U.S. patent application number 12/797132 was filed with the patent office on 2010-11-18 for optofluidic microscope device.
This patent application is currently assigned to CALIFORNIA INSTITUTE OF TECHNOLOGY. Invention is credited to Demetri Psaltis, Changhuei Yang.
Application Number | 20100290049 12/797132 |
Document ID | / |
Family ID | 35449126 |
Filed Date | 2010-11-18 |
United States Patent
Application |
20100290049 |
Kind Code |
A1 |
Yang; Changhuei ; et
al. |
November 18, 2010 |
OPTOFLUIDIC MICROSCOPE DEVICE
Abstract
An optofluidic microscope device is disclosed. The device
includes a fluid channel having a surface and an object such as a
bacterium or virus may flow through the fluid channel. Light
imaging elements in the bottom of the fluid channel may be used to
image the object.
Inventors: |
Yang; Changhuei; (Pasadena,
CA) ; Psaltis; Demetri; (St. Sulpice, CH) |
Correspondence
Address: |
Sheila Martinez-Lemke
110 Pacific Avenue, Suite 240
San Francisco
CA
94111-1900
US
|
Assignee: |
CALIFORNIA INSTITUTE OF
TECHNOLOGY
Pasadena
CA
|
Family ID: |
35449126 |
Appl. No.: |
12/797132 |
Filed: |
June 9, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11125718 |
May 9, 2005 |
7773227 |
|
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12797132 |
|
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60590768 |
Jul 23, 2004 |
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60577433 |
Jun 4, 2004 |
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Current U.S.
Class: |
356/436 ;
977/773 |
Current CPC
Class: |
B82Y 10/00 20130101;
G01N 21/6458 20130101; B82Y 20/00 20130101 |
Class at
Publication: |
356/436 ;
977/773 |
International
Class: |
G01N 21/00 20060101
G01N021/00 |
Claims
1. An optofluidic microscope device comprising: a body comprising a
fluid channel having a surface; light transmissive regions in the
body; an illumination source adapted to provide illumination
through the light transmissive regions; and an optical detector
adapted to receive light from the illumination source through the
light transmissive regions.
2. The optofluidic microscope device of claim 1 wherein the surface
is the bottom of a fluid channel.
3. The optofluidic microscope device of claim 1 wherein the light
transmissive regions are holes.
4. The optofluidic microscope device of claim 1 wherein the optical
detector comprises a charge coupled device.
5. The optofluidic microscope device of claim 1 wherein the optical
detector includes a plurality of discrete light detecting elements,
wherein the light detecting elements respectively correspond to the
light transmissive regions.
6. The optofluidic microscope device of claim 1 wherein the fluid
channel has a bottom with a width of less than about 1 micron.
7. The optofluidic microscope device of claim 1 wherein the light
transmissive regions comprise an optically transparent
material.
8. The optofluidic microscope device of claim 1 wherein the array
of holes extends from a first side of the surface to a second side
of the surface.
9. The optofluidic microscope device of claim 1 wherein the array
of light transmissive regions is a first array of light
transmissive regions and the optical device comprises a second
array of light transmissive regions, wherein the second array of
light transmissive regions form a reference point.
10. The optofluidic microscope device of claim 1 wherein the
surface is part of a bottom wall, and wherein optical detector is
attached to the bottom wall.
11. The optofluidic microscope device of claim 1 wherein the array
of light transmissive regions forms a slanted line.
12. The optofluidic microscope device of claim 1 wherein
illumination source provides white light.
13. The optofluidic microscope device of claim 1 wherein the body
comprises a polymeric material.
14. A method of using the optofluidic microscope device of claim 1,
wherein the method comprises: flowing a fluid comprising a cell
through the fluid channel.
15. An optofluidic microscope device comprising: a body comprising
a fluid channel having surface; a plurality of discrete light
emitting regions on or under the surface; and an optical detector
adapted to receive light generated by the plurality of discrete
light-emitting regions.
16. The optofluidic microscope device of claim 15 wherein the
light-emitting regions comprise quantum dots.
17. The optofluidic microscope device of claim 15 wherein the
plurality of discrete light emitting regions is in the form of a
two-dimensional array.
18. A method of using the optofluidic microscope device of claim 15
comprising: flowing a fluid comprising a cell through the fluid
channel.
19. An optofluidic microscope device comprising: a body comprising
a fluid channel having surface; at least one light imaging element
on or under the surface; and an optical detector adapted to receive
light generated by the at least one light imaging elements.
20. The optofluidic microscope device of claim 19 wherein the at
least one light imaging element is in the form of a diagonal line.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
patent application Ser. No. 11/125,718, filed on May 9, 2005, which
is a non-provisional of and claims priority to U.S. provisional
Patent Application No. 60/590,768 filed on Jul. 23, 2004, entitled
"Fluorophore array based microscopy" and to U.S. provisional Patent
Application No. 60/577,433 filed on Jun. 4, 2004, entitled
"Fluorophore array based microscopy." All of these applications are
herein incorporated by reference in their entirety for all
purposes.
BACKGROUND OF THE INVENTION
[0002] The field of microfluidics has advanced rapidly. These
advancements relate to the development of large-scale integration
of microfluidic circuits, and numerous applications of
microfluidics to life science research. Currently, optical
microscopy is employed in microfluidic research as a technique to
study fundamental microscale flow physics as well as biological
targets. It is also used to study processes that are performed
within these integrated microfluidic systems. In general, these
devices rely on a macro-scale infrastructure (e.g. bulk
microscopes, chip readers) to analyze biological targets.
[0003] Near field scanning optical microscopes (NSOMs) are
extensively used to study biological targets. NSOMs can optically
resolve structures with spatial resolutions of .about.50 nm. An
NSOM uses a strongly enhanced and tightly confined optical field at
the end of an NSOM probe tip to optically probe a specific location
on a target sample. NSOMs are especially useful for profiling
bacteria, because bacteria cannot be easily imaged with
conventional optical microscopy. In comparison to other high
resolution imaging devices, such as scanning electron microscopes,
NSOMs are able to selectively map the distribution of proteins or
biochemicals in samples via fluorescence. In addition, NSOM imaging
methods are non-destructive and can be used to image bioentities
that are immersed in buffer media. Given all these advantages, one
would expect that NSOMs would be widely used in clinical
applications to distinguish bacteria types. However, the lack of
publications on this suggests that significant technical barriers
exist to using NSOMs. One such barrier is the difficulty of
performing high throughput imaging with an NSOM. High throughput
imaging requires raster scanning the probe tip over a target
bioentity.
[0004] Embodiments of the invention are directed to devices which
are improvements over NSOMs and conventional microfluidic systems
that use bulky optics.
SUMMARY OF THE INVENTION
[0005] Embodiments of the invention are directed to optofluidic
microscope devices or OFM devices. The optofluidic microscope
devices according to embodiments of the invention are able to
achieve resolutions similar to those of NSOMs. However, unlike
NSOMs, embodiments of the invention can be used for high throughput
imaging.
[0006] One embodiment of the invention is directed to an
optofluidic microscope device comprising: a body comprising a fluid
channel having a surface; light transmissive regions in the body;
an illumination source adapted to provide illumination through the
light transmissive regions; and an optical detector adapted to
receive light from the illumination source through the light
transmissive regions.
[0007] Another embodiment of the invention is directed to an
optofluidic microscope device comprising: a body comprising a fluid
channel having surface; a plurality of discrete light emitting
regions on or under the surface; and an optical detector adapted to
receive light generated by the plurality of discrete light-emitting
regions.
[0008] Another embodiment of the invention is directed to an
optofluidic microscope device comprising: a body comprising a fluid
channel having surface; at least one light imaging element on or
under the surface; and an optical detector adapted to receive light
generated by the at least one light imaging element.
[0009] The optofluidic microscope devices according to some
embodiments of the invention can use off the shelf detectors such
as CCDs (charge coupled devices). Based on the parameters of
off-the-shelf linear CCD arrays, a 100.times.100 pixel image of a
bacterium can be acquired by an optofluidic microscope device
according to an embodiment of the invention within a time frame as
short as 1 millisecond. In embodiments of the invention, numerous
optofluidic microscope devices may also be operated in parallel on
a single chip.
