U.S. patent application number 17/165930 was filed with the patent office on 2021-06-17 for devices and methods for imaging biomolecules.
The applicant listed for this patent is Azure Biosystems, Inc.. Invention is credited to Diping Che, Zhefu Zhang.
Application Number | 20210181112 17/165930 |
Document ID | / |
Family ID | 1000005420022 |
Filed Date | 2021-06-17 |
United States Patent
Application |
20210181112 |
Kind Code |
A1 |
Che; Diping ; et
al. |
June 17, 2021 |
DEVICES AND METHODS FOR IMAGING BIOMOLECULES
Abstract
The present disclosure provides devices and methods enabling the
analysis of biomolecules. In some embodiments, the biomolecules may
be DNA, RNA, protein, peptide, small molecule, catalyst, precursor,
nucleotide, antibodies, or other biomolecules of interest.
Inventors: |
Che; Diping; (San Ramon,
CA) ; Zhang; Zhefu; (Beijing, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Azure Biosystems, Inc. |
Dublin |
CA |
US |
|
|
Family ID: |
1000005420022 |
Appl. No.: |
17/165930 |
Filed: |
February 2, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16368732 |
Mar 28, 2019 |
10989662 |
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17165930 |
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15880531 |
Jan 26, 2018 |
10281402 |
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16368732 |
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62450699 |
Jan 26, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 21/6428 20130101;
G01N 21/6458 20130101; G01N 33/60 20130101; G06T 7/90 20170101;
G06T 7/0012 20130101; G01N 33/582 20130101; G01N 2021/6441
20130101; G06T 2207/10064 20130101; G01N 2201/06113 20130101; G01J
3/4406 20130101; G01N 21/6456 20130101; G01N 21/6452 20130101; G06T
2207/10024 20130101; H04N 5/372 20130101; G01N 2021/6419 20130101;
C12Q 1/6816 20130101 |
International
Class: |
G01N 21/64 20060101
G01N021/64; C12Q 1/6816 20060101 C12Q001/6816; G01N 33/58 20060101
G01N033/58; G01N 33/60 20060101 G01N033/60; G06T 7/00 20060101
G06T007/00; G01J 3/44 20060101 G01J003/44 |
Claims
1. A detection instrument comprising a first optical path, wherein
the first optical path comprises: a laser light source; a pair of
beam splitters; an objective; an emission filter; a focusing lens;
and a first detector configured to detect light emitted or
reflected from the single substrate.
2. The detection instrument of claim 1, wherein the first optical
path further comprises a dual-band emission filter for
distinguishing between fluorescent emitted light and phosphorescent
emitted light.
3. The detection instrument of claim 1, wherein the first
wavelength is selected from the group consisting of: 488 nm, 520
nm, 658 nm, and 784 nm.
4. The detection instrument of claim 1 further comprising a
photomultiplier tube if the laser light source emits light at 488
nm or 520 nm.
5. The detection instrument of claim 1 further comprising an
avalanche photodiode if the laser light source emits light at 658
nm or 784 nm.
6. The detection instrument of claim 1, wherein the first optical
path is housed within a first scan head.
7. A biological imaging instrument configured to distinguish
between at least two types of molecules in a single substrate, the
biological imaging instrument comprising: a first excitation light
source for illuminating a substrate; a second excitation light
source for illuminating the substrate; a first detector for
detecting light reflected or emitted by the substrate from the
first excitation light source; and a second detector for detecting
light reflected or emitted by the substrate from the second
excitation light source.
8. The biological imaging instrument of claim 7, wherein the first
excitation light source is a laser or LED light.
9. The biological imaging instrument of claim 7, wherein the second
excitation light source is a laser or LED light.
10. The biological imaging instrument of claim 7, wherein the
detector is a CCD camera.
11. The biological imaging instrument of claim 7, wherein the CCD
camera is joined with an associated processor to isolate and scan
the portion of a substrate emitting or reflecting light from an LED
light source.
12. The biological imaging instrument of claim 7 further comprising
an objective through which light from the first excitation light
source and the second excitation light source pass before
illuminating the substrate.
13. The biological imaging instrument of claim 7 further comprising
a first pair of dichroic beam splitters through which light from
the first excitation light source passes before illuminating the
substrate.
14. The biological imaging instrument of claim 7 further comprising
a second pair of dichroic beam splitters through which light from
the second excitation light source passes before illuminating the
substrate.
15. The biological imaging instrument of claim 7 further comprising
a first focus lens through which light reflected from the substrate
passes before reaching the first detector.
16. The biological imaging instrument of claim 7 further comprising
a second focus lens through which light reflected from the
substrate passes before reaching the second detector.
17. The biological imaging instrument of claim 7 further comprising
a first emission filter through which light reflected from the
substrate passes before reaching the first detector.
18. The biological imaging instrument of claim 7 further comprising
a second emission filter through which light reflected from the
substrate passes before reaching the second detector.
19. The biological imaging instrument of claim 7 further comprising
a first cleanup filter through which light from the first
excitation light source passes before illuminating the
substrate.
20. The biological imaging instrument of claim 7 further comprising
a second cleanup filter through which light from the second
excitation light source passes before illuminating the substrate.
Description
PRIORITY CLAIM
[0001] This application is a continuation application of U.S.
patent application Ser. No. 16/368,732, filed on Mar. 28, 2019, and
entitled "DEVICES AND METHODS FOR IMAGING BIOMOLECULES," now U.S.
patent Ser. No. ______, which is a continuation of U.S. patent
application Ser. No. 15/880,531, filed on Jan. 26, 2018, now U.S.
Pat. No. 10,281,402, which claims priority to U.S. Provisional
Patent Application Ser. No. 62/450,699, filed Jan. 26, 2017, the
entire contents of each of which are incorporated herein by
reference and relied upon.
BACKGROUND
[0002] Spatially resolving biomolecules in two dimensions on a
substrate is an important tool in molecular biology. Components of
complex mixtures of biomolecules can be spatially resolved on a gel
or blot and imaged or scanned to measure levels of specific
biomolecules of interest. Likewise, biomolecules may be bound to an
array, or spatially segregated in wells of a microtiter plate and
may again be measured by imaging or scanning. Different methods of
labeling biomolecules require different measurement techniques to
read out the analysis.
[0003] One method of imaging fluorescent molecules is to use an
optical scan head, where an objective lens is linked to a light
source and detectors through a series of beam splitters and filters
designed to enable detection of a certain fluorophore or phosphor.
This directs light of a particular wavelength range to excite a
fluorophore or phosphor, and directs the emitted light selectively
through to a detector. This scan head may be moved along the
2-dimensional axes of the substrate in order to get a
high-resolution readout of fluorescence for the entire substrate.
Or in some cases, the substrate may be moved in 2-dimensions in
front of the objective.
[0004] It is not unusual to combine fluorescent and phosphorescent
detection into a single device. Having a dedicated instrument for
each technique can be expensive to acquire and maintain, and will
take up precious space in a laboratory. Having multiple readouts
creates a flexible instrument, also allows interrogation of
multiple biomolecules simultaneously. However, enabling the
detection of multiple "colors" of fluorescence and phosphor can
increase the number of scanning heads, adding complexity and cost
to the instrument, and can lead to reduced sensitivity if there is
overlap between the excitation and emission wavelengths of 2
fluorophores measured with the same optical scan head.
[0005] Scanning often involves placing a substrate on a scanning
bed that is considerably larger than the substrate. Scanning the
entire scan bed results in wasted time and data storage for the
scanning of the region surrounding the substrate.
[0006] A need therefore continues to exist for improved biological
substrate analyzers and methods of analyzing biological substrates.
The present disclosure meets this need.
SUMMARY
[0007] The present disclosure provides devices and methods enabling
the analysis of biomolecules. In particular, the instruments
disclosed herein enable convenient and efficient scanning and
imaging of a biological substrate using multiple wavelengths of
light (e.g., two or more of fluorescent, chemiluminescent,
colorimetric, and phosphor light). In some embodiments, the
biomolecules may be DNA, RNA, protein, peptide, small molecule,
catalyst, precursor, nucleotide, antibodies, or other biomolecules
of interest.
[0008] In some embodiments, the biomolecules will be contained on
or in a substrate. In some embodiments, these biomolecules will be
separated and encased in a gel. This gel can be made of any polymer
such as agarose, polyacrylamide, starch, or any other polymer which
can act as a sieving matrix or as a support, for the separation of
biomolecules. In some embodiments, the biomolecules will be
transferred from a gel to a membrane made of nitrocellulose,
polyvinylidene fluoride (PVDF), or other material capable of
non-specifically binding the biomolecules of interest, before the
imaging step. In some embodiments, the biomolecules will be in a
2-dimensional array, bound to the surface, or within a microtiter
plate.
[0009] In some embodiments biomolecules of interest will be labeled
with fluorescent moieties.
[0010] In some embodiments, an enzyme will be linked to the
biomolecules which can generate a chemiluminescent, fluorescent, or
chromogenic/colorimetric label. In some embodiments, biomolecules
will be directly labeled with radioactive atoms. In some
embodiments, a substrate containing radiolabeled biomolecules
placed on a photostimulable luminescence plate, or, phosphorimaging
screen, which can then be scanned. In some embodiments, labeled
antibodies will be used to selectively label biomolecules of
interest.
[0011] In some embodiments, more than one scanning or imaging
technique will be enabled in a single device. In some embodiments,
a single device will enable scanning fluorescent dyes, phosphor
imaging, optical densitometry, chemiluminescence, and colorimetric
analysis. In some embodiments, a single device will enable scanning
of four different fluorescent dyes simultaneously with 2 scan
heads, phosphor imaging, optical densitometry, chemiluminescence,
and colorimetric analysis.
