here, the separatory process can include
single processes, such as columns giving rise to fractions, and/or
multiple processes, such as parallel lanes on a gel giving rise to
sets of bands.
[0106] The moiety of interest also can be the subject of a
sequencing process, such as a peptide, protein, oligonucleotide,
and/or nucleic acid (RNA or DNA) sequencing process. Here, the
sequence can include amino acid sequence, nucleotide or base
sequence (e.g., G, C, T, A, U, etc.), and so on, and the sequencing
process can include generating fragments (or other derivatives) of
the moiety to be sequenced and labeling those fragments (before or
after their generation) with different luminophores. Thus, in
nucleic acid sequencing, the presence of a G, C, T, A, or U at a
particular position in a moiety of interest, or in a fragment or
derivative thereof, can be determined by the identity of an
associated luminophore.
[0107] The moiety of interest also can be the subject of an
identification, or affinity, process, such as a northern, western,
and/or southern blot.
[0108] In some cases, the effect of some condition on the moiety of
interest can be determined, for example, by comparing results in
the presence of the condition with predicted and/or measured
results in the absence of the condition and/or the presence of
another condition. Exemplary conditions can include presence or
absence of a modulator (agonist or antagonist) or cofactor, and/or
changes in temperature, concentration, pH, osmolarity, ionic
strength, and/or the like.
[0109] The sample can include any appropriate material, with any
suitable origin. For example, the sample can include and/or be
derived from a biomolecule, organelle, virus, cell, tissue, organ,
and/or organism. The sample can be biological in origin and/or
synthetically prepared. A sample optionally can be, or can be
derived from, a biological sample, such as a sample prepared from a
blood sample, urine sample, fecal sample, saliva sample, and/or
mucous sample, obtained using any suitable physiological sampling
method, such as a swipe or a smear, among others. A sample
optionally can be, or can be derived from, an environmental sample,
such as an air sample, a water sample, or a soil sample. A sample
can be aqueous, and yet can contain biologically compatible organic
solvents, buffering agents, inorganic salts, or other components
known in the art for assay solutions. Suitable samples (or
compositions) can include compounds, mixtures, surfaces, solutions,
emulsions, suspensions, cell cultures, fermentation cultures,
cells, suspended cells, adherent cells, tissues, secretions, and/or
derivatives and/or extracts thereof. Depending on the assay, the
term "sample" can refer to the contents of a single sample site
(e.g., microplate well) or of two or more sample sites.
IV. EXAMPLES
[0110] The following examples describe selected aspects of the
present teachings. These selected aspects include, among others,
exemplary apparatus, methods, and compositions for detecting light.
The selected aspects can be combined with other aspects in the same
and/or other examples, and/or in other portions of these teachings,
as suitable and/or desired. These examples are included for
illustration and are not intended to limit or define the entire
scope of the disclosed concepts.
Example 1
Exemplary Subtraction-Based Reference Standards
[0111] This example describes an exemplary subtraction-based
reference standard 500, in accordance with aspects of the present
teachings; see FIGS. 5-9. The exemplary standard can be used with
multi-luminophore photoluminescence systems, among others,
including but not limited to carboxyfluorescein (FAM) and
carboxy-X-rhodamine (ROX) (or similar green and red emitting)
dual-luminophore systems.
[0112] FIG. 5 is a schematic sectional view of reference standard
500. This exemplary standard, which was described above in more
detail in Section I.D, can include several components: (1) a
support element 502, (2) a broadband photoluminescent element 504,
(3) at least one subtractive or spectral filter element 506a,b,c,
(4) an intensity filter element 508, (5) a coating element 510,
and/or (6) a light-blocking element (or mask) 512, among others.
Here, these components are arranged as a series of at least
substantially planar layers, in the indicated order; however, more
generally, these components can have any suitable form, and any
suitable order. Moreover, here, the broadband photoluminescent
component includes Ultem.RTM. 1000 polyetherimide (PEI) plastic
polymer, and the spectral filter components include cold-coating
filter materials configured to transmit light with FAM and ROX
spectra. Overall, the reference standard can take any suitable
form, for example, a 3.10-inch width.times.6.60-inch length.times.