[0010] The high speed processing capability of embodiments of the
invention and the ability to use multiple optofluidic microscope
devices in embodiments of the invention allow embodiments of the
invention to have substantially higher imaging throughput rates
than NSOMs. For example, in an NSOM device, the acquisition time
for a 100.times.100 pixel image is about 10 milliseconds. In
comparison, a parallel series of 10 optofluidic microscope devices
built on a single 1000 pixel linear CCD array can provide up to one
hundred 100.times.100 pixel images in the time that an NSOM creates
a single image. The high throughput imaging capability and high
resolution of the optofluidic microscope devices according to
embodiments of the invention make them highly suited for various
clinical applications. Such applications include differentiating
between different bacteria types.
[0011] Also, the optofluidic microscope devices according to
embodiments of the invention eliminate the bulky optics (e.g., sets
of objective lenses and complex microscope setups) that are used to
obtain biological images in conventional microfluidic devices.
Unlike conventional imaging systems, the optofluidic microscope
devices according to embodiments of the invention are portable and
compact.
[0012] Methods of making and methods of using the optofluidic
microscope devices are also disclosed.
[0013] These and other embodiments of the invention are described
in further detail below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 shows a schematic, perspective drawing of some
components of an optofluidic microscope device according to an
embodiment of the invention.
[0015] FIG. 2 shows a photograph of a top view of a working
embodiment of an optofluidic microscope device according to an
embodiment of the invention. In the photograph, the width of a
fluid channel in the optofluidic microscope device is about 44
microns. A schematic drawing of the fluid channels in the
optofluidic microscope device is also shown to the left of the
photograph.
[0016] FIG. 3(a) is another schematic drawing of an optofludic
microscope device according to embodiment of the invention from a
close up, perspective view.
[0017] FIG. 3(b) is a schematic drawing of a top view of the fluid
channel in the optofluidic microscope device shown in FIG. 3(a).
The light transmissive regions in the bottom of the fluid channel
form a line that is slanted with respect to its walls.
[0018] FIG. 3(c) is a schematic drawing of a side view of the
optofluidic microscope device shown in FIG. 3(a).
[0019] FIG. 3(d) shows collected time traces from each light
detecting element in the optofluidic microscope device, wherein
each light detecting element can produce an output that can be used
to create an object image.
[0020] FIGS. 4(a)-4(e) show cross-sections of an optofluidic
microscope device as it is being made.
[0021] FIG. 5 shows a microscope image of an optofluidic microscope
device and two graphs of transmission intensity vs. time. The left
graph shows the change in transmission intensity through the left
hole as indicated by a first corresponding arrow. The right graph
shows the intensity change through the right hole as indicated by a
second corresponding arrow.
[0022] FIG. 6(a) shows a top view transmission image of an
optofluidic microscope device. The light transmissive regions in
the optofluidic microscope device are in the form of holes (about
0.5 microns in diameter).
[0023] FIG. 6(b) shows occlusion time traces through adjacent holes
in an optofluidic microscope device according to an embodiment of
the invention.
[0024] FIG. 6(c) shows a microscope image of a chlamoydomonas.
[0025] FIG. 6(d) shows preliminary data showing an optofluidic
microscope device image of a chlamoydomonas.
[0026] FIG. 7 shows interspersed reference hole array sets along
the length of an optofluidic microscope device.
[0027] FIG. 8 shows another optofluidic microscope device according
to an embodiment of the invention. In this embodiment, light
transmissive regions with two different sizes are used in the
optofluidic microscope device.
[0028] FIGS. 9(a)-9(c) show a schematic of system that uses
optofluidic microscope devices. These Figures show a blood
differential unit in various operational stages. Active elements
are shown. FIG. 9(a) shows input and mixing where a sample is mixed
with a reagent. FIG. 9(b) shows optofluidic microscope device
measurements where a mixture is imaged through a series of
optofluidic microscope devices with different transmission optical
filters. FIG. 9(c) shows reprocessing, where any given fraction of
an analyte may be re-mixed with a different reagent and then
re-analyzed.
[0029] FIG. 10(a) shows a top plan view of another optofluidic
microscope device according to an embodiment of the invention. In
this embodiment, instead of holes in a fluid channel, distinct
quantum dots are used to image a biological entity such as a
cell.
[0030] FIG. 10(b) shows another embodiment of an optofluidic
microscope device according to an embodiment of the invention from
a top view. The quantum dots are in a line, rather than in an
array.
[0031] FIGS. 11(a) and 11(b) show exemplary illumination source and
detector configurations for embodiments like those shown in FIGS.
10(a) and 10(b).
[0032] FIG. 12 shows a top view of another embodiment of the
invention using quantum dots. In this embodiment, two dimensional
arrays of quantum dots are in a fluid channel and they are oriented
differently with respect to each other.
[0033] FIG. 13 shows a schematic perspective view of the
optofluidic microscope device shown in FIG. 10(b).
[0034] FIG. 14 shows a side view of an optofluidic microscope
device using quantum dots.
[0035] FIG. 15 shows a top view of another embodiment of the
invention using quantum dots. In this embodiment, the quantum dots
are arranged in lines and the lines are slanted with respect to
each other.
[0036] FIG. 16 shows a top view of another embodiment of the
invention using quantum dots. The quantum dots in this embodiment
are spaced apart by larger distances than prior embodiments.
[0037] FIG. 17 shows a top view of another embodiment of the
invention using quantum dots. The quantum dots in this example are
in the form of diagonal lines.
[0038] FIG. 18 shows an embodiment where a thin transparent or
reflective trench structure is formed in a substrate.
[0039] In the Figures, like numerals designate like elements.
DETAILED DESCRIPTION
[0040] Embodiments of the invention are directed to optofluidic
microscopes that can use light transmissive regions (e.g., spaced
holes) or discrete light emitting elements (e.g., quantum dots) in
a body defining at least a portion of a fluid channel. The light
transmissive regions or the light emitting elements (in conjunction
with other elements) can be used to image entities such as
biological entities passing through the fluidic channel. Other
embodiments are directed to optofluidic microscope devices that
have at least one light imaging element in or on a surface of a
bottom wall defining a fluid channel. The light imaging elements
may be in the form of one or more light transmissive regions such
as holes, one or more light emitting elements such as quantum dots,
one or more linear structures such as reflective lines or lines of
closely adjacent quantum dots, or even one or more light scattering
bodies such as nanoparticles.
[0041] In the specifically described embodiments, the imaging of
cells is mentioned. It is understood, however, that embodiments of
the invention are not limited thereto. For example, instead of
cells, any suitable object can be imaged. The object can be a
chemical or biological entity. Examples of biological entities
include whole cells, cell components, microorganisms such as
bacteria or viruses, cell components such as proteins, etc.
Chemical entities such as macromolecules may also be imaged by
embodiments of the invention.
[0042] I. Optical Devices Using Light Transmissive Regions
[0043] A. Exemplary Embodiments Using Light Transmissive
Regions
[0044] One embodiment of the invention is directed to an
optofluidic microscope device. The optofluidic microscope device
comprises a body defining at least a portion of a fluid channel.
Light transmissive regions are in the body, and the body may have a
surface that coincides with the bottom surface of the fluid
channel. An illumination source provides light which passes through
the light transmissive regions and is received by an optical
detector. The optical detector is disposed on the opposite side of
the surface as the illumination source, and may have discrete
individual light detecting elements (e.g., pixels) that are
respectively associated with the light transmissive regions. The
optofluidic microscope device is much more compact that
conventional microfluidic optical systems.
[0045] In some embodiments, the light transmissive regions are
micro-holes or nano-holes defined in an opaque or semi-opaque
layer, which may form part of the body. The holes may be defined
using any suitable hole formation process including electron beam
lithography. In embodiments of the invention, each hole (or light
transmissive region) may have a size (e.g., diameter) less than
about 5, 1, or 0.5 microns. The holes may also have any suitable
shape, and may be circular, square, etc.
[0046] The holes many also form an array. The array may be one or
two dimensional. For example, in some embodiments, the holes in the
array may form a slanted line extending from one lateral side of
the fluid channel to the other lateral side of the fluid
channel.