[0012] In some embodiments, one optical scan head will be used to
detect multiple labels on a substrate. In some embodiments, a
dual-band emission filter will enable detection of light of
multiple wavelengths emitting from substrate. In some embodiments,
two fluorescent wavelengths can be scanned simultaneously with one
scan head. In some embodiments, a dual-band emission filter will
allow phosphor emission and fluorescence emission to be detected
with the same optical path and components. In some embodiments,
more than one optical scan head will be used.
[0013] In some embodiments, multiple scan heads will allow for
scanning several wavelengths at once. In some embodiments, 2 scan
heads will be used to scan up to 4 channels (e.g., four different
wavelengths) at once.
[0014] In some embodiments, the substrate will be placed on a
scanning bed that is larger than the substrate. In some
embodiments, a digital image will be taken of the scan bed and
analyzed to define the boundaries of the substrate before scanning
with optical scan head. In some embodiments, a digital image will
be acquired using a charge-coupled device (CCD), a complementary
metal-oxide semiconductor (CMOS), digital camera, or other
appropriate digital imaging technology.
[0015] In some embodiments, multiple types of detectors will be
used to optimize sensitivity. In some embodiments, a
photomultiplier tube (PMT) will be used for wavelength range
300-700 nm, avalanche photodiode (APD) for 650-900 nm, and CCD for
area, colorimetric, chemiluminescence from 400-900 nm.
[0016] In some embodiments, multiple types of light sources will be
used. In some embodiments lasers and LEDs will be used to
illuminate substrates.
[0017] In some embodiments, the present disclosure provides a
detection instrument capable of distinguishing between at least two
types of molecules in a single substrate, the detection instrument
comprising: a first optical path comprising a laser light source
and a first detector for scanning the substrate with light having a
first wavelength; and a second optical path comprising an LED light
source and a second detector for capturing an image of the
substrate at visible wavelengths.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0019] FIG. 1 shows a scan head optical path for simultaneous
excitation and detection of two fluorescent labels according to one
embodiment of the present disclosure.
[0020] FIG. 2 shows a schematic of a scanning device consistent
with one embodiment of the present disclosure.
[0021] FIG. 3 shows a scan head optic setup, and wavelength
profile, capable of scanning 2 fluorescent dyes simultaneously,
according to one embodiment of the present disclosure.
[0022] FIG. 4 shows a scan head optic setup, and wavelength
profile, capable of phosphorimaging or scanning 2 fluorescent dyes
simultaneously, according to one embodiment of the present
disclosure.
[0023] FIG. 5 shows a scan head optical path for simultaneous
excitation and detection of two fluorescent labels according to one
embodiment of the present disclosure.
[0024] FIG. 6 shows a scan head optical path for illumination by
LED and detection by CCD camera of a substrate placed on a glass
bed according to one embodiment of the present disclosure.
[0025] FIG. 7A shows a schematic view of a scanning instrument
comprising an LED-illuminated CCD camera and at least one
laser-illuminated scan head consistent with one embodiment of the
present disclosure.
[0026] FIG. 7B shows a schematic view of the scanning instrument of
FIG. 7A wherein the at least one laser-illuminated scan head has
been retracted to enable imaging by the LED-illuminated CCD
camera.
[0027] FIG. 8A shows an image obtained from simultaneous detection
of two fluorescent dyes (Cy3 and Cy5) from a two-dimensional
electrophoresis gel consistent with one embodiment of the present
disclosure.
[0028] FIG. 8B shows an image obtained from simultaneous detection
of chemiluminescent and markers from an electrophoresis gel
consistent with one embodiment of the present disclosure.
[0029] FIG. 8C shows an image obtained from simultaneous detection
of two NIR dyes, one detectable at 700 nm and the other detectable
at 800 nm, from a Western blot consistent with one embodiment of
the present disclosure.
[0030] FIG. 8D shows images obtained from detection of Coomassie
dye from an electrophoresis gel consistent with one embodiment of
the present disclosure.
[0031] FIG. 8E shows an image obtained from detection of ethidium
bromide dye from a DNA gel consistent with one embodiment of the
present disclosure.
[0032] FIG. 8F shows an image obtained from detection of
phosphorimaging dye from an electrophoresis gel consistent with one
embodiment of the present disclosure.
[0033] FIG. 8G shows an image obtained from simultaneous detection
of three fluorescent dyes, one detectable at 520 nm, one detectable
at 658 nm, and one detectable at 785 nm, from an in-cell Western
gel consistent with one embodiment of the present disclosure.
[0034] FIG. 8H shows an image obtained from simultaneous detection
of four fluorescent dyes from a Western blot gel consistent with
one embodiment of the present disclosure.
[0035] FIG. 8I shows an image obtained from simultaneous detection
of two fluorescent dyes, one detectable at 520 nm and one
detectable at 658 nm, from a microarray consistent with one
embodiment of the present disclosure.
[0036] FIG. 8J shows an image obtained from simultaneous detection
of three tissue dyes from a rat brain cross-sectional sample
consistent with one embodiment of the present disclosure.
[0037] FIG. 8K shows an image obtained from simultaneous detection
of two tissue dyes from a pelagorium stem cross-sectional sample
consistent with one embodiment of the present disclosure.
[0038] FIG. 8L shows an image obtained from simultaneous detection
of two fluorescent dyes, one detectable at 488 nm and the other
detectable at 785 nm, from a bee head sample according to one
embodiment of the present disclosure.
[0039] FIG. 8M shows an image obtained from simultaneous detection
of three fluorescent dyes, one detectable at 488 nm, one detectable
at 520 nm, and one detectable at 658 nm, from a chicken liver
tissue sample consistent with one embodiment of the present
disclosure.
[0040] FIG. 8N shows an image obtained from simultaneous detection
of two fluorescent dyes, one detectable at 658 nm and the other
detectable at 785 nm, from a mixed tissue sample consistent with
one embodiment of the present disclosure.
[0041] The figures depict various embodiments of this disclosure
for purposes of illustration only. One skilled in the art will
readily recognize from the following discussion that alternative
embodiments of the structures and methods illustrated herein may be
employed without departing from the principles of embodiments
described herein.
DETAILED DESCRIPTION
[0042] Biomolecule imaging instruments disclosed herein offer
convenient multiplex imaging of biological substrates heretofore
not possible using conventional substrate imaging technologies. In
some embodiments, a biomolecule imaging instrument disclosed herein
comprises a scanner and an large-area imager that, collectively,
are capable of scanning and imaging a biological substrate that
includes a plurality of differently-labeled biomolecules. In some
embodiments, the biomolecule imaging instrument comprises a display
for viewing a composite image comprising a plurality of individual
images of a substrate, wherein each individual image includes
visual information corresponding to a single type of labeled
biomolecule.
[0043] In other embodiments, the instrument causes a separate
display device to display a composite image comprising a plurality
of individual images of a substrate, wherein each individual image
includes visual information corresponding to a single type of
labeled biomolecule. In some embodiments, each individual image is
displayed in a single unique color in the displayed composite
image.
[0044] The biomolecule imaging instruments disclosed herein are
capable of distinguishing multiple types of labeled biomolecules.
For example, in some embodiments, a biomolecule imaging instrument
disclosed herein can distinguish between molecules is labeled with
two, three, or four types of biomolecules wherein each type of
biomolecule is labeled with a different fluorescent tag.
[0045] In some embodiments, a biomolecule imaging instrument
disclosed herein can distinguish between a first type of molecule
in a substrate and a second type of molecule in the substrate. In
some embodiments, the first type of molecule in the substrate is
labeled with a fluorescent label, and the second type of molecule
in the substrate is selected from the group consisting of:
molecules labeled with a second, different fluorescent label,
chemiluminescent molecules, colorimetric molecules, and
phosphorescent molecules. In some embodiments, the first type of
molecule in the substrate is a chemiluminescent molecule and the
second type of molecule in the substrate is a colorimetric molecule
or a phosphorescent molecule. In some embodiments, the first type
of molecule is a colorimetric molecule and the second type of
molecule is a phosphorescent molecule.
[0046] In some embodiments, a biomolecule imaging instrument
disclosed herein can distinguish between a first type of molecule
in a substrate, a second type of molecule in the substrate, and a
third type of molecule in a substrate. In some embodiments, the
first type of molecule is a molecule labeled with a first
fluorescent label, the second type of molecule is a molecule
labeled with a second, different fluorescent label, and the third
type of molecule is a molecule labeled with a third, different
fluorescent label. In some embodiments, the first type of molecule
is a molecule labeled with a first fluorescent label, the second
type of molecule is a molecule labeled with a second, different
fluorescent label, and the third type of molecule is a
chemiluminescent molecule. In some embodiments, the first type of
molecule is a molecule labeled with a first fluorescent label, the
second type of molecule is a molecule labeled with a second,
different fluorescent label, and the third type of molecule is a
colorimetric molecule. In some embodiments, the first type of
molecule is a molecule labeled with a first fluorescent label, the
second type of molecule is a molecule labeled with a second,
different fluorescent label, and the third type of molecule is a
phosphorescent molecule. In some embodiments, the first type of
molecule is a molecule labeled with a fluorescent label, the second
type of molecule is a chemiluminescent molecule, and the third type
of molecule is a colorimetric molecule. In some embodiments, the
first type of molecule is a molecule labeled with a fluorescent
label, the second type of molecule is a chemiluminescent molecule,
and the third type of molecule is a phosphorescent molecule. In
some embodiments, the first type of molecule is a molecule labeled
with a fluorescent label, the second type of molecule is a
colorimetric molecule, and the third type of molecule is a
phosphorescent molecule. In some embodiments, the first type of
molecule is a chemiluminescent molecule, the second type of
molecule is a colorimetric molecule, and the third type of molecule
is a phosphorescent molecule.