1/16-inch height sheet, corresponding to the approximate size of a
Society for Biomolecular Screening (SBS) standard microplate used
in common fluorescence detection systems, among others.
[0113] FIG. 6 is a plot of an exemplary emission spectrum for
broadband photoluminescent component 504, from reference standard
500. The emission spectrum is a plot of the relative intensity of
photoluminescence emitted from the photoluminescent component as a
function of the wavelength of the photoluminescence, following
excitation with suitable excitation light (i.e., excitation light
having a wavelength (or range of wavelengths) capable of inducing
photoluminescence emission from the photoluminescent component).
The spectrum represents photoluminescence emitted by uncoated
Ultem.RTM. 1000 plastic, following excitation at 488 nm (e.g.,
using an argon ion laser). Ultem.RTM. 1000 plastic can be
translucent, rather than optically clear, with a yellow tint and a
broad photoluminescence emission spectrum. In addition, Ultem.RTM.
1000 plastic can have one or more advantageous mechanical
properties, such as durability and/or high-melting temperature,
among others. The spectrum shows an envelope of values constructed
by displaying the highest and lowest values at each wavelength,
based on measurements of 384 similarly prepared samples.
[0114] FIG. 7 is a plot of individual and composite transmission
spectra for filter elements 506a,b,c, from reference standard 500.
The transmission spectra are plots of relative transmissivity as a
function of wavelength. In these spectra, higher values of the
transmissivity correspond to relatively greater transmission (and
lesser absorption) of light, and lower values of the transmissivity
correspond to relatively lesser transmission (and greater
absorption) of light. Here, there are three filter layers, each
with its own transmission spectrum: (1) layer 1 (dashed line), a
short-pass filter, that generally transmits short-wavelength light,
and generally blocks long-wavelength light, (2) layer 2 (thin solid
line), a first band-reject (or notch) filter, that generally
rejects light at a first intermediate wavelength, while generally
passing light with relatively shorter and longer wavelengths, and
(3) layer 3 (dot-dashed line), a second band-reject (or notch)
filter, that generally rejects light at a second intermediate
wavelength, while generally passing light with relatively shorter
and longer wavelengths. The combined or composite action of these
three filter layers (thick solid line) creates a bandpass filter
that generally transmits light at two distinct wavelengths (or
ranges of wavelengths) and that generally blocks light with other
(nearby) wavelengths. For example, in a FAM/ROX dual-luminophore
system, the optical coating typically will have a relatively narrow
bandpass transmission peak near the emission band of each
luminophore, that is, near about 520-nm (green) wavelength for the
FAM dyes, and near about 600-nm (red) wavelength for the ROX dye,
among others. Moreover, if excitation and emission occur on the
same side of the reference standard, as in epi-fluorescence mode,
the combined or composition action of the three filter layers also
can pass light at wavelengths shorter or longer than those of the
two transmission peaks, for single-photon or multi-photon
excitation, respectively. For example, for single-photon excitation
of FAM and ROX dyes, the optical coating typically will pass light
near about 488-nm (blue-green) wavelength. Suitable filter layers
are available commercially. For example, suitable cold coatings can
be obtained from Chroma Technology Corp. (Rockingham, Vt.) for
application to the support and/or broadband component.
[0115] FIG. 8 is a plot of an exemplary calculated emission
spectrum for reference standard 500. The calculated emission
spectrum (solid line) is a product of the emission spectrum of
broadband photoluminescent component 504 (thick dashed line; see
also FIG. 6) and the composite transmissivity of filter components
506a-c (thin dashed line; see also FIG. 7).
[0116] FIG. 9 is a plot of an exemplary measured emission spectrum
for reference standard 500. The spectrum, measured on an Applied
Biosystems (AB) Sequence Detection System 7900 instrument, includes
two distinct, relatively narrow peaks, as predicted in FIG. 8. In
this embodiment, the intensities of these peaks are about one-sixth
those of the broadband component alone; however, in other
embodiments, the intensities can be increased using other substrate
materials and/or filter components. This spectrum, like that in
FIG. 7, shows an envelope of values defined by the highest and
lowest values at each wavelength, based on measurements of 384
wells.
