U.S. patent number RE44,711 [Application Number 13/287,093] was granted by the patent office on 2014-01-21 for optoelectronic tweezers for microparticle and cell manipulation.
This patent grant is currently assigned to The Regents of the University of California. The grantee listed for this patent is Pei-Yu Chiou, Aaron T. Ohta, Ming Chiang Wu. Invention is credited to Pei-Yu Chiou, Aaron T. Ohta, Ming Chiang Wu.
United States Patent |
RE44,711 |
Wu , et al. |
January 21, 2014 |
Optoelectronic tweezers for microparticle and cell manipulation
Abstract
An optical image-driven light induced dielectrophoresis (DEP)
apparatus and method are described which provide for the
manipulation of particles or cells with a diameter on the order of
100 .mu.m or less. The apparatus is referred to as optoelectric
tweezers (OET) and provides a number of advantages over
conventional optical tweezers, in particular the ability to perform
operations in parallel and over a large area without damage to
living cells. The OET device generally comprises a planar
liquid-filled structure having one or more portions which are
photoconductive to convert incoming light to a change in the
electric field pattern. The light patterns are dynamically
generated to provide a number of manipulation structures that can
manipulate single particles and cells or group of particles/cells.
The OET preferably includes a microscopic imaging means to provide
feedback for the optical manipulation, such as detecting position
and characteristics wherein the light patterns are modulated
accordingly.
Inventors: |
Wu; Ming Chiang (Moraga,
CA), Chiou; Pei-Yu (Los Angeles, CA), Ohta; Aaron T.
(Honolulu, HI) |
Applicant: |
Name |
City |
State |
Country |
Type |
Wu; Ming Chiang
Chiou; Pei-Yu
Ohta; Aaron T. |
Moraga
Los Angeles
Honolulu |
CA
CA
HI |
US
US
US |
|
|
Assignee: |
The Regents of the University of
California (Oakland, CA)
|
Family
ID: |
35150549 |
Appl.
No.: |
13/287,093 |
Filed: |
November 1, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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60561587 |
Apr 12, 2004 |
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Reissue of: |
11105304 |
Apr 12, 2005 |
7612355 |
Nov 3, 2009 |
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Current U.S.
Class: |
250/559.04;
204/603; 204/547 |
Current CPC
Class: |
B03C
5/026 (20130101); B01L 3/502761 (20130101); G01N
2035/1046 (20130101); B01L 2400/0454 (20130101) |
Current International
Class: |
G01N
21/86 (20060101); G01V 8/00 (20060101) |
Field of
Search: |
;204/450,600,603,451,601,547,643 ;250/208.1,551,559.04
;430/58.05,60,58.7,70,159,57.5 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Chiou et al.--"A Novel Optoelectronic Tweezer Using Light Induced
Dielectrophoresis"--Proc. IEEE/LEOS Int. Conf. on Optical MEMS and
their applications (OMEMS 2003) Aug. 2003, pp. 8-9. cited by
applicant .
Chiou et al.--"Cell Addressing and Trapping Using Novel
Optoelectronic Tweezers"--Proc. IEEE MEMS 2004, Jan. 2004, pp.
21-24. cited by applicant .
Chiou et al.--"Light Actuated Microfluidic Devices"--Proc. IEEE
MEMS, Kyoto, Japan, Jan. 2003, pp. 355-358. cited by applicant
.
Chiou et al.--"Light Actuation of liquid by
optoelectrowetting"--Sensors and Actuators A, vol. 104, Mar. 2003,
pp. 222-228. cited by applicant .
Chiou et al.--"Optical Actuation of Microfluidics Based on
Opto-Electrowetting"--Solid State Sensor, Actuator and Microsystems
Workshop, Hilton Head, South Carolina, Jun. 2-6, 2002, pp. 269-272.
cited by applicant .
Chiou et al.--"Pico Liter Droplet Manipulation Based on a Novel
Continuous Opto-Electrowetting Mechanism"--Proc. IEEE Transducers,
2003, pp. 468-471. cited by applicant .
Hayward et al.--"Electrophoretic assembly of colloidal crystals
with optically tunable micropatterns"--Nature, vol. 404, Mar. 2,
2000, pp. 56-59. cited by applicant .
Lui et al.--"Virtual Particle Channels Based on Optical
Dielectrophoresis Forces"--Proc. IEEE/LEOS Int. Conf. on Optical
MEMS, Aug. 2004, pp. 20-21. cited by applicant .
Ozkan et al.--"Optical Addressing of Polymer Beads in
Microdevices"--Sens. Mater., vol. 14, 2002, pp. 189-197. cited by
applicant .
Hossack et al.--"High Speed Holographic Optical Tweezers using a
Ferroelectric Liquid Crystal Microdisplay"--Optics Express, vol.
11, No. 17, 2003, pp. 2053-2059. cited by applicant .
Japanese Patent Office, Notification of Reason for Refusal issued
on Jan. 24, 2011, related Japanese Patent Application No.
2007-507569, original Japanese language copy (pp. 1-4), English
translation (pp. 5-7), with claims (pp. 8-18), counterpart to
PCT/US05/12416, claiming priority to U.S. Appl. No. 61/561,587,
(pp. 1-18). cited by applicant .
Japanese Patent Office, Decision of Refusal issued on Feb. 15,
2012, related Japanese Patent Application 2007-507569, original
Japanese language copy (pp. 1-3), English translation (pp. 4-5),
with claims (pp. 6-17), counterpart to PCT/US05/12416, claiming
priority to U.S. Appl. No. 61/561,587, (pp. 1-17). cited by
applicant .
United States Patent & Trademark Office (USPTO), International
Search Report and Written Option issued on Jun. 17, 2008, including
claims searched, related PCT International Application No.
PCT/US05/12416, pp. 1-18. cited by applicant .
European Patent Office, First Office Action (pp. 1-9) issued on
Feb. 8, 2013 for related European Patent Application No. 05
745418.3-1232 (PCT/US05/12416) with claims examined (pp. 10-16) pp.
1-16 (All references, D1-D5, were submitted with IDS filed on Apr.
22, 2012). cited by applicant .