[0047] Any suitable number or density of holes or light
transmissive regions may be used in embodiments of the invention.
For example, there may be greater than about 10, 50, 100, or even
1000 light transmissive regions per optofluidic microscope device.
There may also be more than 10, 50, 100, or even 1000 light
transmissive regions per square centimeter in some embodiments.
[0048] The light transmissive regions and the optical detector can
form an "imager" in the optofluidic microscope device. The imager
can image entities such as biological targets, which may be present
in biological or non-biological samples. The samples and the
biological targets contained therein can be transported to the
imager using a standard microfluidic focusing arrangement defined
in a molded upper section of the optofluidic microscope device. The
molded upper section of the optofluidic microscope device may
define fluid channels, and may comprise a poly-(dimethylsiloxane)
(PDMS) material. Electrokinetics or pressure can drive the samples
through the fluid channels in the optofluidic microscope device to
and through the imager. As the biological targets pass through the
imager, the light transmission from the illumination source through
each light transmissive region is modulated over time. Using the
modulated light signals passing through the light transmissive
regions, the image of the targets passing through the imager can be
reconstructed.
[0049] FIG. 1(a) shows a schematic illustration of an optofluidic
microscope device 10 according to an embodiment of the invention.
The optofluidic microscope device 10 has a body 16, which may be a
multilayer structure. It could alternatively be a single,
monolithic structure in other embodiments. In the illustrated
example, however, the body 16 includes an opaque or semi-opaque
layer 19, which in turn has light transmissive regions 14 in it. A
transparent protective layer (not shown) may optionally cover the
opaque or semi-opaque layer 19 to isolate the opaque or semi-opaque
layer 19 from a fluid flowing in a fluid channel 22 in the
optofluidic microscope device.
[0050] The body 16 may define or include the fluid channel 22 and a
surface 16(a) of the body 16 may coincide with the bottom wall of
the fluid channel 22. During operation, the fluid channel 22 can
have a fluid including a cell 20 flowing in it. Micropumps,
electrokinetic devices and other devices (not shown) can be used to
cause the fluid to flow through the fluid channel 22.
[0051] The fluid channel 22 may have any suitable dimensions. For
example, the width and/or height of the fluid channel 22 may each
be less than about 10, 5, or 1 micron in some embodiments.
[0052] The light transmissive regions 14 in the body 16 are
preferably holes. For example, the light transmissive regions 14
may be holes that are etched into a metallic layer such as gold. In
the illustrated example, the light transmissive regions 14 form a
diagonal line, which extends from one lateral side of the fluid
channel 22 to the other lateral side of the fluid channel 22. In
other embodiments, the light transmissive regions 14 can be in the
form of an array or a one-dimensional line that extends
perpendicular to the direction of flow within the fluid channel
22.
[0053] An illumination source 12 is on one side of the surface
16(a). Suitable examples of illumination sources include white
light sources, natural lighting, colored light sources, etc. The
illumination source 12 produces light which passes through the
fluid passing through the fluid channel 22. Suitable illumination
sources are commercially available.
[0054] An optical detector 18 is on the other side of the surface
16(a). The optical detector 18 may include a charge coupled device,
and may include an array of discrete light detecting elements that
respectively correspond to the light transmissive regions 14. The
optical detector 18 could also be a diode array (e.g., a linear or
two-dimensional diode array), where each diode in the diode array
corresponds to a light transmissive region 14. Suitable optical
detectors are also commercially available.
[0055] As shown, a fluid including a biological cell 20 may flow
through the fluid channel 22. As the cell 20 passes through the
fluid channel 22, the light transmissive regions 14 can be used to
image the biological cell 20 (or other object). For example, as
shown in FIG. 1(a), a liquid containing a cell 20 may flow through
the fluid channel 22. As it flows through the fluid channel 22,
light from the illumination source 12 passes through the fluid
channel 22 and illuminates the surface 16(a) of the body 16. Light
that is not blocked by the cell 20 passes through some of the light
transmissive regions 14 substantially unaltered. Light that passes
to the cell 20 may be blocked or may be altered in some way (e.g.,
reduced intensity, altered wavelength, etc.) relative to the light
that passes through the cell 20. As noted above, individual light
detecting elements (not shown) in the light detector 18 can be
respectively associated with the light transmissive regions 14.
Each individual light detecting element in the detector 18 is
sampled over time and the changes in light received by the light
detecting elements over time can be used to image the cell 20. This
process is explained in further detail below.
[0056] Referring to FIG. 2, reference number 34(a) shows a
schematic diagram of a microfluidic channel system that can be used
in a microfluidic device. Reference number 34(b) shows an SEM
(scanning electron microscope) image of a portion of the
microfluidic channel system corresponding to reference number
34(a). As shown by reference number 34(b), a line of light
transmissive regions extends from lateral one side of a fluid
channel to the other lateral side.
[0057] The branched fluid channel structure may be used to "focus"
a biological target toward the center of the imager including the
light transmissive regions. For example, biological targets may
flow in the center of the fluid channel of the three upstream fluid
channels shown by reference number 34(a). The three fluid channels
converge into a single fluid channel, the biological targets will
stay confined to the center of the single fluid channel. This helps
to ensure that the biological target will travel in a substantially
straight line as it passes over the light transmissive regions.
[0058] The operation of a optofluidic microscope device according
to an embodiment of the invention can be further described with
respect to FIGS. 3(a)-3(d). FIG. 3(a) is another schematic drawing
of an optical device according to embodiment of the invention from
a close up view. FIG. 3(b) is a top view of the fluid channel in
the optical device in FIG. 3(a). FIG. 3(c) is a side view of the
optofluidic microscope device shown in FIG. 3(a). FIG. 3(d) shows
collected time traces from each pixel in the optical device,
wherein each pixel can be processed to create an object image.
[0059] As shown in FIGS. 3(a)-3(c), a cell 20 passes through the
fluid channel 22 and blocks light transmissive regions 14 as it
passes through the fluid channel 22. The cell 20, or other target
object, flows through the channel 22 at a constant velocity.
Electrokinetic or pressure devices (not shown) can cause the liquid
containing the cell 20 to flow so that the cell 20 is confined to
the center of the imager. As shown in FIG. 3(a), the light
transmissive regions 14 are slanted and extend from one lateral
side of the fluid channel 22 to the other lateral side of the fluid
channel 22. By slanting the light transmissive regions 14, each
lateral position of the fluid channel 22 can be monitored and used
to image the lateral edges of the cell 20. In other embodiments,
the light transmissive regions 14 could be in a straight line that
is perpendicular to the walls of the fluid channel 22.
[0060] As shown in FIG. 3(c), a light detector 18 is under an
opaque or semi-opaque layer 19 including a number of light
transmissive regions 14. The opaque or semi-opaque layer 19 may be
a gold film (which may be present on a transparent layer) and may
be, at 100 nanometers, nearly opaque to white light transmission
(the skin depth of a 632.8 nanometer He--Ne laser in a gold layer
is about 12 nanometers).
[0061] Any suitable commercially available light detector may be
used in embodiments of the invention. The light detector 18
includes a number of discrete light detecting elements (e.g.,
pixels) respectively corresponding to the light transmissive
regions 14. During operation, the light detector 18 may or may not
receive substantially unmodulated light from the illumination
source 12 through the light transmissive regions 14. This depends
upon whether or not the cell 20 is covering the light transmissive
regions 14. Changes in the light signals received through the light
transmissive regions 14 over time can be used to image the cell
20.
[0062] Illustratively, FIG. 3(d) shows collected time traces from
each light detecting element under each light transmissive region
14. More specifically, an output for each the light detecting
element in the detector 18 over time is shown. Using the known
relative positions of the light transmissive regions 14 (and their
associated light detecting pixel) and the time traces generated
from the discrete light detecting elements in the light detector
18, an object image can be formed as shown in FIG. 3(d).
[0063] As a target passes over the light transmissive regions 14,
the characteristics of the light passing through them are changed
in some way. In effect, each light transmissive region 14 functions
like a probe tip of a near field optical microscope (NSOM), or as a
pinhole in confocal microscope. Embodiments of the invention thus
have high sensitivity.