[0047] In some embodiments, a biomolecule imaging instrument
disclosed herein can distinguish between a first type of molecule
in a substrate, a second type of molecule in the substrate, a third
type of molecule in a substrate, and a fourth type of molecule in
the substrate. In some embodiments, the first type of molecule is a
molecule labeled with a first fluorescent label, the second type of
molecule is a molecule labeled with a second, different fluorescent
label, the third type of molecule is a molecule labeled with a
third, different fluorescent label, and the fourth type of molecule
is a molecule labeled with a fourth, different fluorescent label.
In some embodiments, the first type of molecule is a molecule
labeled with a first fluorescent label, the second type of molecule
is a molecule labeled with a second, different fluorescent label,
the third type of molecule is a molecule labeled with a third,
different fluorescent label, and the fourth type of molecule is a
chemiluminescent molecule. In some embodiments, the first type of
molecule is a molecule labeled with a first fluorescent label, the
second type of molecule is a molecule labeled with a second,
different fluorescent label, the third type of molecule is a
molecule labeled with a third, different fluorescent label, and the
fourth type of molecule is a colorimetric molecule. In some
embodiments, the first type of molecule is a molecule labeled with
a first fluorescent label, the second type of molecule is a
molecule labeled with a second, different fluorescent label, the
third type of molecule is a molecule labeled with a third,
different fluorescent label, and the fourth type of molecule is a
phosphorescent molecule. In some embodiments, the first type of
molecule is a molecule labeled with a first fluorescent label, the
second type of molecule is a molecule labeled with a second,
different fluorescent label, the third type of molecule is a
chemiluminescent molecule, and the fourth type of molecule is a
colorimetric molecule. In some embodiments, the first type of
molecule is a molecule labeled with a first fluorescent label, the
second type of molecule is a molecule labeled with a second,
different fluorescent label, the third type of molecule is a
chemiluminescent molecule, and the fourth type of molecule is a
phosphorescent molecule. In some embodiments, the first type of
molecule is a molecule labeled with a fluorescent label, the second
type of molecule is a chemiluminescent molecule, the third type of
molecule is a colorimetric molecule, and the fourth type of
molecule is a phosphorescent molecule.
[0048] In some embodiments, a biomolecule imaging instrument
disclosed herein can distinguish between a first type of molecule
in a substrate, a second type of molecule in the substrate, a third
type of molecule in a substrate, a fourth type of molecule in the
substrate, and a fifth type of molecule in the substrate. In some
embodiments, the first type of molecule is a molecule labeled with
a first fluorescent label, the second type of molecule is a
molecule labeled with a second, different fluorescent label, the
third type of molecule is a molecule labeled with a third,
different fluorescent label, the fourth type of molecule is a
molecule labeled with a fourth, different fluorescent label, and
the fifth type of molecule is a chemiluminescent molecule. In some
embodiments, the first type of molecule is a molecule labeled with
a first fluorescent label, the second type of molecule is a
molecule labeled with a second, different fluorescent label, the
third type of molecule is a molecule labeled with a third,
different fluorescent label, the fourth type of molecule is a
molecule labeled with a fourth, different fluorescent label, and
the fifth type of molecule is a colorimetric molecule. In some
embodiments, the first type of molecule is a molecule labeled with
a first fluorescent label, the second type of molecule is a
molecule labeled with a second, different fluorescent label, the
third type of molecule is a molecule labeled with a third,
different fluorescent label, the fourth type of molecule is a
molecule labeled with a fourth, different fluorescent label, and
the fifth type of molecule is a phosphorescent molecule.
[0049] In some embodiments, a biomolecule imaging instrument
disclosed herein can distinguish between a first type of molecule
in a substrate, a second type of molecule in the substrate, a third
type of molecule in a substrate, a fourth type of molecule in the
substrate, a fifth type of molecule in the substrate, and a sixth
type of molecule in the substrate. In some embodiments, the first
type of molecule is a molecule labeled with a first fluorescent
label, the second type of molecule is a molecule labeled with a
second, different fluorescent label, the third type of molecule is
a molecule labeled with a third, different fluorescent label, the
fourth type of molecule is a molecule labeled with a fourth,
different fluorescent label, the fifth type of molecule is a
chemiluminescent molecule, and the sixth type of molecule is a
colorimetric molecule. In some embodiments, the first type of
molecule is a molecule labeled with a first fluorescent label, the
second type of molecule is a molecule labeled with a second,
different fluorescent label, the third type of molecule is a
molecule labeled with a third, different fluorescent label, the
fourth type of molecule is a molecule labeled with a fourth,
different fluorescent label, the fifth type of molecule is a
chemiluminescent molecule, and the sixth type of molecule is a
phosphorescent molecule. In some embodiments, the first type of
molecule is a molecule labeled with a first fluorescent label, the
second type of molecule is a molecule labeled with a second,
different fluorescent label, the third type of molecule is a
molecule labeled with a third, different fluorescent label, the
fourth type of molecule is a molecule labeled with a fourth,
different fluorescent label, the fifth type of molecule is a
colorimetric molecule, and the sixth type of molecule is a
phosphorescent molecule.
[0050] In some embodiments, a biomolecule imaging instrument
disclosed herein can distinguish between a first type of molecule
in a substrate, a second type of molecule in the substrate, a third
type of molecule in a substrate, a fourth type of molecule in the
substrate, a fifth type of molecule in the substrate, a sixth type
of molecule in the substrate, and a seventh type of molecule in the
substrate. In some embodiments, the first type of molecule is a
molecule labeled with a first fluorescent label, the second type of
molecule is a molecule labeled with a second, different fluorescent
label, the third type of molecule is a molecule labeled with a
third, different fluorescent label, the fourth type of molecule is
a molecule labeled with a fourth, different fluorescent label, the
fifth type of molecule is a chemiluminescent molecule, the sixth
type of molecule is a colorimetric molecule, and the seventh type
of molecule is a phosphorescent molecule.
[0051] FIG. 1 discloses a representative diagram of the optical
path for detection of biomolecules by a scan head. In a typical
mode of using the scan head, a substrate S is scanned using the
optical scan head 100. Light from light source 118, is reflected
off beam splitter 114, and then beam splitter 112, before being
focused through objective 102, onto substrate S. In the case where
the substrate is labeled with a fluorescent molecule, the
fluorescent molecule is excited by the laser light, and fluoresces,
emitting light of a different wavelength than the laser. This light
can pass through objective 102, be reflected by beam splitter 112,
and then pass-through beam splitter 114, which is selected to
reflect light of the laser wavelength, and allow light of the
fluorescent emission wavelength to pass through. An emission filter
108 is used to eliminate light not coming from the intended
fluorophore. In some embodiments, the emission filter 108 is a
dual-band emission filter that eliminates light not coming from
either of two intended fluorophores. Lastly, the fluorescent
emission light (e.g., one or two fluorescent wavelengths of light)
is focused through focusing lens 104 onto detector 122.
[0052] Likewise, a second fluorophore can be read with the same
scan head by light from a second light source 120, reflecting off
beam splitter 116, and passing through beam splitter 112 before
being focused through objective 102 and exciting a second
fluorophore on substrate S. Light emitted from this second
fluorophore will pass through the objective 102, pass through beam
splitter 112 and beam splitter 116 before finally passing through
emission filter 110, focusing lens 106 and onto detector 124.
[0053] By selecting appropriate beam splitters 112, 114, and 116
two fluorophores may be read simultaneously using one scanning
head. In some embodiments, the optical paths for two fluorophores
have minimal overlap in emission spectra so cross-talk between the
emitted fluorescent light wavelengths is minimized.
[0054] FIG. 2 discloses a representative diagram of a biomolecule
scanner 200. A substrate, 202, is placed onto the scan bed 204. The
substrate can be interrogated several different ways. The CCD 206
can be used to image the substrate using a particular color of LED
210 (colorimetric/optical densitometry), in darkness
(chemiluminescence) or in white or colored light to establish
substrate position on bed before scanning. The dual scan head 208
shown can scan 4 different fluorescent dyes simultaneously, or can
be used for scanning a phosphorimaging screen. In some embodiments,
the CCD 206 is used to identify a region of interest within the
area of the scan bed 204 that requires scanning. For example, in
some embodiments, the CCD 206 captures an image (e.g., a
low-resolution image) of the entire scan bed 204, and an associated
processor identifies a smaller region of the scan bed 204 that is
emitting light or reflecting light from the LED 210. The dual scan
head 208 then performs a scan of the identified smaller region of
the scan bed 204.
[0055] FIG. 3 provides a representative example of selecting
filters, beam splitters, light sources and detectors to enable two
color scanning in a single optical scan head. In this case,
components were selected to scan "green excitable" dyes, which can
be excited by a 525 nm light source and emit light at a wavelength
between 555 to 585 nm, and near-infra-red dyes ("NIR excitable")
which can be excited by a 780 nm light source and emit light at a
wavelength between 805 to 855 nm. Optical components can of course
be selected to allow detection of any two fluorophores which do not
overlap in excitation or emission wavelength.
[0056] FIG. 4 provides a representative example of selecting
filters, beam splitters, light sources and detectors to enable both
phosphor imaging and two color scanning in a single optical scan
head. In this case, components were selected to scan "red
excitable" dyes, which can be excited by a 635 nm light source and
emit light at a wavelength between 675 to 735 nm, and "blue
excitable" dyes which can be excited by a 473 nm light source and
emit light at a wavelength between 495 to 525 nm. By selecting
appropriate filters and beam splitters, this same optical head can
be used to read phosphorimaging plates which can be excited using
an 635 nm light source and emit light at a wavelength between 370
and 410 nm.