W.J. Hossack et al., "High Speed Holographic Optical Tweezers Using
a Ferroelectric Liquid Crystal Microdisplay," Optics Express, vol.
11, No. 17, pp. 2053-2059 (2003). cited by applicant .
R.D. Hayward et al., "Electrophoretic Assembly of Colloidal
Crystals with Optically Tunable Micropatterns," Nature, vol. 404,
Mar. 2, 2000, pp. 56-59. cited by applicant .
M. Ozkan et al., "Optical Addressing of Polymer Beads in
Microdevices," Sens. Mater., vol. 14, pp. 189-197 (2002). cited by
applicant .
Y.-S. Lui et al., "Virtual particle Channels Based on Optical
Dielectrophoresis Forces," Proceedings IEEE/LEOS International on
Optical MEMS, pp. 20-21, Aug. 2004. cited by applicant .
Pei Yu Chiou et al., "Cell Addressing and Trapping Using Novel
Optoelectronic Tweezers," Proc. IEEE MEMS 2004, pp. 21-24 (2004).
cited by applicant .
Pei Yu Chiou et al., "A Novel Optoelectronic Tweezer Using Light
Induced Dielectrophoresis," Proceedings IEEE/LEOS International
Conference on Optical MEMS and Their Applications (OMEMS'03), 2003,
pp. 8-9. cited by applicant .
Pei Yu Chiou et al., "Optical Actuation of Microfluidics Based on
Opto-Electrowetting," Solid-State Sensor, Actuator and Microsystems
Workshop, Hilton Head, South Carolina, Jun. 2-6, 2002, pp. 269-272.
cited by applicant .
Pei Yu Chiou et al., "Light Actuated Microfluidic Devices," Proc.
IEEE MEMS, Kyoto, Japan, Jan. 2003, pp. 355-358. cited by applicant
.
Pei Yu Chiou et al., "Light Actuation of Liquid by
Optoelectrowetting," Sensors and Actuators A, vol. 104 (2003), pp.
222-228. cited by applicant .
Pei Yu Chiou et al., "Pico Liter Droplet Manipulation Based on a
Novel Continuous Opto-Electrowetting Mechanism," Pro. IEEE
Transducers 2003, pp. 468-471. cited by applicant.
|
Primary Examiner: Legasse, Jr.; Francis M
Attorney, Agent or Firm: O'Banion; John P.
Government Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
This invention was made with Government support under Grant No.
442521-WM-22622/NCC2-1364, awarded by NASA. The Government has
certain rights in this invention.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority from U.S. provisional application
Ser. No. 60/561,587 filed on Apr. 12, 2004, incorporated herein by
reference in its entirety.
Claims
What is claimed is:
1. An apparatus for manipulating cells or particles by light
induced dielectrophoresis (DEP), the apparatus comprising: a first
surface and a second surface configured for retaining a liquid
comprising particles or cells to be manipulated; at least one
photoconductive area on said first or said second surface
configured for conversion of received light to a local electric
field in the vicinity of the received light; a light source to
provide the light received by the photoconductive area; wherein the
local electric field selectively repels or attracts particles or
cells; a microvision-based pattern recognition subsystem which is
configured for controlling the output of said light source in
response to registering the position of, and optionally the
characteristics of, particles or cells as determined from
microscopic imaging.
2. An apparatus as recited in claim 1, wherein said characteristics
are selected from the group of particle and cell characteristics
consisting essentially of size, color, shape, texture, viability,
motility, conductivity, permeability, capacitance and response to
changes in the environment of the particle or cell.
3. An apparatus for manipulating cells and particles using optical
image-driven light induced dielectrophoresis (DEP) over a
two-dimensional area, comprising: a first surface and second
surface configured for retaining a liquid containing particles, or
cells to be manipulated; at least one photoconductive area on said
first or second surface which is configured for inducing a local
electric field, virtual electrode, in the vicinity of received
light; an optical projector or scanning laser configured for
generating dynamic sequential two-dimensional light patterns onto
said photosensitive surface thereby inducing dynamic localized
electric fields for DEP manipulation of particles or cells; and a
microscopic imaging subsystem which is configured for controlling
the output of said optical projector in response to registering the
position of, and optionally the characteristics of, particles or
cells as determined from analyzing microscopic images.
4. An apparatus as recited in claim 3, wherein said characteristics
are selected from the group of particle and cell characteristics
consisting essentially of size, color, shape, texture, viability,
motility, conductivity, permeability, capacitance and response to
changes in the environment of the particle or cell.
.Iadd.5. An apparatus as recited in claim 1, wherein said
microvision-based pattern recognition subsystem is further
configured for controlling said light source to project a pattern
of light onto said photoconductive area that traps ones of said
particles or cells..Iaddend.
.Iadd.6. An apparatus as recited in claim 5, wherein said pattern
of light comprises traps each of which traps an individual one of
said particles or cells..Iaddend.
.Iadd.7. An apparatus as recited in claim 6, wherein each of ones
of said traps comprises an enclosure light pattern enclosing an
individual one of said particles or cells, said enclosure light
pattern creating an electric field cage that repels by
dielectrophoresis said particle or cell, thereby trapping said
particle or cell..Iaddend.
.Iadd.8. An apparatus as recited in claim 7, wherein said enclosure
light pattern is a ring..Iaddend.
.Iadd.9. An apparatus as recited in claim 6, wherein said
microvision-based pattern recognition subsystem is further
configured for controlling said light source to change said pattern
of light projected onto said photoconductive area to move ones of
said traps, thereby moving ones of said particles or
cells..Iaddend.
.Iadd.10. An apparatus as recited in claim 3, wherein said
microscopic imaging subsystem is further configured for controlling
said optical projector or scanning laser to generate a pattern of
light onto said photoconductive area that traps ones of said
particles or cells..Iaddend.
.Iadd.11. An apparatus as recited in claim 10, wherein said pattern
of light comprises traps each of which traps an individual one of
said particles or cells..Iaddend.
.Iadd.12. An apparatus as recited in claim 11, wherein each of ones
of said traps comprises an enclosure light pattern enclosing an
individual one of said particles or cells, said enclosure light
pattern creating an electric field cage that repels by
dielectrophoresis said particle or cell, thereby trapping said
particle or cell..Iaddend.