[0064] The time trace associated with each light transmissive
region is dependent on the profile of the target being imaged as
well as its optical properties. For example, a pixel output that
corresponds to low intensity at a predetermined position for a
predetermined period of time provides data regarding the width of
the object at a particular position in the fluid channel. The data
for each pixel can be processed using a computer to form an image
of the object. In this example, it is presumed that the cell 20
moves in a straight line as it passes through the fluid channel 22
and over the pixels in the detector and over the light transmissive
regions 14.
[0065] Embodiments of the invention have a number of advantages.
Embodiments of the invention provide high resolution, are
inexpensive to fabricate, use small sample volumes, are easy to
view, and have high throughput. Embodiments of the invention can be
as small as a matchbox, are capable of high throughput processing,
and are easy to mass produce. As an alternative to conventional
bulky microscopes, they are easy and inexpensive to fabricate, and
they are compact. Embodiments of the invention can also reach the
sub wavelength resolution regime, thus opening up a new field of
optical on-chip imaging of small bacteria and viruses. A high
throughput approach for imaging and distinguishing different
viruses or bacterium types can be important and convenient for
biological and clinical usage.
[0066] B. Methods for Making the Optofluidic Microscope Devices
[0067] The optofluidic microscope devices according to embodiments
of the invention can be fabricated in any suitable manner. An
exemplary method for fabricating an optofluidic microscope device
according to an embodiment of the invention can be described with
reference to FIGS. 4(a)-4(e). Any suitable combination of well
known processes including etching, lamination, and soft lithography
can be used to fabricate the optofluidic microscope devices
according to embodiments of the invention.
[0068] Fabrication of the imaging array is shown in FIG. 4(a) and
begins by first evaporating a layer of gold 34 (approximately 100
nanometers thick) on the transparent surface of a glass plate 32.
The glass plate 32 could alternatively be some other transparent
layer. The layer of gold 34 could alternatively be any other
suitable opaque or semi-opaque layer.
[0069] As shown in FIG. 4(b), a poly(methylmethacrylate) (PMMA)
resist layer 36 is then spun on the gold layer 34 and standard
electron-beam lithography used to form a hole pattern in the PMMA
resist 36. Instead of a PMMA resist 36, any other suitable type of
photoresist may be used.
[0070] As shown in FIG. 4(c), after developing, the gold layer 34
is wet etched thereby defining the imager holes 39. Alternatively,
a dry etching process may be used to form the imager holes 39.
[0071] In other embodiments, etching need not be used. For example,
a laser ablation process can be used to form the holes 39. In this
case, a photoresist layer is not needed to form the holes 39.
[0072] As shown in FIG. 4(d), the remaining PMMA layer 36 is then
removed and replaced with a new PMMA film 37 (about 200 nanometers
thick) which serves to electrically and mechanically isolate the
imager from the fluidics portion of the optofluidic microscope
device. Alternatively, instead of a PMMA film 37, a different type
of transparent or semi-transparent isolating material can be
used.
[0073] The new PMMA film 37, the prior PMMA layer 36, and any other
layer formed in the optofluidic microscope device may be deposited
using any suitable process. Exemplary processes include roller
coating, spin coating, vapor deposition, etc.
[0074] In the final assembly stage, as shown in FIG. 4(e), a PDMS
(poly dimethylsiloxane) structure 40 is pre-formed and is then
attached to the PMMA film 37. Access holes (not shown) are then
punched in the PDMS (poly dimethylsiloxane) structure 40 to form
fluid inlets and outlets. The PDMS structure 37 may be formed using
a soft lithography technique (well known in the art) and is then
exposed to air plasma for about 30 seconds. The PDMS layer 40 and
the PMMA film 37 may be laminated together. After assembly, an 80
degree C. post bake can be used to help improve bonding strength
between the various components of the optofluidic microscope
device.
[0075] Also, as shown in FIG. 4(e), a detector 43 including
discrete light detecting elements 43(a) is then attached to the
glass plate 32 using an adhesive or other suitable bonding
mechanism to form an optofluidic microscope device according to an
embodiment of the invention. As noted above, the light detector 43
may be a commercially available part.
[0076] Suitable electronics (not shown) may be connected to the
attached detector. Such electronics may comprise a computer
comprising image processing software, software for distinguishing
between different biological entities, signal processing software
and electronics, etc. Those of ordinary skill in the art know what
types of electronics can be used in embodiments of the invention to
form images from the imager in the optofluidic microscope device.
In addition, computer code for performing any of the signal
processing or other software related functions may be created by
those of ordinary skill in the art. The computer code in any
suitable programming language including C, C++, Pascal, etc.
[0077] C. Hole Spacing and Resolution
[0078] As noted above, in an exemplary optofluidic microscope
device, the transmission of light passing through holes in an
opaque gold layer is monitored by a detector such as a linear CCD
or photodiode array which is directly underneath the opaque layer.
This arrangement makes the optofluidic microscope device compact
and free of bulk optics. Each hole and the transmission of light
through each hole can uniquely map to a single a single CCD pixel
or photodiode. For example, the inter-hole spacing may be on the
order of about 13 microns so that the spacing is the same as the
spacing of the discrete light detecting elements in a commercially
available detector (e.g. a line scan sensor such as a Dalsa tall
pixel sensor IL-C6-2048).
[0079] The pixel resolution in y-direction, r.sub.y, perpendicular
to the flow direction, depends on the spacing of adjacent holes in
this direction. The more holes etched through the gold layer per
unit width, the higher the achievable resolution as defined by Eq.
(1) below,
r y = w n h ( 1 ) ##EQU00001##
where n.sub.h equals to the number of holes and w is the channel
width. For example, if the channel width is 40 .mu.m, the
y-direction pixel size would be 1 micron if there are 40 holes
across the fluid channel. In the x (flow) direction, the pixel size
is determined by the acquisition rate of the optical measurement
unit and the net velocity of the target (i.e., the resolution in
x-direction is equal to target moving speed, u, times the pixel
acquisition time .DELTA.t) as defined by Eq. (2),
r.sub.x=u.DELTA.t (2)
For example, if the target flow speed is 100 microns per second,
and the detector's reading rate is 1 KHz, the maximum resolution in
the x direction would be equal to about 0.1 micron.
[0080] The sensitivity of the optofluidic microscope device depends
on the total amount of light transmitted through each hole.
Assuming the opaque layer is perfectly conductive, two different
regimes of hole size (S.sub.h) are examined. They are as
follows.
S.sub.h>.lamda., Large hole limit--In this regime, the effective
transmission area A.sub.T is simply equal to the physical cross
section of the hole. S.sub.h<.lamda., Small hole limit--In this
regime, assuming that the hole is infinitesimally thin, Bethe
(Bethe H A, `Theory of diffraction by small holes`, physics Review,
66, 163 (1944)) showed that the effective transmission area is
proportional to the sixth power of the pinhole diameter.
[0081] In a recent work, De Abajo (F. de Abajo, 2002, "Light
transmission through a single cylindrical hole in a metallic film",
Optics Express, vol. 10, pp. 1475-1484) observed that the
transmission is further attenuated exponentially as a function of
the hole depth. Combining these two effects, the effective
transmission area can be expressed as:
A T = ( 16 .pi. 3 27 ) ( S h 6 .lamda. 4 ) exp ( - 4 .pi. d 0.586 2
S h 2 - 1 .lamda. 2 ) ( 3 ) ##EQU00002##
This formulation agrees well with the simulation data that De Abajo
reported. However, for the sake of better estimating optofluidic
microscope device performance, the finite conductivity of the
material and thus reply on the optics simulation needs to be taken
into account. The total transmission photon count for a pixel dwell
time .tau. (also equivalent to the inverse of frame rate) is given
by,
N T = IA T .tau. h c .lamda. ( 4 ) ##EQU00003##
where
h c .lamda. ##EQU00004##
is the energy that one single photon carries; I is illumination
intensity; and .epsilon. is the quantum efficiency of CCD
camera.