[0057] FIG. 5 discloses a representative diagram of the optical
path for detection of biomolecules by a scan head 550. In a typical
mode of using the scan head 550, a substrate S is scanned by light
from light source 518, which may pass through a cleanup filter 528,
reflect off beam splitter 514, and then beam splitter 512, before
being focused through objective 502, onto substrate S. In the case
where the substrate is labeled with a fluorescent molecule, the
fluorescent molecule is excited by the laser light, and fluoresces,
emitting light of a different wavelength than the laser. This light
can pass through objective 502, be reflected by beam splitter 512,
and then pass-through beam splitter 514, which is selected to
reflect light of the laser wavelength, and allow light of the
fluorescent emission wavelength to pass through. An emission filter
508 is used to eliminate light not coming from the intended
fluorophore. Lastly, the fluorescent emission light is focused
through focusing lens 504 onto detector 522.
[0058] Likewise, a second fluorophore can be read with the same
scan head by light from a second light source 520, which passes
through a cleanup filter 526 reflects off beam splitter 516, and
passes through beam splitter 512 before being focused through
objective 502 and exciting a second fluorophore on substrate 501.
Light emitted from this second fluorophore will pass through the
objective 502, pass through beam splitter 512 and beam splitter 516
before finally passing through emission filter 510, focusing lens
506 and onto detector 524.
[0059] By selecting appropriate beam splitters 512, 514, and 516
two fluorophores may be read simultaneously using one scanning
head. By designing the optical path for two fluorophores that have
minimal overlap in emission spectrum, cross-talk will be
minimized.
[0060] In some embodiments, light source 518 is a laser that emits
light at 520 nm (e.g., PL-520, OSRAM Opto Semiconductors GmbH), and
light source 520 is a laser that emits light at 784 nm (e.g.,
GH0781 RA2c, Sharp). In such embodiments, the objective 502 may
have a focus length of 20 mm (e.g., GCL-010612, Daheng). In such
embodiments, the dichroic beam splitter 516 may be a single-edge
laser-flat dichroic beamsplitter (e.g., Di02-R785, Semrock), the
dichroic beam splitter 514 may be a single-edge laser-flat dichroic
beamsplitter (e.g., FF552-Di02, Semrock), and the dichroic beam
splitter 512 may be a single-edge laser-flat dichroic beamsplitter
(e.g., FF757-Di01, Semrock). The cleanup filter 528 may be a
single-band bandpass filter (e.g., FF01-514/30, Semrock), while
cleanup filter 526 may be a single-band bandpass filter (e.g.,
FF01-769/41, Semrock). Emission filter 508 may be a single-band
bandpass filter (e.g., FF01-565/24, Semrock), while emission filter
510 may be a single-band bandpass filter (e.g., FF01-832/37,
Semrock). Detector 522 in such embodiments, may be an avalanche
photodiode (e.g., S12023-10, Hamamatsu), while detector 524 may be
an avalanche photodiode that is the same or different than detector
522 (e.g., S12023-10, Hamamatsu). Focus lens 504, 506 may be a
plano-convex lens having a focal length of 50 mm (e.g., GCL-010107,
Daheng). The scan bed 599 may be a glass scan bed of suitable
thickness, such as 5 mm (e.g., BK7 glass, Glass Dynamics LLC).
[0061] In some embodiments, light source 518 is a laser that emits
light at 520 nm (e.g., FL-520, OSRAM Opto Semiconductors GmbH), and
light source 520 is a laser that emits light at 658 nm (e.g.,
ML101J25, Mitsubishi). In such embodiments, the objective 502 may
have a focus length of 20 mm (e.g., GCL-010612, Daheng). In such
embodiments, the dichroic beam splitter 516 may be a single-edge
laser-flat dichroic beamsplitter (e.g., FF677-Di01, Semrock), the
dichroic beam splitter 514 may be a single-edge laser-flat dichroic
beamsplitter (e.g., BCOME-0015, Semrock), and the dichroic beam
splitter 512 may be a single-edge laser-flat dichroic beamsplitter
(e.g., FF593-Di03, Semrock). The cleanup filter 528 may be a
single-band bandpass filter (e.g., FF01-475/28, Semrock), while
cleanup filter 526 may be a single-band bandpass filter (e.g., 658
nm short pass, Filtech Photonics). Emission filter 508 may be a
single-band bandpass filter (e.g., BCOME-0016, Semrock), while
emission filter 510 may be a single-band bandpass filter (e.g.,
FF01-710/40, Semrock). Detector 522 in such embodiments, may be a
photomultiplier tube (e.g., H10721-110, Hamamatsu), while detector
524 may be an avalanche photodiode (e.g., S12023-10, Hamamatsu).
Focus lenses 504,506 may be a plano-convex lens having a focal
length of 50 mm (e.g., GCL-010107, Daheng). The scan bed 599 may be
a glass scan bed of suitable thickness, such as 5 mm (e.g., BK7
glass, Glass Dynamics LLC).
[0062] Referring now to FIG. 6, a biomolecule scanner 600
consistent with the present disclosure comprises a scan bed 699, an
LED light source 610, mirror 602, a lens 604, and a CCD camera 606.
A substrate, S, is placed onto the scan bed 699. LED light emitted
by the LED light source 610 penetrates the scan bed 699 to
illuminate the substrate S. The LED light reflected from the
substrate S then reflects off of the mirror 602. The lens 604
focuses the reflected light to the CCD camera 606 for capture. The
biomolecule scanner 600 can be used to image the substrate S using
a particular color of LED 610 (colorimetric/optical densitometry),
in darkness (chemiluminescence) or in white or colored light to
establish substrate position on bed before scanning. In some
embodiments, the lens 604 has a focal length of 25 mm and maximum
aperture of f/0.95 (e.g., Nokton 25 mm, f/0.95, Voigtlander), and
the camera 606 is configured to capture at least 4 million pixels
per scan, such as at least 4 million pixels, at least 5 million
pixels, or at least 6 million pixels per image. In some
embodiments, the CCD camera 606 is used to identify a region of
interest within the area of the scan bed 699 that requires
scanning. For example, in some embodiments, the CCD camera 606
captures an image (e.g., a low-resolution image) of the entire scan
bed 699, and an associated processor identifies a smaller region of
the scan bed 699 that is emitting light or reflecting light from
the LED 610. The biomolecule scanner 600 then performs a scan of
the identified smaller region of the scan bed 699.
[0063] As shown representatively in FIGS. 7A-7B, the present
disclosure provides a scanning instrument 700 comprising one or
more laser-illuminated scan heads 100/550, an LED-illuminated
Camera 750, a transparent scan bed 799, and an enclosure 780. In
some embodiments, the one or more laser-illuminated scan heads
100/550 is consistent with optical scan head 100 described above
and shown representatively in FIG. 1. In other embodiments, the one
or more laser-illuminated scan heads 100/550 is consistent with
optical scan head 550 described above and shown representatively in
FIG. 5.
[0064] In some embodiments, the LED-illuminated camera head 750
comprises an LED light source 710, a mirror 702, a camera lens 704,
and a camera 706. In operation, the one or more laser-illuminated
scan heads 100/550 is moved in line with the substrate S, as shown
in FIG. 7A. The substrate S is then scanned with the one or more
laser-illuminated scan heads 100/500 as described above with
respect to FIG. 1 or FIG. 5.
[0065] Once the one or more laser scan heads 100/550 have completed
scanning the substrate S, the one or more laser scan heads 100/550
is moved out of line with the substrate S as shown in FIG. 7B, so
that LED light from the LED light source 710 is not blocked by the
one or more laser-illuminated scan heads 100/550.
[0066] Scanning of the substrate S by the LED camera head 750 then
proceeds. LED light emitted by the LED light source 710 penetrates
the scan bed 799 to illuminate the substrate S. The LED light
reflected from the substrate S then reflects off of the mirror 702.
The lens 704 focuses the reflected light to the CCD camera 706 for
capture. In this mode, the scanning instrument 700 can be used to
image the substrate S using a particular color of LED 710
(colorimetric/optical densitometry), in darkness
(chemiluminescence) or in white or colored light to establish
substrate position on bed before scanning. In some embodiments, the
lens 704 has a focal length of 25 mm and maximum aperture of f/0.95
(e.g., Nokton 25 mm, f/0.95, Voigtlander), and the camera 706 is
configured to capture at least 4 million pixels per scan, such as
at least 4 million pixels, at least 5 million pixels, or at least 6
million pixels per scan.
[0067] In other embodiments, scanning of the substrate S by the LED
scan head 750 occurs first, followed by scanning by the one or more
laser-illuminated scan heads 100/550. In such embodiments, the one
or more laser-illuminated scan heads 100/550 is moved out of line
with the substrate S as shown in FIG. 7B, so that LED light from
the LED light source 710 is not blocked by the one or more
laser-illuminated scan heads 100/550. LED light emitted by the LED
light source 710 penetrates the scan bed 799 to illuminate the
substrate S. The LED light reflected from the substrate S then
reflects off of the mirror 702. The lens 704 focuses the reflected
light to the CCD camera 706 for capture. In this mode, the scanning
instrument 700 can be used to image the substrate S using a
particular color of LED 710 (colorimetric/optical densitometry), in
darkness (chemiluminescence) or in white or colored light to
establish substrate position on bed before scanning. In some
embodiments, the lens 704 has a focal length of 25 mm and maximum
aperture of f/0.95 (e.g., Nokton 25 mm, f/0.95, Voigtlander), and
the camera 706 is configured to capture at least 4 million pixels
per scan, such as at least 4 million pixels, at least 5 million
pixels, or at least 6 million pixels per scan.
[0068] Thereafter, the LED light source 710 is turned off, and
scanning of the substrate S by the one or more laser-illuminated
scan heads 100/550 proceeds. The one or more laser-illuminated scan
heads 100/550 is moved in line with the substrate S, as shown in
FIG. 7A. The substrate S is then scanned with the one or more
laser-illuminated scan heads 100/500 as described above with
respect to FIG. 1 or FIG. 5.