Example 2
Exemplary Addition-Based Reference Standards
[0117] This example describes exemplary addition-based reference
standards, in accordance with aspects of the present teachings; see
FIGS. 10 and 11. The exemplary standards can be used with
multi-luminophore photoluminescence systems, among others,
including but not limited to carboxyfluorescein (FAM) and
carboxy-X-rhodamine (ROX) (or similar green and red emitting)
dual-luminophore systems. These standards generally can include any
combination of two or more luminophores, with different emission
spectra, and optionally with similar or overlapping excitation
spectra (such that the combinations of luminophores can be excited
using light of similar wavelength). The exemplary standards can
include (A) photoluminescent nanocrystals ("quantum dots") and/or
(B) photoluminescent glass, among others, as described below.
[0118] A. Photoluminescent Nanocrystal-Based Reference
Standards
[0119] This section describes exemplary photoluminescent
nanocrystal-based reference standards; see FIG. 10.
Photoluminescent nanocrystals, as used herein, generally can
include any small, solid-state photoluminescent compositions,
including but not limited to "quantum dots."
[0120] Photoluminescent nanocrystals can include (1) a core, (2) a
shell, and (3) an optional coating, among others. The core
typically is the primary source of the nanocrystal's
photoluminescence, with the composition and size of the core
(primarily) determining the properties of this photoluminescence.
The composition of the core can coarsely determine emission
properties. For example, cadmium sulfide (CdS) cores can be
particularly suitable for ultraviolet-blue emission, cadmium
selenide (CdSe) can be particularly suitable for visible emission,
and cadmium telluride (CdTe) can be particularly suitable for
far-red and near-infrared emission, among others. The size of the
cores can more finely determine emission properties. For example,
relatively large (>6-nm diameter) CdSe cores can be used to
prepare 655-nm emitting nanocrystals, and relatively small
(<3-nm diameter) CdSe cores can be used to prepare 525-nm
emitting nanocrystals. The cores generally can have any suitable
shape, including spheres, rods, and/or pyramids, among others. The
shell typically surrounds the core, functioning to strengthen and
stabilize photoluminescence emission. The shell can be formed of
any suitable material(s). The coating typically surrounds the shell
(and thus the core), functioning to determine the hydrophilicity
and/or reactivity of the nanocrystal, among others. The coating can
be formed of any suitable material(s), including hydrophobic and/or
hydrophilic materials, reactive groups, binding groups (e.g.,
antibodies or antigens. avidin or biotin, lectins or sugars, etc.),
and so on.
[0121] Photoluminescent nanocrystal-based optical reference
standards (like other optical reference standards in accordance
with the present teachings) can take any suitable format,
including, among others, (1) discrete formats, in which the
luminophores are disposed at discrete sites (e.g., corresponding to
microplate wells), and/or (2) continuous formats, in which the
luminophores are disposed continuously (e.g., in and/or on a
microscope slide), as described below.
[0122] A.1. Discrete Embodiments
[0123] Discrete formats correspond to reference standards in which
the luminophores are disposed at discrete sites in and/or on the
reference standard. Exemplary discrete formats can include
microplate-based embodiments, among others, including wet and dry
embodiments. In wet embodiments, soluble or suspendable
luminophores such as photoluminescent nanocrystals can be dissolved
or suspended in an appropriate (e.g., aqueous) solution, before or
after being dispensed into wells of a microplate. Suitable
microplates can have 96, 384, and/or 1536 wells, among others. Wet
microplate-based standards could provide various benefits. For
example, wet standards could position the luminophores in the
reference standard in the same or similar environment (solution
and/or sample holder) as the luminophore(s) in the sample, to
account for any contribution to the signal from the sample holder.
Moreover, wet standards could position the luminophores in the
standard at the designed focal point of the optical detection
system used in the assay.