[0082] The dominating noise source includes the photon counting
noise (shot noise) and the receiver noise (n.sub.r.tau.). Thus, the
sensitivity can be expressed as:
SNR = N T N T + ( n r .tau. ) 2 ( 5 ) ##EQU00005##
Therefore, object imaging with a micron level resolution and 30 dB
sensitivity can be readily performed with the use of natural light
illumination. In principal, sub-wavelength resolution can be
achieved in an optofluidic microscope device by simply spacing the
adjacent holes in the y-direction at the desired resolution limit.
Since the holes are separated in x-direction by tens of microns,
their transmission contributions will be distinguishable from each
other on the CCD camera. State of the art nanofabrication
technology enables the creation of etching patterns with a
resolution within the tens of nanometers. Optofluidic microscope
devices with resolutions of below 100 nanometers can be
created.
[0083] D. Microfluidic Transport of Targets
[0084] On the micro and nano scale, fluid flow and particulate
transport can be accomplished using numerous different techniques,
the most popular of which include traditional pressure driven flow,
electrokinetic transport, discrete droplet translocation via
electrowetting, or thermocapillarity techniques.
[0085] While imaging features created with electron beam
lithography can be made as small as 20 nanometers, the ultimate
resolution of the optofluidic microscope can be limited by the
vertical and horizontal confinement stability of the targets.
Physical confinement requires that the channel size be on the order
of that of the target being imaged, which for smaller targets
(<0.5 micron) could mean channel sizes on the order of hundreds
of nanometers.
[0086] Electroosmotic transport results from the interaction of an
externally applied electric field with an electrical double layer.
The electrical double layer is a very thin region of non-zero
charge density near the interface (in this case a solid-liquid
interface) and is generally the result of surface adsorption of a
charged species and the resulting rearrangement of the local free
ions in solution so as to maintain overall electroneutrality. As it
is a surface driven effect, the electrokinetic velocity is nearly
independent of channel size. Therefore, electroosmotic transport is
better for the physical confinement and imaging ranges that are
ultimately desired.
[0087] In addition to physical confinement, fluidic confinement of
the targets will be equally desirable for final imaging stability.
A number of researchers have studied Brownian particle dynamics and
have demonstrated that particles in a shear flow tend to migrate to
a particular location where the various hydrodynamic forces acting
on the particle equilibrate. For pressure driven flow in low aspect
ratio microchannel systems, such as those described here, there
exists a strong shear gradient in the vertical direction which will
tend to confine the targets at a location roughly 40% of the
distance from either the upper or lower surface of the channel to
the midpoint (i.e., there are two vertical equilibrium positions).
In the horizontal direction, however, there is no significant
velocity gradient (i.e., in low aspect ratio channels the parabolic
velocity profile in the horizontal direction tends to be very weak)
and thus no mechanism to stabilize the targets against Brownian
diffusion after it is initially focused into the channel. Recent
advancements in localized modification of the electroosmotic
mobility in microfluidic devices could allow for velocity profile
tuning thereby creating a single shear equilibrium position for
confinement against Brownian motion in both the vertical and
horizontal directions. Such tuning is difficult to perform with
traditional pressure driven flow. Brownian motion effects and ways
to address these effects are discussed in further detail below.
[0088] An electrokinetic driven microfluidic setup is shown in FIG.
2. As seen in FIG. 2, the fluid from the two side branches acts as
a focusing force. The central channel contains the biological
targets which are confined to the center of the imager through
upstream focusing. By dynamically adjusting the voltage applied to
the fluid, biological targets can be driven faster to get into the
detection region but slower when they are passing the detection
region for the purpose of obtaining more pixel information for
horizontal direction. As noted above, the opaque or semi-opaque
layer (e.g., perforated gold layer) is isolated from the electrical
ports by a thin layer of PMMA. The PMMA layer and the PDMS channels
are treated with oxygen plasma for tighter sealing and better flow
properties. Embodiments of the invention require a voltage gradient
no higher than 50 V in the target transportation channels and less
than 30 V in the focusing channels to drive the biological targets
with an appropriate speed for image acquisition.
[0089] E. Exemplary Experimental Results
[0090] As mentioned above and as illustrated in FIG. 3, with the
full information of the transmission time traces, the geometrical
profiles of the imaging targets can be reconstructed. A biological
target that was imaged was Chlamydomonas provided by Carolina
biological supply. Chlamydomonas is a single-celled, biflagellate,
green alga. It is roughly circular in shape and has a diameter
ranging from about 10-20 microns. It contains several species that
have become popular as research tools, because it has well defined
genetics that can transformed by well developed techniques. In
these initial experiments, an OFM configuration similar to the one
shown in FIG. 1 was used. The transmission changes through each
hole were recorded by an 8-bit Sony XCD-X710 firewire CCD camera,
mounted on Olympus IX-71 inverted microscope.
[0091] FIG. 5 shows the transmission change of two adjacent holes
when a single Chlamydomonas cell passes over them. Ten continuous
picture frames are streamed into a computer program from which the
pixel information from each hole is extracted. It can be seen from
FIG. 5 that light transmission through holes does respond
dynamically to the object flowing across the imaging region. If
more frames are rapidly collected, more detailed pixel information
can be obtained, and a two dimensional image of a biological sample
can be subsequently regenerated.
[0092] FIG. 6(a) shows a transmission image of the optofluidic
microscope device. The holes are smaller than they appear (0.5
microns). FIG. 6(b) shows occlusion time traces through adjacent
holes. FIG. 6(c) Microscope image of a chlamoydomonas. FIG. 6(d)
shows preliminary data showing an optofluidic microscope device
image of a chlamoydomonas.
[0093] F. Brownian Motion
[0094] The imaging method employed in optofluidic microscope
devices assumes that the flow of the target object across the hole
array is straight and undeviated during the entire trajectory. Any
deviation from a straight trajectory can distort the processed
object image.
[0095] The achievable image resolution is bounded by the effective
deviation of the object along its trajectory. Unintended object
trajectory deviations can be caused by temperature gradients, bulk
system vibrations, and Brownian motions. While the first two can be
minimized by careful system design, there is little that can be
done to suppress Brownian motions directly.
[0096] The root mean square deviation along a single dimension
{square root over (x.sup.2)}, of a spherical particle of diameter l
in a fluid of viscosity .eta. on a time scale of t is given by:
x 2 = 2 k B T 3 .pi..eta. l t ( 6 ) ##EQU00006##
where k.sub.B is the Boltzmann constant and T is the system
temperature. An object of diameter 10 microns drifting in room
temperature water will experience a mean deviation of 29 nanometers
in one dimension for a time period of about 10 milliseconds. There
is also a Brownian motion actuated rotation. However, the relative
extent of the rotation and its effect on resolution is small
compared with the translational Brownian motion deviation
effect.
[0097] The deviation's inversion dependency on particle size
implies that the degrading effect of Brownian motion on the
achievable resolution increases with smaller objects. Indeed, an
optofluidic microscope device with 30 nanometer resolution and line
scan acquisition rate of 10 kHz can be expect to achieve a
100.times.100 pixel image with image resolution of 70 nanometers
when it is used to image a microbe of size 4 microns. It is noted
that when high resolution optofluidic microscope devices are used
to image relatively large bioentities that are not as pronouncedly
impacted by Brownian motion artifacts, the optofluidic microscope
devices can easily achieve their predicted resolution.
[0098] It is possible to experimentally study and verify a method
for correcting motional artifacts attributable to Brownian motions.
The method involves building a tracking system into the optofluidic
microscope device that is capable of tracking the motional drifts
of the target object as it passes through the detection array.
[0099] FIG. 7 shows an exemplary embodiment including tracking
light transmissive regions. In FIG. 7, tracking sets of light
transmissive regions 42 may be interleaved with imaging light
transmissive regions 44. In this example, the tracking light
transmissive regions 42, each have identical sets of five or six
holes and are generally oriented along the center of the fluid
channel 22. In comparison, imaging light transmissive regions 44
form of a slanted array of light transmissive regions that extends
from one wall of the fluid channel 22 to the other wall of the
fluid channel 22. The configuration of the illumination source (not
shown) and the detector (not shown) can be as in the previously
described embodiments.