.Iadd.13. An apparatus as recited in claim 12, wherein said
enclosure light pattern is a ring..Iaddend.
.Iadd.14. An apparatus as recited in claim 11, wherein said
microscopic imaging system is further configured for controlling
said optical projector or scanning laser to change said pattern of
light generated onto said photoconductive area to move ones of said
traps, thereby moving ones of said particles or cells..Iaddend.
Description
INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ON A COMPACT
DISC
Not Applicable
NOTICE OF MATERIAL SUBJECT TO COPYRIGHT PROTECTION
A portion of the material in this patent document is subject to
copyright protection under the copyright laws of the United States
and of other countries. The owner of the copyright rights has no
objection to the facsimile reproduction by anyone of the patent
document or the patent disclosure, as it appears in the United
States Patent and Trademark Office publicly available file or
records, but otherwise reserves all copyright rights whatsoever.
The copyright owner does not hereby waive any of its rights to have
this patent document maintained in secrecy, including without
limitation its rights pursuant to 37 C.F.R. .sctn. 1.14.
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention pertains generally to cell and microparticle
manipulation, and more particularly to optoelectronic tweezers
(OET).
2. Description of Related Art
The ability to manipulate biological cells and micrometer scale
particles plays an important role in many biological and colloidal
science applications. However, conventional manipulation
techniques, including optical tweezers, electrokinetic forces
(electrophoresis, dielectrophoresis (DEP), traveling-wave
dielectrophoresis), magnetic tweezers, acoustic traps, and
hydrodynamic flows, cannot simultaneously achieve high resolution
and high throughput.
DEP is a well established technique that has been widely used to
manipulate micrometer and sub-micrometer particles as well as
biological cells. Traveling-wave dielectrophoresis (TWD) is
particularly attractive for high throughput cell manipulation
without external liquid pumping. The traveling electric field
produced by multi-phase alternating current (AC) bias on a parallel
array of electrodes levitates and transports many particles
simultaneously. However, the TWD cannot resolve individual
particles. Recently, a programmable DEP manipulator with
individually addressable two-dimensional electrode array has been
realized using complementary metal-oxide-semiconductor (CMOS)
integrated circuit (IC) technology. Parallel manipulation of a
large number (i.e., approximately 10,000) of individual cells was
demonstrated. The CMOS DEP manipulator has two potential drawbacks.
The need of on-chip IC increases the cost of the chip, making it
less attractive for disposable applications. The trap density
(i.e., approximately 400 sites/mm.sup.2) is also limited by the
size of the control circuits.
Consequently, the use of electrokinetic forces and similar
mechanisms provide high throughput, but lack the flexibility or the
spatial resolution for controlling individual cells, or groups of
cells. In addition, these techniques require structures formed
through numerous lithographic steps.
Optical tweezers, however, offer high resolution for trapping
single particles, yet provide limited manipulation area due to
tight focusing requirements. The optical tweezers use direct
optical force for the manipulating purpose, and require highly
focused coherent light sources used with an objective lens having a
high numerical-aperture (N. A.) value and a small field of view. To
generate multiple optical traps or special optical patterns, it
also requires techniques such as holography. These techniques
require intense calculation for creating even simple optical
patterns.
Accordingly a need exists for a particle and cell manipulation
apparatus and method which provides parallel processing capability
while still providing selectivity down to the single particle
level. The present invention fulfills those needs, as well as
others, and provides for manipulation of particles and cells at low
light levels without the need of complex lithography or 3D beam
control.
BRIEF SUMMARY OF THE INVENTION
The present invention is directed to performing particle and cell
manipulation using optical image-driven light induced
dielectrophoresis (DEP). The term "particle" will be used herein to
reference microparticles, nanoparticles, cells, and other organic
and inorganic material having a diameter generally between a few
nanometers up to the order of approximately 100 .mu.m. The
techniques allow for the use of moderate intensity incoherent light
sources, which create dynamic patterns that can be controlled in
response to image detection and processing of actual particle
composition and position.
In accordance with one embodiment of the invention, an optical
image-driven dielectrophoresis apparatus and method is described
for patterning electric fields on a photoconductive surface for
manipulating single particles, or collections of particles. A wide
variety of light sources can be utilized, such as incoherent light,
and single or multistage manipulation of particles can be readily
achieved.
One embodiment comprises optoelectronic tweezers (OET) configured
for cell and microparticle manipulation using optical control. The
OET permits functions such as cell trapping, repelling, collecting,
transporting, and sorting of cells and microparticles by using
sequentially projected images controlled by a spatial light
modulator (e.g., microdisplay or DMD mirrors, and so forth). With
optical actuating power as low as 1 mW optical manipulation can be
performed using incoherent lightly focused light and a direct image
projection system.
In one embodiment, dynamic DMD-driven optoelectronic tweezers
perform dynamic manipulation of microscopic particles using a DMD
produced projection image. Single-particle trapping and movement
(up to 40 .mu.m/sec) via optically-induced dielectrophoresis were
observed in this embodiment.
Another embodiment is described in which dynamic array manipulation
of particles and microparticles is performed using optoelectronic
tweezers. One demonstration details the individual trapping of
polystyrene particles with 20 .mu.m and 45 .mu.m diameters trapped
by light patterns generated by a digital light projector with
digital micromirror device (DMD). Self-organization and individual
addressing of the particles are demonstrated. Movement of 45 .mu.m
polystyrene particles is measured to be 35 .mu.m/sec (a force of 15
pN).
Another embodiment provides for the manipulation of live red and
white blood cells with optoelectronic tweezers. Optically-induced
dielectrophoresis is enabled within the optoelectronic tweezers
(OET) to manipulate live mammalian cells demonstrated by
concentrating bovine red blood cells in solution. A spatial light
modulator and an incoherent light source integrated in combination
with the OET provide the ability to easily create reconfigurable,
complex manipulation patterns. This capability is also demonstrated
in the patterning of human white blood cells into complex
patterns.