[0124] Discrete (microplate and/or other well or fluid based)
embodiments can be covered or sealed, using any suitable mechanism
and material, to increase their robustness and durability. Suitable
seal mechanisms can include pressure-sensitive adhesive (PSA), heat
seal material (where one or more layers melts, flows, and bonds to
the microplate (or other sample holder)), ultrasonic welding,
and/or similar devices and processes. Exemplary embodiments can
include a silicone-based adhesive on a clear polyolefin laminate
backing (polypropylene/polyethylene terephthalate (PP/PET)) with a
white polyester film release liner, and/or a heat-sealable
polyester-based film laminate of PET/MR, in which the seal
optionally can fuse with the microplate material. In some
embodiments, with or without a cover or seal, an evaporation
inhibitor such as DMSO can be added to the aqueous solution to
further prevent evaporation. In these embodiments, if any air leaks
do occur in the seal, the DMSO will act to absorb ambient
moisture.
[0125] Luminophores can be suspended, in some embodiments, in a
low-fluorescing gel, polymer, epoxy, and/or other solid or
semi-solid material medium, before or after being dispensed into a
well or other embodiment. The fluid compartment can again be
sealed, transiently or permanently, by an acceptable sealing
material. The suspension material and optional sealing would lower
the risk of unwanted leaks and spills. Specific examples of
alternative suspension media include polymer gels, agarose gels,
acrylamide gels, optical grade epoxies, and/or others.
[0126] Luminophores can be "dried down," in some embodiments, after
being dispensed--suspended or dissolved--into a fluid compartment,
by evaporating off the carrier solvent(s). The dried-down
luminophores, such as photoluminescent nanocrystals, still would
exhibit their stable fluorescence spectra, but without the risk of
liquid spills. This approach is particularly suitable as a standard
for adherent cell assays, because it positions the luminophores in
the standard at the same focal height as the cells in the assay.
The microplates could be sealed, transiently or permanently, by an
acceptable seal material, as above.
[0127] FIG. 10 is an exemplary emission spectrum for an
addition-based, photoluminescent-nanocrystal-containing reference
standard, showing relative emission intensity as a function of
emission wavelength, following excitation at a fixed excitation
wavelength. The shaded envelope is constructed from the lowest and
highest intensities measured at each emission wavelength, based on
analysis of 20 identically prepared samples. Here, exemplary
photoluminescent nanocrystals with emission at 525 nm and 608 nm
were mixed and suspended in aqueous solution, and pipetted into
wells in a 384-well microplate, for measurement of the emission
spectrum. Specifically, the 525-nm and 608-nm nanocrystals were
suspended at concentrations of 0.55 .mu.M and 0.034 .mu.M,
respectively, and then 10 .mu.L of the resulting solution was added
to each well measured. The molar concentration of the suspended
nanocrystals optionally can be correlated or matched to the
appropriate molar concentrations of dyes, such as FAM and ROX dyes,
commonly used in biological assays. Suitable microplates (for any
discrete embodiments) can include, among others, the AB 96-well
microplate, the AB 384-well microplate, and the AB 384-well Micro
Fluidic Card (MFC) (Applied Biosystems, Foster City, Calif.).
Suitable light-detection systems (for any embodiments) can include,
among others, the AB Sequence Detection Systems Models 7000, 7300,
7500, 7700, and 7900 (Applied Biosystems), among others.
[0128] In various embodiments, instruments for thermal cycling can
include a heated lid with apertures corresponding to wells to be
interrogated and a thermal block to provide thermal contact with
the wells. The luminescence reference plate can contact to the
block to establish the appropriate temperature for the
light-producing elements. The luminescence reference plate includes
an insulator on the upper side of the carrier with light-blocking
elements to prevent contact with the heated lid that can avoid
thermal short. The carrier can be configured as apertures on the
top portion forming the light-blocking elements. The apertures in
the carrier can be configured to align with the apertures in the
heated lid. The carrier can be configured with recesses on the
bottom portion where the recesses on the bottom portion correspond
to the apertures on the top portion. The top portion and bottom
portion of the carrier can be configured to bound a layer of media
forming the light-producing elements. The recesses on the bottom
portion of the carrier can include black paint to provide a
background correction. A user of the luminescence reference plate
can obtain the luminescent calibration from the top portion on the
carrier and then flip the luminescence reference plate over to
obtain a black background correction.