[0100] Using the tracking light transmissive regions 42, it is
possible to track the lateral drift of the object 20 with each
tracking set of light transmissive regions 42 as the object flows
across the hole array. As the tracking sets are identically placed
in the middle of the channel, any signal drift across adjacent set
of light transmissive regions 42 can be correlated to the net drift
of the object in the y-direction. This drift information can be
used to modify any images that are formed using the optofluidic
microscope device. The change in the arrival time of the object 20
between tracking sets of light transmissive regions 42 can be
correlated to the net drift of the object 20 in the
x-direction.
[0101] The performance of the optofluidic microscope device can be
expected to improve if more tracking sets are used. The resolution
uncertainty associated with Brownian motion artifacts should
decrease as the square root of the number of tracking set used
(assuming the tracking set is regularly spaced). The construction
and implementation of optofluidic microscope devices with tracking
systems is straightforward.
[0102] G. Embodiments Using Light Transmissive Regions and
Fluorescence
[0103] Some embodiments of the invention use light transmissive
regions and fluorescence to image objects. As background for these
embodiments, a hole in a thick opaque or semi-opaque conductive
layer will effectively transmit only light of wavelength shorter
than the cutoff wavelength for zero mode propagation. Light of
longer wavelengths is transmitted less efficiently. The approximate
formulation of this transmission behavior as a function of the
effective transmission area is given by:
A transmission = ( 16 .pi. 3 27 ) ( s h 6 .lamda. 4 ) exp ( - 4
.pi. d 0.586 2 s h 2 - 1 .lamda. 2 ) . ( 7 ) ##EQU00007##
where .lamda. is the wavelength, d is the conductive layer
thickness and s.sub.h is the diameter of the hole. This equation is
mentioned above. For a sufficiently thick conductive layer, the
transmission drops very sharply when .lamda. exceeds s.sub.h/0.586.
If "d" is large, the transmission curve will look like a step
function.
[0104] The approach for performing simultaneous fluorescence and
transmission imaging of the object involves illuminating the sample
with the appropriate excitation light field (at wavelength
.lamda..sub.ex). Fluorophores within the sample will absorb and
re-emit at a different wavelength .lamda..sub.f;
.lamda..sub.f>.lamda..sub.ex. Assuming a uniform illumination
field, the transmission at wavelength .lamda..sub.ex will project a
transmission image of the object, while the fluorescence pattern
from the sample projects a fluorescence image of the object. By
using both fluorescence and transmission imaging modes, more
accurate object images can be produced.
[0105] Referring to FIG. 8, it is possible to acquire both images
by using an optofluidic microscope device with a pair of interlaced
lines of holes 214. The hole size for the first line (or set) of
holes 211 can be chosen so that the holes 212 will transmit light
of both wavelength .lamda..sub.f and .lamda..sub.ex. The hole size
for the second line of holes 212 will be chosen so that it will
only transmit light of wavelength .lamda..sub.ex. The lines of
holes 211, 212 can be arranged such that each pair of holes is at
the same lateral displacement from the channel walls, so that they
will interrogate the same line across the object. The transmission
through the second line of holes 211 can then be used to generate a
transmission microscope image of the object 20 flowing through the
fluid channel. The fluorescence image of the object can be
generated by taking the difference of the transmission through the
first line of holes 211 and the second line of holes 212. The
configuration of the illumination source (not shown) and the
detector (not shown) can be as in the previously described
embodiments.
[0106] H. Optofluidic Microscope Device Applications
[0107] Schematic diagrams of a system using optofluidic microscope
devices according to embodiments of the invention is shown in FIGS.
9(a)-9(c). Any of the previously described optofluidic microscope
devices can be used in a system such as the one illustrated in
FIGS. 9(a)-9(c).
[0108] As shown in FIG. 9(a), the system is used as a blood
differential unit that is capable of performing blood cell
identification and counting. The system includes a number of fluid
inlets (e.g., as designated by "sample in" and "reagent in" and
fluid outlets (e.g., as designated by "waste out". As shown, there
is a mixing changer, and three optofluidic microscope devices in
series. The outlets to the optofluidic microscope devices converge
into a single recycle stream that is fed back into a "sample in"
fluid inlet. FIG. 9(b) shows fluids as they pass into the system.
FIG. 9(c) shows fluids during operation when fluid is recycled back
to a sample in fluid stream.
[0109] An exemplary operational procedure is as follows. First, the
blood sample of interest and the differential reagent are flowed
into a mixing chamber. The differential reagent employed may be
varied based on the blood fraction of interest and may include a
red blood cell lysing agent, a chemical stain (such as Chlorazole
Black), fixing reagent or a diluent. Given the relatively large
volume (.about.1 microliter) of the blood sample and reagent used
in this step, the flow may be pressure driven or electrokinetically
actuated. Second, the mixture is allowed a sufficient period of
time to mix and react (about 10 seconds). Third, the mixture is
then driven across the optofluidic microscope devices by
electrokinetics. The proposed flow channel will be 20 microns in
dimension and each optofluidic microscope device will be designed
to have a resolution of 500 nanometers. The optimal flow rate would
be about 6.0 millimeters/second.
[0110] Each optofluidic microscope device may be fabricated to
incorporate a bandpass filter between the device and the associated
CCD array. The bandpass filters can provide filtering at 500-550
nm, 550-600 nm, 600-650 nm and 650-700 nm, in order to span a
reasonable portion of the visible spectrum. The specifics of the
filter may be readjusted based on the spectral components that are
of the greatest interest.
[0111] In the illustrated embodiment, the expected imaging speed of
1 cell per 1.6 milliseconds. The acquired multi-spectral image data
for each cell is processed manually or through an automated
program. The processed information can be used to differentiate
cells from each other and processed cells may then be channeled to
the appropriate collection or disposal reservoir by biasing the
correct output voltage at the correct reservoir.
[0112] As an additional processing step, any given sorted blood
fraction may be reprocessed through the entire system by simply
channeling the desired fraction to the input of the system. This
fraction may then be mixed with a different differential reagent
and imaged and resorted to further distinguish different components
in the fraction or simply to collect more information about the
fraction as a whole. The ability to reprocess sorted fractions with
little or no loss is unique to a microfluidic based cell sorter (a
conventional cell cytometry system is generally not designed for
reprocessing). This advantage is likely to be a significant factor
for future cell sorting applications that use multi-stage
processing.
[0113] The speed of cell identification and sorting is comparable
to that of a commercial blood differential unit (which processes
cells at a speed of 1 cell per 200 microseconds). However, the
processing speed of embodiments of the invention can be
significantly increased by an order of magnitude by simply
increasing the number of systems in operation simultaneously on the
same chip. Further, embodiments of the invention are more precise
and accurate with respect to sorting. In particular, embodiments of
the invention can outperform commercial blood differential units in
distinguishing between band cells and mature neutrophils, and in
the counting of nucleated red blood cells.
[0114] The optofluidic microscope device according to embodiments
of the invention can potentially be used to identify circulating
tumor cells (CTC) in blood. Given that circulating tumor cells
(CTC) are generally distinguished and classified based on a visual
inspection of their cell or nucleus morphology, an optofluidic
microscope based cell sorting system can be used for automated CTC
cell counting. The optofluidic microscope device imaging method
will be useful in a wide variety of applications, ranging from its
incorporation into microfluidic based flow cytometer as an on-chip
imaging system, to its use as a high throughput analysis system for
the identification and counting of different bacteria types in
urine.
[0115] II. Optofluidic Devices Using Light Emitting Elements
[0116] In conventional optical microscopy, the maximum achievable
resolution, as defined by the Rayleigh criterion, is theoretically
limited to .lamda./2 where .lamda. is the optical wavelength of the
light involved. (It is also noted that the hole-based method
described above will allow for a resolution greater than the
.lamda./2 limit.) Through the use of novel techniques, such as near
field microscopy, entangled photon microscopy, Stimulated Emission
Depletion (STED) microscopy, and structured illumination
microscopy, it is possible to overcome this limitation to some
extent. However, the computational costs, system complexity and
optical power requirements rise exponentially as a function of the
desired resolution.
[0117] Disclosed are other embodiments which can provide images of
entities with sub-wavelength resolution in a cost effective manner.