[0069] In some embodiments, a method of imaging a biological sample
comprises placing the sample on a scanning bed of an imaging
instrument consistent with the present disclosure, illuminating the
biological sample with a first light having a first wavelength,
capturing a first light emitted from the biological sample,
illuminating the biological sample with a second light having a
second wavelength, and capturing a second light emitted from the
biological sample, wherein the biological sample is not moved after
capturing the first light emitted from the biological sample and
before illuminating the biological sample with the second light. In
some embodiments, the method further comprises combining a pattern
from the first captured light with a pattern from the second
captured light to form a composite image.
[0070] In some embodiments, a method of imaging a biological sample
comprises placing the sample on a scanning bed of an imaging
instrument consistent with the present disclosure, illuminating the
biological sample with a first light having a first wavelength,
capturing a first light emitted from the biological sample,
illuminating the biological sample with a second light having a
second wavelength, capturing a second light emitted from the
biological sample, illuminating the biological sample with a third
light having a third wavelength, and capturing a third light
emitted from the biological sample, wherein the biological sample
is not moved after capturing the first light emitted from the
biological sample and before illuminating the biological sample
with the second light, and wherein the biological sample is not
moved after capturing the second light emitted from the biological
sample and before illuminating the biological sample with the third
light. In some embodiments, the method further comprises combining
a pattern from the first captured light with a pattern from the
second captured light and with a pattern from the third captured
light to form a composite image.
[0071] In some embodiments, a method of imaging a biological sample
comprises placing the sample on a scanning bed of an imaging
instrument consistent with the present disclosure, illuminating the
biological sample with a first light having a first wavelength,
capturing a first light emitted from the biological sample,
illuminating the biological sample with a second light having a
second wavelength, capturing a second light emitted from the
biological sample, illuminating the biological sample with a third
light having a third wavelength, capturing a third light emitted
from the biological sample, illuminating the biological sample with
a fourth light having a fourth wavelength, and capturing a fourth
light emitted from the biological sample, wherein the biological
sample is not moved after capturing the first light emitted from
the biological sample and before illuminating the biological sample
with the second light, wherein the biological sample is not moved
after capturing the second light emitted from the biological sample
and before illuminating the biological sample with the third light,
and wherein the biological sample is not moved after capturing the
third light emitted from the biological sample and before
illuminating the biologicals sample with the fourth light. In some
embodiments, the method further comprises combining a pattern from
the first captured light with a pattern from the second captured
light, with a pattern from the third captured light, and with a
pattern from the fourth captured light to form a composite
image.
[0072] In some embodiments, the present disclosure provides a
composite image comprising a first pattern having a first color and
a second pattern having a second color, wherein the first pattern
corresponds to a pattern of first labeled biomolecules present in a
2D or 3D biological sample, and wherein the second pattern
corresponds to a pattern of second labeled biomolecules present in
the 2D or 3D biological sample, wherein the first labeled
biomolecules and the second labeled biomolecules are different. In
some embodiments, the first labeled biomolecules are excitable at a
first wavelength of light and the second labeled biomolecules are
excitable at a second, different wavelength of light. The images
shown in FIGS. 8A, 8B, 8C, 8I, 8K, 8L and 8N are each consistent
with such embodiments.
[0073] In some embodiments, the present disclosure provides a
composite image comprising a first pattern having a first color, a
second pattern having a second color, and a third pattern having a
third color, wherein the first pattern corresponds to a pattern of
first labeled biomolecules present in a 2D or 3D biological sample,
the second pattern corresponds to a pattern of second labeled
biomolecules present in the 2D or 3D biological sample, and wherein
the third pattern corresponds to a pattern of third labeled
biomolecules present in the 2D or 3D biological sample, wherein the
first labeled biomolecules, the second labeled biomolecules, and
the third labeled biomolecules are each different from each other.
In some embodiments, the first labeled biomolecules are excitable
at a first wavelength of light, the second labeled biomolecules are
excitable at a second, different wavelength of light, and the third
labeled biomolecules are excitable at a third, different wavelength
of light. The images shown in FIGS. 8G, 8J and 8M are each
consistent with such embodiments.
[0074] In some embodiments, the present disclosure provides a
composite image comprising a first pattern having a first color, a
second pattern having a second color, a third pattern having a
third color, and a fourth pattern having a fourth color, wherein
the first pattern corresponds to a pattern of first labeled
biomolecules present in a 2D or 3D biological sample, the second
pattern corresponds to a pattern of second labeled biomolecules
present in the 2D or 3D biological sample, the third pattern
corresponds to a pattern of third labeled biomolecules present in
the 2D or 3D biological sample, and the fourth pattern corresponds
to a pattern of fourth labeled biomolecules present in the 2D or 3D
biological sample, wherein the first labeled biomolecules, the
second labeled biomolecules, the third labeled biomolecules, and
fourth labeled biomolecules are each different from each other. In
some embodiments, the first labeled biomolecules are excitable at a
first wavelength of light, the second labeled biomolecules are
excitable at a second, different wavelength of light, the third
labeled biomolecules are excitable at a third, different wavelength
of light, and the fourth labeled biomolecules are excitable at a
fourth, different wavelength of light. The image shown in FIG. 8H
is consistent with such an embodiment.
[0075] In some embodiments, the present disclosure provides a
detection instrument comprising a first excitation light source for
illuminating a substrate; a second excitation light source for
illuminating the substrate; a first detector for detecting light
reflected or emitted by the substrate from the first excitation
light source; and a second detector for detecting light reflected
or emitted by the substrate from the second excitation light
source. In some embodiments, the detection instrument further
comprises an objective through which light from the first
excitation light source and the second excitation light source pass
before illuminating the substrate. In some embodiments, the
detection instrument further comprises a first pair of dichroic
beam splitters through which light from the first excitation light
source passes before illuminating the substrate. In some
embodiments, the detection instrument further comprises a second
pair of dichroic beam splitters through which light from the second
excitation light source passes before illuminating the substrate.
In some embodiments, the detection instrument further comprises a
first focus lens through which light reflected from the substrate
passes before reaching the first detector. In some embodiments, the
detection instrument further comprises a second focus lens through
which light reflected from the substrate passes before reaching the
second detector. In some embodiments, the detection instrument
further comprises a first emission filter through which light
reflected from the substrate passes before reaching the first
detector. In some embodiments, the detection instrument further
comprises a second emission filter through which light reflected
from the substrate passes before reaching the second detector. In
some embodiments, the detection instrument further comprises a
first cleanup filter through which light from the first excitation
light source passes before illuminating the substrate. In some
embodiments, the detection instrument further comprises a second
cleanup filter through which light from the second excitation light
source passes before illuminating the substrate.
[0076] In some embodiments, the present disclosure provides a
method of simultaneously detecting multiple fluorescent dyes in a
biological substrate, the method comprising: contacting a substrate
with more than one fluorescent dye; simultaneously illuminating the
substrate with laser light comprising at least two wavelengths; and
detecting an intensity of at least two wavelengths of light
reflected from the substrate. In some embodiments, the method
further comprises passing the laser light through at least two
dichroic beam splitters before illuminating the substrate. In some
embodiments, the method further comprises passing the laser light
through an objective before illuminating the substrate. In some
embodiments, the light reflected from the substrate passes through
at least two emission filters before reaching at least two
detectors. In some embodiments, the method further comprises
passing the laser light through at least two cleanup filters before
illuminating the substrate. In some embodiments, the light
reflected from the substrate passes through at least two focus
lenses before reaching at least two detectors. In some embodiments,
the step of contacting the substrate comprises contacting the
substrate with at least two dyes selected from the group consisting
of: cyanine 3 (Cy3), cyanine 5 (Cy5), a near infrared (NIR) dye,
Coomassie blue dye, ethidium bromide, and phosphor-imaging dye. In
some embodiments, the substrate is an electrophoresis gel. In some
embodiments, the method comprises simultaneously illuminating the
substrate with laser light comprising two wavelengths; and
detecting an intensity of two wavelengths of light reflected from
the substrate. In some embodiments, the method comprises
simultaneously illuminating the substrate with laser light
comprising three wavelengths; and detecting an intensity of three
wavelengths of light reflected from the substrate. In some
embodiments, the method comprises simultaneously illuminating the
substrate with laser light comprising four wavelengths; and
detecting an intensity of four wavelengths of light reflected from
the substrate. In some embodiments, the step of detecting an
intensity of at least two wavelengths of light reflected from the
substrate comprises detecting an intensity of at least two
wavelengths of light reflected from a plurality of points on a
surface of the substrate. In some embodiments, the method further
comprises displaying a first visual representation of the detected
intensities of the at least two wavelengths of light as a first
two-dimensional image. In some embodiments, the method further
comprises illuminating the substrate with light from an LED light
source; and detecting an intensity of at least one wavelength of
light reflected or emitted off of the substrate. In some
embodiments, the step of detecting an intensity of at least one
wavelength of light reflected from the substrate comprises
detecting an intensity of at least one wavelength of light
reflected from a plurality of points on a surface of the substrate.
In some embodiments, the method further comprises displaying a
second visual representation of the detected intensities of the at
least one wavelength of light as a second two-dimensional image. In
some embodiments, the method further comprises combining the first
visual representation and the second visual representation to form
a composite two-dimensional image. In some embodiments, the method
further comprises placing the substrate on a scanning bed in
optical communication with the laser light; illuminating the
substrate with light from an LED light source; and detecting
boundaries of the substrate using a CCD camera. In some
embodiments, the step of detecting boundaries of the substrate
occurs before the step of simultaneously illuminating the substrate
with laser light comprising at least two wavelengths, wherein the
step of simultaneously illuminating the substrate with laser light
comprising at least two wavelengths comprises illuminating only an
area within the detected boundaries of the substrate with the laser
light.