Another embodiment provides for using light induced
dielectrophoresis to optically trap and transport micro particles
with optical power in the microwatt range. This embodiment
comprises two pattern-less (unpatterned) surfaces: a bottom glass
substrate coated with photoconductive material and a top
transparent indium-tin-oxide (ITO) glass. To achieve optical
trapping, the liquid-immersed micro-particles are sandwiched
between these two surfaces and an AC electric bias is supplied. A
633 nm He--Ne laser focused by a 40.times. objective lens is used
to transport the particles. Negative dielectrophoretic trapping is
demonstrated and the experimental results show that optical beams
with power as low as 1 .mu.W are sufficient to transport 25 .mu.m
diameter latex particles at a speed of 4.5 .mu.m/sec. The transport
speed increases with higher optical power. A maximum speed of 397
.mu.m/sec is observed at 100 .mu.W.
In this embodiment, an optical sorting mechanism is described based
on a dynamic electric field patterned by the scanning
optoelectronic tweezers (OET). The sorting mechanism is based on
the force balance between the hydrodynamic viscous force and the
dynamic light-induced dielectrophoretic force. Randomly distributed
particles with different sizes are sorted out and positioned in
size-dependent deterministic positions relative to a line-shaped
scanning laser beam. A 240 .mu.m laser beam moving at a speed of 10
.mu.m/sec can sort polystyrene beads with diameters of 5 .mu.m, 10
.mu.m, and 20 .mu.m to relative positions of 17 .mu.m, 29 .mu.m,
and 60 .mu.m from the beam center.
This embodiment also provides for moving toward an all optical
lab-on-a-chip system requiring optical manipulation tools for both
microparticles and microfluids. Although optical tweezers are
important for manipulating cells or particles, they are not
effective in handling microfluid. The typically high optical power
requirements have also limited the applicability in high throughput
bioanalysis system. In this embodiment two novel mechanisms are
demonstrated: (1) optoelectrowetting (OEW) for handling
microdroplets, and (2) optoelectronic tweezers (OET) for optical
manipulation of microscopic particles with low optical power
actuation. Instead of using direct optical force, both mechanisms
rely on light induced electrical force for optical manipulation.
Optoelectrowetting (OEW) enables control of microfluids in droplet
form by optical beams and is based on light induced electrowetting,
which changes surface tension at solid-liquid interface at
illuminated area. It is realized by integrating a layer of
photoconductive material with electrowetting electrodes. By
programming the illumination pattern, we have successfully
demonstrated various functions for droplets, such as moving,
splitting, and merging. A 100 .mu.L droplet was transported at a
speed of 785 .mu.m/sec by an optical beam with an optical power of
100 .mu.W.
Optoelectronic tweezers (OET) manipulate cells or particles based
on light induced dielectrophoresis (DEP). Trapping or repelling of
microscopic particles is achieved with a light intensity of 2
W/cm.sup.2, which is five orders of magnitudes lower than that
required by optical tweezers (approximately 105 W/cm.sup.2 to 107
W/cm.sup.2). The liquid containing cells or particles is sandwiched
between a photosensitive surface and a transparent ITO glass, with
an AC bias between them. When the laser beam is focused on the
photosensitive layer, it creates a virtual electrode on the
illuminated area, resulting a nonuniform electric field at the
aqueous layer. Cells or particles in the liquid layer are polarized
by this non-uniform electric field and driven by the DEP force. The
force could be attractive or repulsive, depending on the dielectric
properties of the particles and the bias frequency. Using OET, we
have demonstrated concentration of polystyrene particles and live
E. coli cells using an optical power less than approximately 10
.mu.W.
According to another embodiment of the invention, an apparatus for
manipulating cells or particles by light induced dielectrophoresis
(DEP) comprises: (a) a first surface and a second surface
configured for retaining a liquid comprising particles or cells to
be manipulated; (b) at least one photoconductive area on the first
or the second surface configured for conversion of received light
to a local electric field in the vicinity of the received light;
and (c) means for directing light patterns for receipt on the
photoconductive area to selectively repel or attract particles or
cells in response to the induced local electric field. The light
pattern directing means preferably comprises a light source
configured for generating two-dimensional light patterns.
In accordance with another embodiment of the invention, an
optoelectronic tweezers (OET) apparatus for manipulating cells and
particles using optical image-driven light induced
dielectrophoresis (DEP) over a two-dimensional area comprises: (a)
a first surface and second surface having sufficient separation for
retaining a liquid which contains particles, or cells, to be
manipulated; (b) at least one photoconductive area on the first or
second surface which is configured for conversion of optical energy
to an electric field in the photoconductive area to create a local
electric field, or virtual electrode, in the vicinity of the
received light; and (c) means for dynamic optical image positioning
on the at least one photoconductive area to generate moving virtual
electrode patterns for manipulating the positioning of particles or
cells using light-induced dielectrophoresis (DEP).
Another embodiment of the invention provides an apparatus for
manipulating cells or particles by light induced dielectrophoresis
(DEP), the apparatus comprising: (a) a first surface and a second
surface configured for retaining a liquid comprising particles or
cells to be manipulated; (b) at least one photoconductive area on
the first or the second surface configured for conversion of
received light to a local electric field in the vicinity of the
received light; and (c) a light source to provide the light
received by the photoconductive area, wherein the local electric
field selectively repels or attracts particles or cells.
The light source preferably comprises an optical projection system
configured for generating two dimensional light patterns, such as
in the form of image sequences or streams upon the photoconductive
area.
In a further embodiment of the invention, an optoelectronic
tweezers (OET) apparatus for manipulating cells and particles using
optical image-driven light induced dielectrophoresis (DEP) over a
two-dimensional area, comprises: (a) a first surface and second
surface having sufficient separation for retaining a liquid which
contains particles, or cells, to be manipulated; (b) at least one
photoconductive area on the first or second surface which is
configured for inducing an electric field, thereby creating a
virtual electrode, in the vicinity of the received light (or
similarly converting optical energy to an electric field) and (c)
an optical projector configured for generating dynamic sequential
two-dimensional light patterns onto the photosensitive surface
thereby inducing dynamic localized electric fields for DEP
manipulation of particles or cells.
The optical projector, or similar means of dynamically projecting
light images is preferably directed at the OET through a lens
assembly, so that a sequence of images can be formed onto the
photoconductive area. In one preferred embodiment of the invention
electrodes are coupled to the first and second surfaces so that a
bias signal can be applied to the liquid with the contained
particles or cells.