[0129] A.2. Continuous Embodiments
[0130] Continuous formats correspond to reference standards in
which luminophores are disposed continuously throughout part or all
of the reference standard. This "continuous" distribution can be
uniform or variable, in one, two, or three dimensions.
[0131] Exemplary embodiments can include (1) a support element,
such as a planar support element, and (2) a light-producing layer
(or layers) of fluorescence reference standard material, such as
photoluminescent nanocrystals. The light-producing layer(s) can be
formed on an outer surface of the support element, and/or
sandwiched between layers (or other portions) of the support
element. The support element and/or light-producing layer(s)
optionally can be covered with a coating, for example, as described
above.
[0132] The light-producing layer(s) can be produced by any suitable
mechanism, such as a solid layer than can be transferred intact to
the support structure (like a cellophane), or as a liquid or vapor
applied to or contacted to the support element. For example, in the
latter case, to produce a thin sheet of fluorescence reference
standard material with a continuous and uniform fluorescent
surface, the luminophores can be suspended in an appropriate
solution and spin coated onto the support element. For a glass
substrate, appropriately charged luminophores can be spin coated
and adhered onto the glass surface. For a more durable solution
having a plastic substrate, luminophores that are soluble, or
suspended, in organic solvents can be used. The appropriately
coated luminophores can be suspended in organic solvents and put
into a fluorinated polymer. In this case, the organic solvents can
be dried off, leaving the luminophores in the plastic polymer.
[0133] B. Photoluminescent-Glass-Based Reference Standards
[0134] This example describes exemplary photoluminescent glass
reference standards; see FIG. 11. Glasses, as used herein,
generally can include any super-cooled liquids or noncrystalline
solids, with intrinsic luminescence and/or with "added"
luminophores. The glass can be used as a continuous slab, alone or
in conjunction with a mask that limits excitation and/or emission
to discrete locations. Alternatively, or in addition, the glass can
be disposed at discrete locations, for example, in microplate
wells. Glasses can have a number of potential advantages, including
sensitivity (efficiency), durability (e.g., to temperatures above
100.degree. C.), wide excitation band, large Stokes' shift, and so
on. The number of bands and the relative intensities of the bands
can be adjusted by using suitable filter materials (just like with
the subtraction-based standards).
[0135] FIG. 11 shows an exemplary emission spectrum obtained with
Lumilass G9.TM. glass, a commercially available photoluminescent
glass (SUMITA Optical Glass, Inc.). Suitable photoluminescent
glasses can include (1) Lumilass R7.TM. glass, which can be excited
with ultraviolet light in the 200-420 nm range, and which emits red
fluorescence at about 610 nm, among others, (2) Lumilass G9 glass,
which can be excited with ultraviolet light in the range 200-390
nm, and which emits green florescence at about 540 nm, among
others, and (3) Lumilass B.TM. glass, which can be excited with
ultraviolet light in the range 200-400 nm, and which emits blue
fluorescence at about 405-410 nm, among others.
Example 3
Exemplary Light-Detection System
[0136] This example describes an exemplary light-detection system
1200, including an associated optical reference standard, in
accordance with aspects of the present teachings; see FIG. 12. This
exemplary embodiment is described with reference to specific
components; however, these components can be omitted or substituted
with other components, as appropriate or feasible.
[0137] Light-detection system 1200 includes (1) a light source 1202
(e.g., a vertically oriented tungsten halogen lamp), (2) a detector
1204 (e.g., a charge-coupled device (CCD) camera), and (3) an
optical relay structure 1206 for directing light from the light
source onto one or more samples 1208 disposed in wells 1210 of a
multiwell sample holder 1212 such as a microplate at an examination
site 1214, and from the sample(s) to the detector. The optical
relay structure, in the pictured embodiment, includes a (long-pass)
dichroic beamsplitter 1216 for separating and properly routing
excitation and emission light, and a fold mirror 1218 for turning
excitation and emission light toward and away from the samples,
respectively. The light-detection system further may include an
infrared-blocking "hot mirror" 1220 for filtering out infrared
light, an excitation filter 1222 for filtering out additional
unwanted (e.g., selected visible) excitation light, a Fresnel lens
1224 and well lenses 1226 for directing light onto the samples, and
an emission filter 1228 for filtering out stray excitation light
mixed with the emission light.