[0077] In some embodiments, the present disclosure provides a
detection instrument capable of distinguishing between at least two
types of molecules in a single substrate, the detection instrument
comprising: a first optical path comprising a laser light source
and a first detector for scanning the substrate with light having a
first wavelength; and a second optical path comprising an LED light
source and a second detector for capturing an image of the
substrate at visible wavelengths. In some embodiments, the first
optical path further comprises a dual-band emission filter for
distinguishing between fluorescent emitted light and phosphorescent
emitted light. In some embodiments, the detection instrument
further comprises a third optical path comprising a second laser
light source and a second detector for scanning the substrate with
light having a second, different wavelength. In some embodiments,
the detection instrument further comprises a fourth optical path
comprising a third laser light source and a third detector for
scanning the substrate with light having a third, different
wavelength. In some embodiments, the detection instrument further
comprises a fifth optical path comprising a fourth laser light
source and a fourth detector for scanning the substrate with light
having a fourth, different wavelength. In some embodiments, the
first wavelength is selected from the group consisting of: 488 nm,
520 nm, 658 nm, and 784 nm. In some embodiments, the first
wavelength and the second wavelength are different and
independently selected from the group consisting of: 488 nm, 520
nm, 658 nm, and 784 nm. In some embodiments, the first wavelength,
the second wavelength, and the third wavelength are different and
independently selected from the group consisting of: 488 nm, 520
nm, 658 nm, and 784 nm. In some embodiments, the first wavelength,
the second wavelength, the third wavelength, and the fourth
wavelength are different and independently selected from the group
consisting of: 488 nm, 520 nm, 658 nm, and 784 nm. In some
embodiments, the first wavelength and the second wavelength are
different and independently selected from the group consisting of:
658 nm and 784 nm. In some embodiments, the first wavelength, the
second wavelength, and the third wavelength are different and
independently selected from the group consisting of: 488 nm, 520
nm, and 658 nm. In some embodiments, the detection instrument
further comprises a photomultiplier tube if any one of the optical
paths comprises a laser light source that emits light at 488 nm or
520 nm. In some embodiments, the detection instrument further
comprises an avalanche photodiode if any of the optical paths
comprises a laser light source that emits light at 658 nm or 784
nm. In some embodiments, the first optical path and the third
optical path are housed within a first scan head, and wherein the
fourth optical path and the fifth optical path are housed within a
second scan head that is optically distinct from the first scan
head. In some embodiments, the second detector is a CCD camera.
[0078] In some embodiments, the present disclosure provides a
composite image derived from a substrate, the composite image
comprising: a first pattern having a first color and corresponding
to a first pattern of a first type of molecule in the substrate; a
second pattern having a second color and corresponding to a second
pattern of a second type of molecule in the substrate; a third
pattern having a third color and corresponding to a third pattern
of a third type of molecule in the substrate, wherein the first
color, the second color, and the third color are each different,
wherein the first type of molecule is selected from the group
consisting of: a molecule including a fluorescent label, a
chemiluminescent molecule, a colorimetric molecule, and a
phosphorescent molecule, wherein, the second type of molecule is
different than the first type of molecule and is selected from the
group consisting of: a molecule including a fluorescent label, a
chemiluminescent molecule, a colorimetric molecule, and a
phosphorescent molecule, with a first proviso that, if the first
type of molecule is a chemiluminescent molecule, the second type of
molecule is not a chemiluminescent molecule and the third type of
molecule is not a chemiluminescent molecule, with a second proviso
that, if the first type of molecule is a colorimetric molecule, the
second type of molecule is not a colorimetric molecule and the
third type of molecule is not a colorimetric molecule, and with a
third proviso that, if the first type of molecule is a
phosphorescent molecule, the second type of molecule is not a
phosphorescent molecule and the third type of molecule is not a
phosphorescent molecule. In some embodiments, the first pattern,
the second pattern, and the third pattern are each derived from the
substrate without moving the substrate. In some embodiments, at
least two of the first type of molecule, the second type of
molecule, and the third type of molecule includes a fluorescent
label. In some embodiments, the first type of molecule includes a
first fluorescent label, the second type of molecule includes a
second fluorescent label, and the third type of molecule includes a
third fluorescent label. In some embodiments, the composite image
further comprises a fourth pattern having a fourth color and
corresponding to a fourth pattern of a fourth type of molecule in
the substrate, wherein the fourth type of molecule is selected from
the group consisting of: a molecule including a fluorescent label,
a chemiluminescent molecule, a colorimetric molecule, and a
phosphorescent molecule, with a fourth proviso that, if any one of
the first, second, or third types of molecules is a
chemiluminescent molecule, then the fourth type of molecule is not
a chemiluminescent molecule, with a fifth proviso that, if any one
of the first, second, or third types of molecules is a colorimetric
molecule, then the fourth type of molecule is not a colorimetric
molecule, and with a sixth proviso that, if any one of the first,
second, or third types of molecules is a phosphorescent molecule,
then the fourth type of molecule is not a phosphorescent molecule.
In some embodiments, the first type of molecule includes a first
fluorescent label, the second type of molecule includes a second
fluorescent label, the third type of molecule includes a third
fluorescent label, and the fourth type of molecule includes a
fourth fluorescent label. In some embodiments, at least one of the
first pattern, the second pattern, and the third pattern is
captured by an optical path comprising a laser light source and a
photomultiplier tube or an avalanche photodiode. In some
embodiments, the optical path further comprises a dual-band
emission filter. In some embodiments, at least one of the first
pattern, the second pattern, and the third pattern is captured by
an optical path comprising an LED light source and a CCD camera. In
some embodiments, at least one of the first pattern, the second
pattern, and the third pattern has a resolution of 10 .mu.m or
less. In some embodiments, the substrate is an electrophoresis gel.
In some embodiments, the substrate is a PVDF membrane. In some
embodiments, the substrate is a multi-welled plate. In some
embodiments, the substrate is a plant, an animal, or a portion of a
plant or animal.
EXAMPLES
[0079] Aspects of embodiments may be further understood in light of
the following examples, which should not be construed as limiting
in any way.
[0080] Example 1. Using dual scan head to scan 4 fluorescent dyes
simultaneously. Cell lysate prepared from HT-29 cell culture grown
under standard conditions and treated with 500 ng/mL insulin are
lysed in sodium dodecyl sulfate (SDS) containing Laemmle buffer and
polyacrylamide gel electrophoresis (SDS-PAGE) is run on a 10%
polyacrylamide gel for 1 hour at 1000V. Afterwards, separated
proteins are transferred from gel onto PVDF membrane by
electroblotting. The membrane is washed and blocked using standard
procedures, and incubated with antibodies for 4 different cellular
proteins, each labeled with a different fluorescent dye (Alexa
Fluor.RTM. 488, Alexa Fluor.RTM. 532 Alexa Fluor.RTM. 633 and Alexa
Fluor.RTM. 790, Thermo Fisher Scientific Inc., Pittsburgh, Pa.) for
2 hours at room temperature. The membrane is then rinsed with
buffer and ready to load into detection instrument 200.
[0081] Optical components in scan head 1 of dual scan head 208 are
outlined in FIG. 2. In this this example, Alexa Fluor.RTM. 532 will
be excited using 525 nm diode laser as light source 118 and
detected with a photomultiplier tube (PMT) as detector 122. Having
an emission filter that is a single-bandpass 570/40 will allow
light of wavelengths between 550 nm and 590 nm to reach the
detector. And lastly beam splitter 114 is selected to pass
wavelengths above 540 nm, and reflect light below 540 nm.
Similarly, Alexa Fluor.RTM. 790 will be excited using the
components in FIG. 3 listed for NIR excitable dyes. By selecting a
bandpass filter 112 that reflects light below 640 nm and passes
light above 640 nm, these two dyes can be excited and detected
simultaneously with the one scan head.
[0082] Scan head 2 is built using the components in FIG. 4 to
excite and measure the Alexa Fluor.RTM. 488 and Alexa Fluor.RTM.
633.
[0083] The CCD detector 206 takes an image of the entire scan bed
and embedded software processes the image to find the location of
the blot on the scan bed. The optical scan heads then move to the
location of the blot and begin to move along the length of the blot
to measure fluorescence across the entire blot. The resulting
scanned image is presented to the operator for analysis.
[0084] Example 2. RNA binding assay using FIG. 2 optical setup. RNA
samples separated on agarose gel by standard methods are
transferred to nitrocellulose membrane and blocked according to
standard methods before being probed with DNA labeled with
.alpha..sup.32P labeled nucleotide. The membrane is then washed,
wrapped in plastic wrap and placed on a phosphorimaging screen for
120 minutes to allow phosphor on plate to absorb energy from
radiolabel. The phosphorimaging screen is then loaded onto scan bed
of the instrument described in Example 1.
[0085] Optical scan head 2 has components outlined in FIG. 4. This
setup was used as in the previous example to read red and blue
dyes, but also can be used to scan a phosphorimaging screen. Here
the phosphor on the screen will be excited using the 625 nm diode
laser in light source position 120. The phosphor emits light at 400
nm after stimulation, which is captured through the objective,
reflected by beam splitter 112, passes through beam splitter 114,
and the dual band pass emission filter 108 through the focus lens
104 onto PMT detector 122.
[0086] The CCD detector 206 takes an image of the entire scan bed
which is used to find the location of the blot on the scan bed. The
optical scan heads then move to the location of the blot and begin
to scan along both axes of the blot to measure fluorescence across
the entire blot. The resulting scanned image is presented to the
operator for analysis.