According to one aspect of the invention, a means is provided to
perform microscopic imaging of the particles and/or cells and to
register the position, and optionally the characteristics, of
particles and/or cells to provide feedback for controlling optical
image positioning and dynamic image movement.
Another embodiment of the invention provides an optoelectronic
tweezers (OET) apparatus for manipulating cells and particles
(typically on the order of 100 .mu.m diameter or less) using
optical image-driven light induced dielectrophoresis (DEP) over a
two-dimensional area, comprising: (a) a first surface and second
surface having sufficient separation for retaining a liquid which
contains particles and/or cells to be manipulated; (b) at least one
photoconductive area on the first or second surface which is
configured for conversion of optical energy to an electric field in
the photoconductor to create a local electric field, or virtual
electrode, in the vicinity of the received light; (c) an optical
projector coupled to a lens assembly configured for generating
dynamic sequential images (a sequence of light patterns) through
the lens assembly onto the photosensitive surface for creating
dynamic localized electric fields for the DEP manipulation of
nearby particles and/or cells.
The first surface and second surface preferably form a continuous
film upon which DEP manipulation is performed in response to images
received from the optical projector. In this way lithographic
patterning with conductive electrodes is not necessary for
performing DEP manipulation.
In one mode of the invention at least one electrode is coupled to
each of the first and second surface so that a bias signal can be
applied to the liquid with its particles and/or cells. The liquid
preferably comprises a conductive or semiconductive fluid.
Typically a thin dielectric layer is joined to the interior surface
of the electrodes and configured to have an impedance that is less
than the impedance across the liquid.
In a preferred mode of the invention a microvision-based pattern
recognition subsystem is configured for controlling the output of
the optical projector in response to registering the position of,
and optionally the characteristics of, particles and/or cells as
determined from microscopic imaging. The characteristics can
comprise anything which is directly detectable by the microscopic
imaging system or which can be determined in response to detecting
changes in the direct characteristics over time. By way of example
the characteristics can include size, color, shape, texture,
viability, motility, conductivity, permeability, capacitance and
response to changes in the environment of the particle or cell.
Another embodiment of the invention is an apparatus for
manipulating cells by light induced dielectrophoresis (DEP), the
apparatus comprising: (a) a first surface and a second surface
configured for retaining a liquid comprising cells to be
manipulated; (b) at least one photoconductive area on the first or
the second surface configured for inducing a local electric field
in response to received light; (c) a light source to provide the
light received by the photoconductive area, wherein the local
electric field induced by the light selectively repels or attracts
cells and wherein the light received is of sufficiently low optical
intensity that it does not damage the cells being manipulated in
the apparatus. In one preferred mode the embodiment further
comprises a microscopic imaging subsystem configured for
controlling the output of the light source in response to
registering the position of, and optionally the characteristics of
cells within the apparatus.
Another embodiment provides a method of manipulating particle or
cellular objects retained within a liquid, the method comprising
the steps consisting essentially of: (a) confining the liquid
comprising the particle objects or cellular objects within a
structure comprising at least a first and second surface; (b)
applying a bias voltage to the liquid by applying a bias signal to
electrodes coupled to the first and second surfaces; (c) directing
light to a photoconductive portion of the structure, wherein the
light induces a local electric field in the vicinity of the portion
receiving light thereby dielectrophoretically repelling or
attracting the particles or cells.
Another embodiment provides a method of manipulating biological
objects retained within a liquid, the method comprising: (a)
confining the liquid comprising the particle objects or cellular
objects within a structure comprising at least a first and second
surface; (b) applying a bias voltage to the liquid by applying a
bias signal to electrodes coupled to the first and second surfaces;
(c) generating control signals in response to registering the
characteristics and positions of biological objects within the
structure; (d) directing light in response to the control signals
upon a photoconductive portion of the structure to induce a local
electric field in the vicinity of the received light to
dielectrophoretically repel or attract cellular objects, wherein
the light is of sufficiently low intensity that live cells being
manipulated by the method remain alive and viable.
Another embodiment of the invention generally provides a method of
dynamically manipulating particle and cellular objects retained
within a liquid, comprising: (a) confining a liquid containing
particle objects, and/or cellular objects within a structure having
at least a first and second surface; (b) applying a bias voltage to
the liquid by applying a bias signal to electrodes coupled to the
first and second surfaces; (c) focusing a light pattern on a
photoconductive portion of the first surface and/or second
surfaces, so that the optical energy of the light is converted to a
local electric field to create a virtual electrode in the vicinity
of the received light; and (d) dynamically positioning the light
pattern in response to feedback received from registering the
position, and optionally characteristics, of the particles and/or
cells.
Another embodiment of the invention provides a method of
dynamically sorting cells retained within a liquid, comprising: (a)
confining a liquid contains cells within a structure having at
least a first and second surface; (b) applying a bias voltage to
the liquid by applying a bias signal to electrodes coupled to the
first and second surfaces; (c) generating control signals in
response to registering the characteristics and positions of cells
within the structure and determining into which category cells are
to be sorted; (d) directing light, of sufficient low intensity to
prevent cellular damage, in response to the control signals upon a
photoconductive portion of the structure to induce a local electric
field in the vicinity of the received light to
dielectrophoretically repel or attract the cells; and (e)
sequentially directing light in response to the control signals to
move categorized cells into different sort groups within the
structure or for conveyance outside of the structure.
A still further embodiment of the invention provides a method of
dynamically sorting particles or cells retained within a liquid,
comprising: (a) confining a liquid containing particles or cells
within a structure having at least a first and second surface; (b)
applying a bias voltage to the liquid by applying a bias signal to
electrodes coupled to the first and second surfaces; and (c)
directing a moving pattern of light across a photoconductive
portion of the structure to induce a local electric field in the
vicinity of the pattern to dielectrophoretically repel particles or
cells displacing them from the pattern according to their relative
size.
Embodiments of the present invention can provide a number of
beneficial aspects which can be implemented either separately or in
any desired combination without departing from the present
teachings.