These embodiments use a fluorophore array or grid, in which each
array or grid point will have a fluorophore that possesses a narrow
and distinct fluorescence or Raman emission spectra from the other
fluorophores in the array or grid. Suitable fluorophores include
quantum dots. Quantum dots can be engineered with distinct, narrow
emission spectra and are strong and compact fluorescence emitters.
Quantum dots are commercially available and are described in the
following exemplary publications: J. K. Jaiswal, H. Mattoussi, J.
M. Mauro, S. M. Simon, Long-term multiple color imaging of live
cells using quantum dot bioconjugates Nature Biotechnology 21, 47
(January 2003); and M. Dahan, T. Laurance, F. Pinaud, D. S. Chemla,
A. P. Alivisatos, M. Sauer, S. Weiss, Time-gated biological imaging
by use of colloidal quantum dots Optics Letters 26, 825 (2001)).
While embodiments of the invention are described using quantum
dots, embodiments of the invention are not limited to quantum
dots.
[0118] There are a number of potential applications for the
embodiments of the invention that use light producing elements. For
example, embodiments of the invention can be used as a means to
profile bacteria or virus shape as a way to identify their species,
as a means to profile a large protein shape, as a means to measure
and obtain the shape of a cell nucleus as a way to identify
cancerous cells, a means to measure the refractive index profile of
a cell, and as a means to identify the shape of small objects for
quality control or other purposes in manufacturing, synthesis and
production.
[0119] An exemplary optofluidic microscope device including light
emitting elements is shown in FIG. 10(a). In FIG. 10(a), a grid of
quantum dots 62 is embedded in or present on a surface 16(a) of a
body. Each quantum dot 62 has a distinct fluorescence emission
spectrum from those of the other quantum dots 62. As shown in FIG.
10(a), an object 20 is on the grid of quantum dots 62 and is imaged
using the quantum dots 62.
[0120] The quantum dots 62 may have sizes on the order of about 10
nanometers, and the spacings between adjacent quantum dots can be
as small as 10 nanometers. Other grid spacings and other sizes of
quantum dots can be used in other embodiments of the invention.
[0121] To image the shape and size of an object 20, the object 20
is placed on top of the grid of quantum dots 62 and is subjected an
uniform illumination field from an illumination source (not shown).
Quantum dots 62 that are under the object 20 will receive less
illumination and will therefore fluoresce less.
[0122] By collecting the fluorescence from the entire grid and
spectrally resolving the spectrum with the use of a commercially
available spectrometer (or other detector), it is possible to
determine which quantum dots 62 are covered. For example, the
positions of the quantum dots 62 and the respective fluorescent
emission spectra associated with quantum dots 62 are known. Using a
spectrometer, a computer can determine which fluorescent spectra
have been received by the spectrometer and can consequently
determine which specific grid points are covered by the object 20.
From this, the shape of the object 20 can be derived with a
resolution that is limited only by spacing of the quantum dots
62.
[0123] Instead of composing a grid of quantum dots, an alternate
configuration for the microscope can be one where a linear array of
distinct quantum dots 62 is employed. The quantum dots 62 are on or
under the surface 16(a). This is shown in FIG. 10(b). In FIG.
10(b), distinct quantum dots 62 are disposed in a line that runs
perpendicular to the direction of the flow of the object 20. As in
the previously described embodiments, it is possible to measure the
fluorescence spectra of the quantum dots 62 over time to determine
which the quantum dots 62 are covered. In this example, the object
20 flows across the array of quantum dots 62 through a fluid
channel 22. The time changing fluorescence spectrum can then be
processed to provide sufficient data to determine the shape of the
object 20.
[0124] The arrangement of the detector and the illumination source
in the optofluidic microscope device embodiments shown in FIGS.
10(a) and 10(b) can vary. Two exemplary configurations are shown in
FIGS. 11(a) and 11(b). FIGS. 11(a) and 11(b) are side schematic
views of embodiments of the invention. In FIGS. 11(a) and 11(b),
like numerals designate like elements.
[0125] In FIG. 11(a), an illumination source 420 may provide light
of a predetermined first wavelength. The light of a first
wavelength 430 may excite a quantum dot 62 disposed on or within
the body 410(a) so that light of a second wavelength 431 is
provided by the quantum dot 62. The light of the second wavelength
431 may be received by the detector 430. When the cell 20 passes
over the quantum dot 62, it blocks or modulates the light of the
first wavelength 20 so that the light emitted from the quantum dot
62 is different than the light of the second wavelength 431 that
was previously received by the detector 430. In this example, the
body 410(a) may be transparent, semi-transparent, or opaque, since
the illumination source 420 and the optical detector 430 are above
the quantum dot 62 and the body 410(a).
[0126] FIG. 11(b) shows another embodiments with a different
configuration. In this embodiment, the detector 430 is under the
body 410(b). In this embodiment, the body 410(b) is transparent or
semi-transparent so that the light of the second wavelength 431 can
pass through it to the detector 430.
[0127] Other yet other embodiments, if the bodies 410(a), 410(b)
are transparent or semi-transparent, the illumination source 420
could alternatively be on the under the bodies 410(a), 410(b). The
detector 430 could be positioned over or under the bodies 410(a),
410(b).
[0128] An alternate microscope having a microfluidic channel can be
fabricated. This is shown in FIG. 12. The quantum dots 62 are laid
down in two or more sets 64 of lines 62(a) on the substrate 22.
Each line 62 has the same distinct quantum dots 62, and each line
will have a different emission spectrum from the other lines 62.
The orientation of each set 64 can be different. In the embodiment
shown in FIG. 14, the fluorescence spectrum can be measured to
determine occlusion still applies. In this case, the extent of
fluorescence diminishment in a specific spectral band is an
indication of the extent to which a specific line of quantum dots
is covered by the object.
[0129] After making a measurement using a set of lines, the object
20 flows to the next set 64 of quantum dot lines. The transport is
performed in a manner that the orientation of the object 20 is
unperturbed. The process is repeated for all line sets 64. By
performing computed tomography computations on the line sets 64,
one can determine the shape of the object 20.
[0130] The configuration in FIG. 12 has advantages over the
previous configurations. For example, the shape of the object 20 on
an N.times.N grid can be found with the use of N distinct types of
quantum dots. In the prior embodiments described above, the same
resolution requires the use of N.sup.2 distinct types of quantum
dots.
[0131] Referring to FIGS. 13 and 14, if the object is too
transparent and does not occlude a sufficient amount of the
excitation light field from the quantum dots to sufficiently
diminish the emission from the quantum dots, embodiments of the
invention can instead rely on the use of an evanescent excitation
field.
[0132] The excitation light field 70 is directed toward the quantum
dots 62 on or under the surface 16(a) of the fluid channel 22 in a
body. It is carefully adjusted such that its angle of incidence on
the glass-channel interface is over the critical angle. In this
situation, aside from an exponentially decaying evanescent field,
very little of the optical excitation field will reach the quantum
dots 62 and the quantum dots 62 will not fluoresce or fluoresce
very weakly. If an object 20 with a higher refractive index than
the liquid medium flowing in the channel were to cross the path of
the excitation light field 70, it will see a change in the
refractive index.
[0133] As shown more clearly in FIG. 14, if the new refractive
index formed by the presence of the object 20 is sufficiently high
enough to change the critical angle to a larger value, then the
optical excitation field will now become a propagating field 82 in
the fluid channel 22. The propagating field 82 can thereafter
excite the quantum dot 62 under the object 20 and an imaging system
122 (which may include an optical filter) and detector 124 can
receive the light produced by the quantum dot 62. In these
embodiments, a fluorescence signal from a particular array grid
point indicates that a particular quantum dot 62 at that grid point
is occluded.
[0134] The optofluidic microscope device embodiments described with
reference to FIGS. 13 and 14 can be used for other purposes. For
example, they can be used to interrogate the object's 20 refractive
index profile. By changing the angle of incidence of the excitation
light field and determining when the transmission becomes a
propagating wave through the object 20, one can accurately measure
its refractive index profile.
[0135] FIG. 15 shows yet another optofluidic microscope device
according to an embodiment of the invention. The embodiments used
quantum dots that were laid down in a staged line pattern. The
difference in fluorescence signal associated with adjacent lines
could be attributed to a specific location on the object.