[0138] The light-detection system optionally includes and/or can be
used with an optical reference standard 1230. This reference
standard is positioned, in the pictured embodiment, in the optical
path between the fold mirror (1218) and the Fresnel lens (1224).
The reference standard, which resembles reference standard 300
(embodiment "a") in FIG. 3, includes a light-producing component
1232 and a light-blocking or mask component 1234. The reference
standard functions like a multi-well version of optical reference
standard 110 in FIG. 1.
Example 4
Miscellaneous Matters
[0139] This example describes miscellaneous aspects of
light-detection systems and optical reference standards, in
accordance with aspects of the present teachings.
[0140] The optical reference standard can be used for any suitable
purpose, consistent with these and/or other applications. These
purposes optionally can include, among others, (1) focusing images
of microplate wells onto a CCD camera by adjusting a focusing lens
for maximum fluorescence intensity from the reference standard, (2)
rotating a CCD camera so that pixel columns are parallel to the
rows or columns of the microplate by aligning the fluorescence
spectra from the reference standard, (3) calibrating the optics to
a known spectral wavelength (or wavelengths), (4) adjusting the
multiple components of a light detection system, such as lasers,
beam splitters, neutral density filters, and/or the like, (4)
verifying instrument performance (alignment and event size and
count) with specific sizes, and with specific numbers, of exemplary
fluorescent targets (particularly for cell counting devices), (5)
locating grids within a microfluidic array device, and/or (6)
correlating instrument response with the concentration of organic
dye. Elaboration on these purposes, and/or additional purposes, are
described elsewhere herein.
[0141] The optical reference standard may have any properties
suitable with its function. These properties optionally can
include, among others, (1) an ability to withstand temperatures
normally achieved in fluorescence detection systems, such as
real-time polymerase chain reaction (PCR) systems, such that it
does not melt at temperatures below about 110.degree. C., (2) few
and/or minor lot-to-lot variations in optical and physical
properties, (3) high-intensity narrow-emission peaks (e.g., full
width at half-max 15 nm) at the wavelengths detected by the
instrumentation, (4) uniform fluorescence emission spectra across
all discrete wells, or positions, or that are continuously uniform
across the entire homogenous surface, (5) stability for long
periods of time (e.g., 5 years), with little or no degradation from
photobleaching, and/or (6) low cost. The emission peaks can be
centered, in some embodiments, at about 520 nm corresponding to FAM
dye and at about 600 nm corresponding to ROX dye, when excited by a
488-nm wavelength light source, and/or in other embodiments at the
fluorescence wavelengths of Cy5 and Cy5.5 dyes, when excited by a
red laser, and/or so on.
[0142] Instrument performance for cell-counting instruments can be
validated using patterns of luminescent targets. Toward this end, a
mask containing the desired shape, size, and quantity of
fluorescence targets can be applied over the fluorescence reference
standard. Each target is merely a hole through the mask material,
which is opaque and non-fluorescing, thereby providing a clear
aperture of the desired shape and size to the fluorescence
reference standard material underneath. The mask can be an etched
or machined chip. The mask can include several wells that mimic the
wells of normally used microplates (or other sample holders), for
example, a 1536-well SBS standard microplate. A particular pattern
of targets can be contained within each well of the mask. The
particular pattern in a well will depend on its function, that is,
whether the standard is used for alignment, and/or to verify event
size and count.
[0143] The disclosure set forth above may encompass multiple
distinct inventions with independent utility. Although each of
these inventions has been disclosed in its preferred form(s), the
specific embodiments thereof as disclosed and illustrated herein
are not to be considered in a limiting sense, because numerous
variations are possible. The subject matter of the inventions
includes all novel and nonobvious combinations and subcombinations
of the various elements, features, functions, and/or properties
disclosed herein.
* * * * *