An aspect of the invention is to provide an apparatus and method
for manipulating cells and particles using optical image-driven
light induced dielectrophoresis (DEP) within a generally planar
liquid-filled structure.
Another aspect of the invention is performing optical image-driven
light induced DEP over a large two-dimensional area adjacent to a
fluid containing single particles and/or cells, collections of
particles and/or cells, or a combination thereof.
Another aspect of the invention is performing optical image-driven
light induced DEP in which single particles and/or cells, or
particle groups and/or cell groups, can be manipulated in parallel
(simultaneously).
Another aspect of the invention is performing optical image-driven
light induced DEP wherein any of the particles or groups of
particles can be manipulated in any desired direction within the
apparatus as they are not constrained by a physical electrode
structure.
Another aspect of the invention allows for performing optical
image-driven light induced DEP using conventional materials and
processing techniques.
Another aspect of the invention allows for performing optical
image-driven light induced DEP on particles which may be
electrostatically neutral.
Another aspect of the invention allows for performing optical
image-driven light induced DEP in an optoelectric tweezers device
(OET) which achieves high resolution and high throughput
simultaneously.
Another aspect of the invention allows for performing optical
image-driven light induced DEP in an optoelectric tweezers device
(OET) which creates dynamic electric fields to manipulate particle
positioning without the assistance of fluidic flow.
Another aspect of the invention allows for performing optical
image-driven light induced DEP in an optoelectric tweezers device
(OET) which is capable of manipulating the position of particles
and cells at less than approximately 10 .mu.W which is about
1/100,000.sup.th of the optical energy level required by
conventional optical tweezers.
Another aspect of the invention allows for an optoelectric tweezers
device (OET) in which tight optical focusing is not required
thereby allowing manipulation over a maximum area on the order of
one square millimeter (1 mm.sup.2), or larger up to, such as 1.3
mm.times.1.0 mm which is many orders of magnitude larger than that
which is achievable using conventional optical tweezers.
Another aspect of the invention allows for performing optical
image-driven light induced DEP in an optoelectric tweezers device
(OET) in combination with continuous optical electrowetting
techniques (COEW).
Another aspect of the invention is an OET device having a first
surface and second surface separated by chamber walls and
configured for retaining a liquid which contains particles, or
cells, being manipulated.
Another aspect of the invention is an OET device having electrodes
on the first and second surface upon which a biasing current and/or
field can be applied through and/or across the retained liquid.
Another aspect of the invention is an OET device having at least
one photosensitive/photoresponsive surface which creates a local
electric field in response to received light, therein creating
virtual electrodes for manipulating particles, cells, and the like
at low optical power levels.
Another aspect of the invention is an OET device having first and
second surfaces formed as a continuous film, wherein lithographic
patterning of the surface is not necessary for practicing the
invention.
Another aspect of the invention is an OET device having surfaces of
amorphous and/or micro/nano-crystalline semiconductor materials,
amorphous Si, or organic photoconductor materials, used with or
without dielectric layers, such as silicon nitride, silicon
dioxide, and so forth.
Another aspect of the invention is an OET device having thin
dielectric layers with a lower impedance than the liquid retained
in the OET device.
Another aspect of the invention is an OET device using a single or
double-sided photosensitive surface in various combinations with a
conductive surface, non-conductive surface, or no opposing surface
(open structure).
Another aspect of the invention is an OET device configured for
implementing traps, combs, sorters, concentrators, loops,
conveyers, joints, particle channels, wedges, sweepers, which can
be implemented separately, in arrays of manipulation elements, and
combinations and sequences of manipulation elements and so
forth.
Another aspect of the invention is an OET device wherein the
surface integrates with microfluidic devices, such as channels,
cavities, reservoirs, and pumps.
Another aspect of the invention is an OET device for implementing a
comb device for separating particles, cells, and other
micro/nano-particles in response to their size.
Another aspect of the invention is an OET device which can be
biased with AC of a desired frequency, and/or DC biasing.
Another aspect of the invention is an OET device in which the
frequency of the AC bias determines whether particles, cells, and
the like are attracted or repelled by the patterned light.
Another aspect of the invention is an OET device in which a
microscopic imaging means operates in combination with the dynamic
light patterning device so that patterns are created in response to
the actual positioning of particles, cells, and the like within the
OET device.
Another aspect of the invention is an OET device in combination
with a microscopic imaging device which is configured to provide
feedback to the OET device during the characterization and
positioning of particles.
Another aspect of the invention is an OET device in which the
microscopic imaging means is configured for analyzing the actual
positioning and composition of particles, cells, and so forth and
controlling the generation of light output sequences for moving,
collecting, and/or dispersing the particles, cells, and so forth in
response to actual positions detected by the imaging means.
Another aspect of the invention is an OET device in which a
conductive or semiconductive fluid is retained in the device.
Another aspect of the invention is an OET device which is
configured for manipulating particles and cells in response to
light patterns, and in particular dynamic lighting patterns.
Another aspect of the invention is an OET device in which dynamic
lighting patterns are generated to sequentially move particles,
cells and so forth, in response to the light pattern motion.
Another aspect of the invention is an OET device in which the
lighting patterns are generated by a laser or more preferably a
low-intensity incoherent light source (i.e., halogen, LEDs, and so
forth).
Another aspect of the invention is an OET device in which the use
of low-intensity lighting is made possible by the conversion of
optical energy to an electric field in the photoconductor.
Another aspect of the invention is an OET device configured for
sorting particles, or biological cells, in response to differences
in viability (i.e., dead or alive), internal conductivity, size,
color, shape, texture, response to changes in the aqueous
environment, and similar distinguishing characteristics.
Another aspect of the invention is an OET device configured for
sorting biological cells in response to differences in membrane
properties (e.g., permeability, capacitance, and so forth),
internal conductivity, and the like.
Another aspect of the invention is an OET device in which the use
of low-intensity lighting allows for manipulating biological
objects without loss of viability from photodamage ("opticution")
which arises when using conventional optical tweezers.
Another aspect of the invention is an OET device in which the use
of low-intensity lighting is made possible by the conversion of
optical energy to an electric field in the photoconductor.