[0136] An optofluidic microscope device like the one shown in FIG.
15 can also be implemented with slits (not shown) instead of lines
of quantum dots. If the slits are sufficiently narrow and they are
illuminated from the bottom, an evanescent field will be set up on
the top of the substrate. The flow of an object over a given slit
region will turn the evanescent wave to a propagating one. By
measuring the total transmission, one can determine a similar set
of information as that obtained using the described quantum dot
approach.
[0137] The optofluidic microscope devices including light emitting
elements can be made in any suitable manner. In one example, to
create the microscope configuration shown in FIG. 15, a
photolithography and etching process can be used to etch out the
requisite lines in a body (e.g., a substrate). Each conductive line
can then be selectively charged, and a solution containing
non-distinct quantum dots that are oppositely charged can be made
to attach to the chosen line by flowing the quantum dots across the
lines. The adhesion of the quantum dots to the charged line can
then be made permanent by coating over the adhered substrate. The
process can then be repeated with a different conductive line and a
different set of non-distinct quantum dots until the complete line
sets are completed. The resolution of the quantum dot array grid is
only limited by the achievable lithography etching resolution.
[0138] In another approach, a fine needle tip actuator system may
be used. In this approach, the creation of a single grid point uses
a mixture of quantum dots and a carrier such as an epoxy. The
deployed needle tip will then be dipped into the mixture and moved
onto the right location on the substrate to deposit a single drop
of the mixture. The process will then be repeated for different
quantum dots-epoxy mixture until the entire grid is completed. The
resolution of the quantum dot grid is only limited by the
achievable fidelity of the needle tip to move to a specific
location.
[0139] Quantum dots can be made with emission linewidths of about
10 nanometers. If one assumes that the accessible emission spectrum
that one can observe the fluorescence is about 300 nanometers, this
will imply that it is possible to distinctively detect 30 different
types of quantum dots. In this situation, the grid dimension
proposed in the microscope configuration shown in FIG. 10 cannot be
larger than 5.times.5. Larger grid sizes can be achieved if
different spectrometers are dedicated to different sub-sections of
the grid. In this scenario, the highest grid density is given by
equating the 5.times.5 subgrid to an area of (1/2
wavelength).times.(1/2 wavelength). A tighter grid arrangement
cannot be adequately resolved by the collection optics of the
spectrometer. This, in turn, implies that the resolution achievable
by this microscope configuration is about 100 nanometers.
[0140] Lastly, the achievable resolution can be a direct function
of the number of distinct quantum dot types that are available. As
such, it is highly desirable to tailor quantum dots with even
narrower emission bandwidth. As an alternative to this,
fluorescence resonance energy transfer spectroscopy (or some
associated phenomena) could be used. For example, dye pairs can be
fabricated whereby one component of the dye pair absorbs light,
while the other emits light. The absorptive component can be
engineered to have a narrow absorption bandwidth and the emittive
component a narrow emittive component. By different permutative
combinations of these dye pairs with distinct absorptive and
emittive wavelengths, it is possible to create a much larger number
of non-degenerate dye types.
[0141] III. Other Optofluidic Device Embodiments
[0142] FIG. 16 shows yet another optofluidic microscope device
embodiment. In this embodiment, the substrate 22 is embedded with
an angled linear array of distinct quantum dots or nanoparticles.
The horizontal separation between the quantum dots 88, distance u
112, is arranged to be sufficiently large so that it is possible to
resolve two distinct dots from each other when the arrangement is
imaged onto a CCD camera or other imaging device. Distance u can be
equal or larger than the pixel size of the CCD camera if one is
performing one-to-one imaging. Distance v 110 sets the resolution
of this microscope and can therefore be as small as possible.
Ideally, it is equal to the size of the nanoparticles involved.
[0143] The object 20 flows through the fluid channel 22 and the
fluorescence spectrum from each quantum dot 88 is monitored on an
imaging CCD camera (not shown). The CCD camera can be mounted
underneath the surface 16(a) if the surface 16(a) forms part of a
transparent body. Otherwise, it is possible to image the object 20
from above. However, this requires some degree of care in ensuring
that the total internal reflected light from the original input
beam isn't detected by the CCD. As the quantum dots 88 are
sufficiently spaced apart, each quantum dot 88 will be distinctly
associated with a pixel on the CCD. Assuming that the shape of the
object 20 is unchanged in its transition across the quantum dots
88, it is possible to determine its shape from time traces of the
CCD signals.
[0144] Whereas the previous microscopy configuration can be thought
of as approaches for spectrally encoding sub-wavelength spatial
information, this method can be interpreted as an approach for
spatially encoding sub-wavelength spatial information into
above-wavelength spatial information.
[0145] There are two distinct advantages associated with this
approach. First, the fluorophores do not have to be distinct. The
entire array of quantum dots can be non-distinct. This simplifies
the fabrication procedure for the device. There is no need for
distinct quantum dots. Eliminating the requirement of distinct
quantum dots allows for the use of other contrast mechanisms. One
contrast mechanism may involve the use of nanoparticles.
Nanoparticles have enhanced scattering cross-sections compared to
their physical cross-sections. If they are used in place of the
quantum dots, their scattering signals can be used in place of
fluorescence signals generated from the quantum dots.
[0146] As a rough calculation, if one wants a resolution of 10
nanometers and the object is one micron in dimension, a
microfluidic channel that is at least 1 millimeter long (assumes
one-to-one imaging onto a 10 micron pixel size CCD, u=10 micron,
v=10 nanometers) can be used.
[0147] Referring to FIG. 17, another configuration is shown. This
configuration is similar to some of the above-described
embodiments. Instead of embedding individual quantum dots in a
spaced grid, it is possible to lay down a line of quantum dots 160
in the bottom surface 16(a) of a fluid channel 22. The line of
quantum dots 160 can have different segments 152, 154 that map onto
discrete light detecting elements in a detector (not shown).
[0148] The embodiment in FIG. 17 can be formed by creating a trench
in a body and then depositing quantum dots into the trench. As long
as each segment of the quantum dot line maps to a single pixel on
the CCD, the resolving capability of this embodiment is comparable
to the previous embodiments. It is also noted that light-scattering
nanoparticles can be used in place of the quantum dots in other
embodiments.
[0149] The embodiment shown in FIG. 17 has advantages. First,
compared to prior embodiments, there is a much larger signal due to
the larger number of fluorophores or quantum dots contributing to
the signal. Second, the embodiment shown in FIG. 17 is easy to
fabricate. Embedding a line of fluorophores should be easier than
laying down regularly spaced fluorophores.
[0150] FIG. 18 shows a configuration is another variant of the
configuration in FIG. 17. In this case, a transmissive or
reflective line 178 across the body of the optofluidic microscope
device. The reflected or transmitted signal is imaged in a similar
fashion onto a CCD or other detector. Different segments 174, 176
of the line 178 may correspond to discrete light detecting elements
in a detector that may be under the surface 16(a) forming the fluid
channel 22. The processing of the signals from the detector to
determine the shape of the object 20 is the same as in the
previously described embodiments. This embodiment is easy to
fabricate.
[0151] Yet other embodiments are also possible. For example, it is
possible to use a set of periodically textured bull's eye ring
patterned transmissive holes, such as those described in H. J.
Lezec, A. Degiron, E. Davaux, R. A. Linke, L. Martin-Moreno, F. J.
Garcia-Vidal, T. W. Ebbesen, Beaming Light from a Subwavelength
Aperture Science 297, 820 (2002). An appropriately designed
patterned hole will be able to transmit light efficiently with a
small divergence angle. The well confined light transmission can
dramatically cut down on any blurring artifact during the
transmission process.
[0152] The terms and expressions which have been employed herein
are used as terms of description and not of limitation, and there
is no intention in the use of such terms and expressions of
excluding equivalents of the features shown and described, or
portions thereof, it being recognized that various modifications
are possible within the scope of the invention claimed.
[0153] Moreover, one or more features of one or more embodiments of
the invention may be combined with one or more features of other
embodiments of the invention without departing from the scope of
the invention.
[0154] All patents, patent applications, publications, and
descriptions mentioned above are herein incorporated by reference
in their entirety for all purposes. None is admitted to be prior
art.
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