[0087] Example 3. Protein gel imaging with optical densitometry of
Coomassie Blue stained gel. Protein sample is separated on SDS-PAGE
gel according to standard protocol and proteins are fixed in gel
using a mixture of 25% isopropyl alcohol, 10% acetic acid for 60
minutes. The gel is then stained in a 60 mg/L solution of Coomassie
Blue R-250 in 10% Acetic acid, and destained in 10% acetic acid to
remove non-specifically bound Coomassie Blue.
[0088] The gel is then placed on the scanner bed in the same
instrument used for examples 1, 2, and 3. Blue LED is used to
illuminate gel, and CCD below gel measures light transmitted though
gel. The resultant image can then be used to determine protein
density at different location on gel.
[0089] Example 4. Chemiluminescent gel imaging. Cell lysate
prepared from HT-29 cell culture grown under standard conditions
and treated with 500 ng/mL insulin are lysed in sodium dodecyl
sulfate (SDS) containing Laemmle buffer and polyacrylamide gel
electrophoresis (SDS-PAGE) is run on a 10% polyacrylamide gel for 1
hour at 1000V. Afterwards, separated proteins are transferred from
gel onto PVDF membrane by electroblotting. The membrane is washed
and blocked using standard procedures, and incubated with anti-ERK
primary antibody (Millipore 06-182) for 2 hours at room
temperature. The membrane is washed and then incubated with
Horseradish peroxidase (H RP) labeled anti-rabbit secondary
antibody (ThermoFisher 81-6120) at 1:10,000 dilution for 30 minutes
at room temperature. The membrane is then rinsed with buffer and
ready to load into detection instrument 200.
[0090] The membrane is placed on scan bed 204, and covered with
luminol and peroxide mixture (Cat #34075, Thermo Fisher Scientific
Inc., Pittsburgh, Pa.) to initiate chemiluminescence. CCD 206 is
used to capture an image of chemiluminescence. The resultant image
can then be used to determine protein density at different location
on gel.
[0091] Example 5. Using dual scan head to scan 4 fluorescent dyes
simultaneously. Cell lysate prepared from HT-29 cell culture grown
under standard conditions and treated with 500 ng/mL insulin are
lysed in sodium dodecyl sulfate (SDS) containing Laemmle buffer and
polyacrylamide gel electrophoresis (SDS-PAGE) is run on a 10%
polyacrylamide gel for 1 hour at 1000V. Afterwards, separated
proteins are transferred from gel onto PVDF membrane by
electroblotting. The membrane is washed and blocked using standard
procedures, and incubated with antibodies for 4 different cellular
proteins, each labeled with a different fluorescent dye (Alexa
Fluor.RTM. 488, Alexa Fluor.RTM. 532 Alexa Fluor.RTM. 633 and Alexa
Fluor.RTM. 790, Thermo Fisher Scientific Inc., Pittsburgh, Pa.) for
2 hours at room temperature. The membrane is then rinsed with
buffer and ready to load into detection instrument 200.
[0092] Optical components in scan head 1 of dual scan head 208 are
outlined in FIG. 2. In this this example, Alexa Fluor.RTM. 532 will
be excited using 525 nm diode laser as light source 518 and
detected with a photomultiplier tube (PMT) as detector 522. Having
an emission filter that is a single-bandpass 570/40 will allow
light of wavelengths between 550 nm and 590 nm to reach the
detector. And lastly beam splitter 514 is selected to pass
wavelengths above 540 nm, and reflect light below 540 nm.
Similarly, Alexa Fluor.RTM. 790 will be excited using the
components in FIG. 3 listed for NIR excitable dyes. By selecting a
bandpass filter 512 that reflects light below 640 nm and passes
light above 640 nm, these two dyes can be excited and detected
simultaneously with the one scan head.
[0093] Scan head 2 is built using the components in FIG. 4 to
excite and measure the Alexa Fluor.RTM. 488 and Alexa Fluor.RTM.
633.
[0094] The CCD detector 206 takes an image of the entire scan bed
and embedded software processes the image to find the location of
the blot on the scan bed. The optical scan heads then move to the
location of the blot and begin to move along the length of the blot
to measure fluorescence across the entire blot. The resulting
scanned image is presented to the operator for analysis.
[0095] Example 6. RNA binding assay using FIG. 2 optical setup. RNA
samples separated on agarose gel by standard methods are
transferred to nitrocellulose membrane and blocked according to
standard methods before being probed with DNA labeled with
.alpha..sup.32P labeled nucleotide. The membrane is then washed,
wrapped in plastic wrap and placed on a phosphorimaging screen for
120 minutes to allow phosphor on plate to absorb energy from
radiolabel. The phosphorimaging screen is then loaded onto scan bed
of the instrument described in Example 5.
[0096] Optical scan head 2 has components outlined in FIG. 4. This
setup was used as in the previous example to read red and blue
dyes, but also can be used to scan a phosphorimaging screen. Here
the phosphor on the screen will be excited using the 625 nm diode
laser in light source position 520. The phosphor emits light at 400
nm after stimulation, which is captured through the objective,
reflected by beam splitter 512, passes through beam splitter 514,
and the dual band pass emission filter 508 through the focus lens
504 onto PMT detector 522.
[0097] The CCD detector 206 takes an image of the entire scan bed
which is used to find the location of the blot on the scan bed. The
optical scan heads then move to the location of the blot and begin
to scan along both axes of the blot to measure fluorescence across
the entire blot. The resulting scanned image is presented to the
operator for analysis.
[0098] Example 7. Simultaneous visualization of Cy3 and Cy5 labeled
lysate components in a 2D gel. Untreated HeLa lysate was labeled
with Cyanine Dye 3 ("Cy3") according to the dye manufacturer's
instructions. The treated HeLa lysate was then labeled with Cyanine
Dye 5 ("Cy5") according to the dye manufacturer's instructions. The
double-labeled HeLa lysate was then loaded into a gel and separated
using isoelectric focusing ("IEF") in one dimension and SDS-PAGE in
a second, orthogonal dimension. The processed gel was imaged using
a biomolecule scanner consistent with the present disclosure
comprising a first laser emitting light at 520 nm and a second
laser emitting light at 658 nm. The scanned gel is reproduced in
FIG. 8A. The shorter wavelength light causes the Cy3-labeled HeLa
lysate components to fluoresce a first, greenish color, while the
longer wavelength light cases the Cy5-labeled HeLa lysate
components to fluoresce a second, orange color.
[0099] Example 8. Simultaneous visualization of chemiluminescent
and fluorescent-labeled proteins. A protein blot was prepared by
performing gel electrophoresis using SDS-PAGE and standard Laemmli
running buffer, followed by transfer to a PVDF membrane using
transfer buffer (Azure Transfer Buffer #AC2127, Azure Biosystems
Inc., Dublin, Calif.). The membrane was blocked for 30 minutes at
room temperature using blot blocking buffer (Azure Chemi Blot
Blocking Buffer #AC2148, Azure Biosystems Inc., Dublin, Calif.).
The blot was then incubated for one hour at room temperature with
chicken anti-Transferrin antibody in blot blocking buffer (Azure
Chemi Blot Blocking Buffer #AC2148, Azure Biosystems Inc., Dublin,
Calif.) with gentle agitation. The blot was then washed twice
quickly, and then three more times for five minutes each, with at
least 0.5 mL/cm.sup.2 membrane blot washing buffer (Azure Blot
Washing Buffer #AC2113, Azure Biosystems Inc., Dublin, Calif.). The
blot was then incubated with anti-chicken secondary antibody in
blot blocking buffer (Azure Chemi Blot Blocking Buffer #AC2148,
Azure Biosystems Inc., Dublin, Calif.) for one hour at room
temperature with gentle agitation, followed by washing twice
quickly and three times for 5 minutes each as described above. A
chemiluminescent HRP substrate (Radiance Chemiluminescent HRP
Substrate #AC2101, Azure Biosystems Inc., Dublin, Calif.) was
prepared according to the manufacturer's instructions and placed on
the blot at 0.1 mL/cm.sup.2 for two minutes. Excess
chemiluminescent HRP substrate was drained and the blot was then
imaged on a biomolecular scanner consistent with the present
disclosure comprising 16.times.13 cm chemiluminescent imaging area
having 16-bit, 2688-2200 resolution. The image is reproduced herein
as FIG. 8B.
[0100] Example 9. Simultaneous detection of two near-infrared
fluorescent labels. A protein blot was prepared by performing gel
electrophoresis using SDS-PAGE and standard Laemmli running buffer,
followed by transfer to a PVDF membrane using transfer buffer
(Azure Transfer Buffer #AC2127, Azure Biosystems Inc., Dublin,
Calif.). The membrane was blocked for 30 minutes at room
temperature using blot blocking buffer (Azure Fluorescent Blot
Blocking Buffer #AC2190, Azure Biosystems Inc., Dublin, Calif.).
The blot was then incubated with mouse anti-STAT1 antibody and
rabbit anti-phosphor-STAT1 antibody in a fluorescent blot blocking
buffer (Azure Fluorescent Blot Blocking Buffer #AC2190, Azure
Biosystems Inc., Dublin, Calif.) for one hour at room temperature
with gentle agitation. The blot was then washed twice quickly, and
then three more times for five minutes each, with at least 0.5
mL/cm.sup.2 membrane fluorescent blot washing buffer (Azure
Fluorescent Blot Washing Buffer #AC2145, Azure Biosystems Inc.,
Dublin, Calif.). The blot was the incubated with anti-mouse
secondary antibody (AzureSpectra anti-mouse 700 secondary antibody
#AC2129, Azure Biosystems Inc., Dublin, Calif.) and anti-rabbit
secondary antibody (AzureSpectra anti-rabbit 800 secondary antibody
#AC2134, Azure Biosystems Inc., Dublin, Calif.) in fluorescent
blocking buffer (Azure Fluorescent Blot Blocking Buffer #AC2190,
Azure Biosystems Inc., Dublin, Calif.) for one hour at room
temperature with gentle agitation. The blot was then washed twice
quickly and three times for five minutes each as described above,
followed by rinsing for five minutes in PBS with at least 0.5
mL/cm.sup.2 membrane. After drying, the membrane was imaged using a
biomolecule scanner consistent with the present disclosure
comprising a first laser emitting light at 658 nm and a second
laser emitting light at 784 nm. The scanned image is reproduced
herein as FIG. 8C, with the components labeled with anti-mouse
secondary antibody appearing as red-orange bands, and the
components labeled with anti-rabbit secondary antibody appearing as
green bands.