Another aspect of the invention is an OET device in which the light
source is patterned by a spatial light modulator, or similar form
of light modulator.
Another aspect of the invention is an OET device on which the light
patterns are varied in response to magnification or
demagnification.
A still further aspect of the invention is an OET device for use in
biological analysis, cell manipulation, colloidal assembly,
particle sorting, particle assembly, and so forth.
Further aspects of the invention will be brought out in the
following portions of the specification, wherein the detailed
description is for the purpose of fully disclosing preferred
embodiments of the invention without placing limitations
thereon.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)
The invention will be more fully understood by reference to the
following drawings which are for illustrative purposes only:
FIG. 1 is a perspective schematic view of optoelectronic tweezers
(OET) according to an aspect of the present invention for
manipulating microscopic particles sandwiched between structure
layers biased with an AC signal.
FIGS. 2A-2D are images of particle manipulation using particle
traps according to an aspect of the present invention, showing
particles being trapped in a am array.
FIGS. 3A-3D are images of an integrated virtual optical machine
according to an aspect of the present invention with a sorter,
conveyor, joints, and a wedge for sorting microparticles.
FIGS. 4A-4D are images of optical sorting of live and dead cells
using the OET according to an aspect of the present invention.
FIG. 5 is a schematic of an OET device according to an aspect of
the present invention, showing the use of light-defined virtual
electrodes which generate non-uniform electric fields in the liquid
layer.
FIG. 6 is a block diagram of an OET-based cell manipulation system
according to an aspect of the present invention, showing the use of
a programmable spatial light modulator for generating the desired
optical image.
FIG. 7A is a perspective view of OET device structure according to
an aspect of the present invention, showing particles retained in
solution between a first layer and an optically responsive second
layer.
FIG. 7B is a schematic of an experimental OET setup according to an
aspect of the present invention, showing the OET device of FIG. 7A
receiving a modulated and directed light source.
FIG. 8 is a 3-D graph of electric-field distribution for a single
particle ring trap according to an aspect of the present
invention.
FIGS. 9A-9C are images of particle manipulation according to an
aspect of the present invention, shown using a dynamic line cage
with two angle sections (forming a square in FIG. 9A) which contain
the particles in successively smaller regions.
FIGS. 9D-9F are images of particle trapping according to an aspect
of the present invention, showing trapping and moving a single
particle while repelling particles outside of the selection area
(ring).
FIG. 10A is an image of a single-particle OET trap according to an
aspect of the present invention, shown retaining a 45 .mu.m
polystyrene sphere.
FIG. 10B is a graph of electric field distribution for the OET of
FIG. 10A.
FIG. 11 is a perspective view of an OET device according to an
aspect of the present invention, showing particles in the liquid
buffer retained between the top and bottom layers of the OET.
FIG. 12 is a block diagram of an experimental OET setup according
to an aspect of the present invention, showing light controlled
from a PC directed using DMD onto the OET device.
FIGS. 13A-13B are images of self-organization of microparticles
into an array configuration according to an aspect of the present
invention.
FIGS. 14A-14B are images of single-particle manipulation within an
OET array according to an aspect of the present invention.
FIGS. 15A-15D are images of particle array flushing within an OET
array according to an aspect of the present invention.
FIG. 16 is an image of an array of single particles trapped in an
OET array according to an aspect of the present invention.
FIGS. 17A-17D are images of dynamic rearrangement of differently
sized particles according to an aspect of the present
invention.
FIG. 18A is a schematic diagram of a microvision-based automatic
optical manipulation system according to an aspect of the present
invention.
FIG. 18B is a schematic of the OET device shown in the system of
FIG. 18A.
FIGS. 19A-19D is an illustration of steps according to an aspect of
the present invention for arranging particles into any desired
pattern.
FIGS. 20A-20D are images from a particle recognition system and a
graph of recognition percentage according to an aspect of the
present invention.
FIGS. 21A-21B are graphs of electric field distribution induced by
a single optical ring pattern according to an aspect of the present
invention.
FIGS. 22A-22C are images of microvision-based automatic optical
manipulation of microscopic particles according to an aspect of the
present invention.
FIG. 23 is a schematic of an OET device according to an aspect of
the present invention, showing modification of the electric-field
patterns within the particle-laden liquid.
FIG. 24 is a schematic of an experimental OET device setup
according to an aspect of the present invention, shown using a
modulated laser light source to direct optical particle
manipulation patterns.
FIGS. 25A-25D are images of OET particle manipulation using a
combination of optical input and AC biasing according to an aspect
of the present invention.
FIG. 26 is an image of OET particle manipulation according to an
aspect of the present invention, shown in the process of forming
the letters "UC" with human white blood cells.
FIG. 27A is a perspective view of an OET device according to an
aspect of the present invention, showing a focused beam directed
through a liquid to a photoresponsive material.
FIG. 27B is a schematic of the operation of the OET of FIG. 27A in
response to one mode of induced dielectrophoresis.
FIG. 28 is a graph of the relationship between particle speed and
optical power for an OET according to an aspect of the present
invention.
FIGS. 29A-29B are images of using an OET to focus/concentrate
multiple particles according to an aspect of the present
invention.
FIGS. 30A-30C are schematics of an OET device according to an
aspect of the present invention, showing how different sized
particles are organized in response to an optical wave induced
electric field.
FIG. 31 is a block diagram of an OET optical setup according to an
aspect of the present invention, showing a laser illumination
source with underside microscopic imaging.
FIGS. 32A-32D are images of OET-based size sorting according to an
aspect of the present invention, showing size sorting of particles
of approximately 10 .mu.m and 20 .mu.m.
FIGS. 33A-33B are images of OET-based size sorting for particles in
a range of sizes according to an aspect of the present
invention.
FIG. 34 is a graph of distance versus speed for the different
particle sizes demonstrated according to an aspect of the present
invention.
FIG. 35 is a flow diagram comparison of the energy transfer paths
according to different optical manipulation methods.
FIGS. 36A-36B are schematics of an OEW device according to an
aspect of the present invention, showing the change in droplet
characteristics when the device is illuminated.
FIG. 37A is a schematic of droplet transport on a continuous OEW
surface according to an aspect of the present invention.