[0101] Example 10. Simultaneous visualization of visible and
chemilumiescent gel bands. Protein markers and a protein-containing
test sample were separated on a gel using SDS-PAGE in Laemmli
running buffer according to standard protocols well known in the
art. The gel was then removed from the electrophoresis cassette and
incubated in a tray with 25 mL of a rapid Coomassie stain (Generon
Quick Coomassie Stain, Cat# GEN-QC-STAIN-1 L, Generon Ltd, Slough
UK) for one hour at room temperature. The gel was then relocated to
ultra pure water for destaining. The destained gel was imaged on a
biomolecule scanner consistent with the present disclosure
comprising a LED lamp and CCD camera configured to capture visible
light, and a laser source emitting light at a wavelength of 658 nm.
The image is reproduced herein as FIG. 8D.
[0102] Example 11. Visualization of small quantities of nucleic
acid material in an agarose gel. A 1:1 dilution series of molecular
weight ladders in water was prepared according to standard
protocols well known in the art. Agarose gels (0.8%) were cast in
1.times.TAE with ethidium bromide ("EtBr") at a ratio of 1:10,000
according to standard protocols well known in the art. 10 .mu.L of
each dilution was loaded into a well of the agarose gel. Separation
of the molecular weight ladder samples occurred at 70V for 120-150
minutes in 1.times.TAE. Once electrophoretic separation was
complete, the gels were imaged using a biomolecule scanner
consistent with the present disclosure comprising a laser emitting
light at 520 nm. A representative scanned image is reproduced
herein as FIG. 8E, with the left lane having a mass of 1250 pg
(arrow) and the far right lane having a mass of 20 pg (arrow).
[0103] Example 12. Flimless autoradiography of
phosphor-radiolabeled biomolecules. A commercially available
carbon-14 standard including slices ranging from 0.004 .mu.Ci/g to
1,000 .mu.Ci/g (Cat # ARC0146F, American Radiolabeled Chemicals,
Inc., St. Louis, Mo.) was exposed to a BAS-MS storage phosphor
screen having a sensitivity of 0.9 DPM/mm.sup.2/hr (Cat #IS1011,
Azure Biosystems Inc., Dublin, Calif.) for three hours. The storage
phosphor screen was then imaged using a biomolecule scanner
consistent with the present disclosure comprising a laser emitting
light at 658 nm and a photomultiplier tube detector. The limit of
detection was determined to be only 0.036 .mu.Ci/g, and the
detection was linear (R.sup.2=0.99) over 5.4 orders of magnitude.
The scanned image is reproduced herein as FIG. 8F.
[0104] Example 13. Detection of proteins in-situ by in-cell Western
blotting. HeLa cells were serially diluted and seeded into a
sterile 96-well tissue culture plate at a volume of 0.2 mL/well,
and grown until approximately 80% confluent. All wells were then
fixed and permeabilized using 100% methanol for 15 minutes at room
temperature. Cells were then rinsed with PBS and blocked with 1%
fish gelatin in PBS for one hour at room temperature. The blocked
cells were then probed with mouse alpha-tubulin and rabbit is
beta-actin overnight at 4.degree. C. Wells were then washed three
times each with PBS prior to incubation with an anti-rabbit
conjugated secondary antibody (AzureSpectra anti-rabbit 550, Cat
#AC2158, Azure Biosystems Inc., Dublin, Calif.) and an anti-mouse
conjugated secondary antibody (AzureSpectra anti-mouse 800
conjugated secondary antibody, Cat #AC2135, Azure Biosystems Inc.,
Dublin, Calif.) for 60 minutes at room temperature. Cells in all
wells were then stained with a far-red cell membrane-permeable
nuclear dye (RedDot.TM.1, Cat #40060-1, Biotium, Inc., Fremont,
Calif.) consistent with the manufacturer's instructions as a
normalization control. Wells were then washed three times with PBS.
The wells were scanned at multiple wavelengths using a biomolecule
scanner consistent with the present disclosure comprising an LED
emitting light at 520 nm, a first laser emitting light at 658 nm,
and a second laser emitting light at 785 nm, a photomultiplier tube
detector, and an avalanche photodiode detector. The captured images
for each of the three excitation wavelengths were combined into a
single composite image, which is reproduced herein as FIG. 8G.
[0105] Example 14. Simultaneous detection of four different
fluorescent probes. A protein blot was prepared from HeLa cell
samples or HeLa cell samples spiked with transferrin by 4-15%
tris-glycine gel electrophoresis using SDS-PAGE and standard
Laemmli running buffer, followed by transfer to low-fluorescence
PVDF membrane (Cat #AC2105, Azure Biosystems Inc., Dublin, Calif.)
using transfer buffer (Azure Transfer Buffer #AC2127, Azure
Biosystems Inc., Dublin, Calif.). The membrane was blocked for 30
minutes at room temperature using blot blocking buffer (Azure
Fluorescent Blot Blocking Buffer #AC2190, Azure Biosystems Inc.,
Dublin, Calif.). The blot was then incubated with anti-transferrin
antibody (Cat #AC2185, Azure Biosystems Inc., Dublin Calif.)
labeled with fluorescent dye excitable at -490 nm that emits at
-515 nm (AzureSpectra 490, Cat #AC2185, Azure Biosystems Inc.,
Dublin, Calif.), goat anti-rat tubulin (Cat #AC2162, Azure
Biosystems Inc., Dublin, Calif.), goat anti-rabbit actin (Cat
#AC2128, Azure Biosystems Inc., Dublin, Calif.), and goat
anti-chicken GAPHD (Cat #AC2137, Azure Biosystems Inc., Dublin,
Calif.) in blot blocking buffer (Azure Fluorescent Blot Blocking
Buffer #AC2190, Azure Biosystems Inc., Dublin, Calif.) for one hour
at room temperature with gentle agitation. The blot was washed
twice quickly and three times for five minutes each with at least
0.5 ml/cm.sup.2 membrane fluorescent blot washing buffer (Azure
Fluorescent Blot Washing Buffer, Cat #AC2190, Azure Biosystems
Inc., Dublin, Calif.). The washed blot was then incubated with
anti-rat, anti-rabbit and anti-chicken secondary antibodies (Cat
Nos. AC2162, AC2128 and AC2137, respectively, Azure Biosystems
Inc., Dublin, Calif.) in fluorescent blot blocking buffer (Azure
Fluorescent Blot Blocking Buffer, Cat #AC2190, Azure Biosystems
Inc., Dublin, Calif.) for one hour at room temperature with gentle
agitation. The labeled blot was then washed twice quickly and three
times for five minutes each as described above, and then rinsed for
five minutes with at least 0.5 mL/cm.sup.2 PBS. The membrane was
then dried and imaged using a biomolecule scanner consistent with
the present disclosure comprising a first laser emitting light at
488 nm and a second laser emitting light at 520 nm, a third laser
emitting light at 658 nm, and a fourth laser emitting light at 784
nm; a photomultiplier tube detector and an avalanche photodiode
detector. The scanned image is reproduced herein as FIG. 8H, with
the GADPH components appearing as green bands, the actin components
appearing as red bands, the tubulin bands appearing as blue bands,
and the transferrin components appearing as white bands (from
bottom to top).
[0106] Example 15. Simultaneous high-resolution imaging of multiple
fluorescent tags. A scanner calibration slide including a first
array of spots in two-fold dilution series of Cy3 fluorescent dye
and a second array of spots in two-fold dilution series of Cy5
fluorescent dye, with a spot center-to-center distance of 350 .mu.m
(Cat #DS01, Full Moon BioSystems, Inc., Sunnyvale, Calif.) was
scanned at 10 .mu.m resolution by excitation at 520 nm and 658 nm
simultaneously using a biomolecule scanner consistent with the
present disclosure comprising a first laser emitting light at 520
nm and a second laser emitting light at 658 nm. The captured images
for each of the two excitation wavelengths were combined into a
single composite image, which is reproduced herein as FIG. 8I.
[0107] The foregoing descriptions of specific embodiments of the
present invention have been presented for purposes of illustration
and description. They are not intended to be exhaustive or to limit
the invention to the precise forms disclosed, and obviously many
modifications and variations are possible in light of the above
teaching. The embodiments were chosen and described in order to
best explain the principles of the invention and its practical
application, to thereby enable others skilled in the art to best
utilize the invention and various embodiments with various
modifications as are suited to the particular use contemplated. It
is intended that the scope of the invention be defined by the
Claims appended hereto and their equivalents.
[0108] It is to be understood that both the foregoing descriptions
are exemplary and explanatory only, and are not restrictive of the
methods and devices described herein. In this application, the use
of the singular includes the plural unless specifically stated
otherwise. Also, the use of "or" means "and/or" unless stated
otherwise. Similarly, "comprise," "comprises," "comprising,"
"include," "includes" and "including" are not intended to be
limiting.
[0109] All patents, patent applications, publications, and
references cited herein are expressly incorporated by reference to
the same extent as if each individual publication or patent
application was specifically and individually indicated to be
incorporated by reference.
* * * * *