FIG. 37B is a schematic of an equivalent electrical circuit for the
OEW of FIG. 37A.
FIG. 37C is a perspective view of the layer structure of the COEW
surface according to an aspect of the present invention.
FIGS. 38A-38D are images of microdroplet transport utilized COEW
according to an aspect of the present invention.
FIG. 39 is a schematic of an OET device according to an aspect of
the present invention, showing particle containing liquid retained
within a structure having a photoconductive surface for converting
optical energy to an electric field.
FIGS. 40A-40B are graphs of electric field distribution and
strength within the liquid layer in response to photoconductor
illumination.
FIG. 41 is a schematic of an experimental setup for trapping E.
coli cells with an OET device according to an aspect of the present
invention.
FIGS. 42A-42B are images of cells being collected, "focused",
according to an aspect of the present invention.
FIGS. 43A-43B are images of cells being transported according to an
aspect of the present invention.
FIG. 44 is a graph of cell movement speed in response to distance
and optical power according to an aspect of the present
invention.
DETAILED DESCRIPTION OF THE INVENTION
Referring more specifically to the drawings, for illustrative
purposes the present invention is embodied in the apparatus
generally shown in FIG. 1 through FIG. 44. It will be appreciated
that the apparatus may vary as to configuration and as to details
of the parts, and that the method may vary as to the specific steps
and sequence, without departing from the basic concepts as
disclosed herein.
The present invention includes numerous embodiments in which
particles, cells, and other elements suspended in a fluid are
manipulated. The application describes these embodiment within nine
sections.
1. Massively Parallel Manipulation Using Optical Images.
An optical image-driven dielectrophoresis technique is described
herein that permits high-resolution patterning of electric fields
on a photoconductive surface for manipulating single particles. The
technique can be performed at substantially lower light intensity
levels than were required using previous techniques, for example
one embodiment requires approximately 100,000 times less optical
intensity than optical tweezers. In addition, the technique can
make use of incoherent light sources. In one example an incoherent
light source (a light emitting diode (LED) or a halogen lamp) is
utilized with a digital micromirror spatial light modulator to
demonstrate parallel manipulation of 15,000 particle traps within a
1.3 mm.times.1 mm area. With direct optical imaging control,
multiple manipulation functions can be easily combined to achieve
complex, multi-step manipulation protocols.
It has not been appreciated in the industry that optically-induced
electrophoresis could be could be controlled with a dynamic optical
addressing mechanism to provide the capability to perform
manipulation down to the single particle level. The optoelectronic
tweezers (OET) of the present invention utilizes direct optical
images to create high-resolution DEP electrodes for the parallel
manipulation of single particles. DEP force results from the
interaction of the induced dipoles in particles subjected to a
non-uniform electric field. The magnitude of the force depends on
the electric field gradient and the polarizability of the particle,
which is dependent on the dielectric properties of the particle and
the surrounding medium.
FIG. 1 illustrates an example embodiment 10 of optoelectronic
tweezers (OET) according to the present invention. Liquid 12 that
contains microscopic particles (or cells) 14 is sandwiched between
the upper layer 16, such as comprising a transparent conductive ITO
glass, and the bottom layer 18, such as comprising a photosensitive
surface fabricated from an ITO-coated glass 20 topped with multiple
preferably featureless layers with 50 nm of heavily doped a-Si:H
22, 1 .mu.m of undoped a-Si:H 24, and 20 nm of silicon nitride 26.
By way of example the lower layer is shown upon a glass substrate
28.
The top 16 and bottom 18 surfaces are coupled to a bias source 30
such as an AC electric signal of 10 V.sub.PP. Alternatively, the
surfaces can be less preferably DC biased, or biased with a
combination of AC and DC, depending on the specific device
structure and manipulation application. It should be appreciated
that the frequency of the AC bias determines whether particles,
cells, and the like are attracted or repelled by the patterned
light, wherein the use of AC provides a number of advantages over
the use of a DC bias.
The photosensitive surface 24 of the lower layer converts the
received optical energy into a corresponding electric field, shown
by way of illustration by the set of concentric rings 32 of FIG. 1.
The illumination source may be any convenient light source, such as
an LED 34 operating at a wavelength of 625 nm (i.e., manufactured
by Lumileds.RTM., Luxeon.RTM. Star/O.RTM.) as depicted in this
example. An optical projection means provides a mechanism for
outputting light patterns onto the photosensitive lower layer 18,
and preferably is configured for outputting dynamic light patterns
having spatial intensity variation over the surface, and typically
structures defined by light or dark regions, which are output in a
pattern stream (i.e., similar to a movie), or a pattern sequence
(i.e., similar to a slide show). One preferred technique for
projecting the light patterns is using a spatial light modulation
means 36, such as a digital micromirror display (DMD) which in
combination with objective 38 focuses the light from LED 34 onto
the photosensitive surface 24 creating the non-uniform electric
field for DEP manipulation.
When projected light illuminates the photoconductive layer, it
turns on the virtual electrodes, creating non-uniform electric
fields and enabling particle manipulation via DEP forces. These
featureless layers can be made without using any lithography or
microfabrication, making the device inexpensive and attractive for
disposable applications. The OET-based optical manipulation has two
operational modes, positive OET and negative OET, as a result of
DEP forces induced for actuation. Particles can be attracted by or
repelled from the illuminated area, depending on the AC electric
field frequency and the internal and surface dielectric properties
of the particle.
As a consequence of the high photoconductive gain, the minimum
optical intensity required to turn on a virtual electrode is 10
nW/.mu.m.sup.2, which is approximately 100,000 times lower than
that of optical tweezers. This low threshold of optical intensity
opens up the possibility of using incoherent optical images to
control the DEP forces over a large area, such as over a maximum
area on the order of one square millimeter (1 mm.sup.2), or even
larger depending on optical configuration. For example, the optical
images are created in one embodiment by combining an LED and a
digital micromirror spatial light modulator (i.e., a DMD device
such as by Texas Instruments.RTM. having 1024.times.768 pixels with
a 13.68 .mu.m.times.13.68 .mu.m pixel size). The pattern is imaged
onto the photoconductive surface through a 10.times. objective. The
resulting pixel size of the virtual electrode is 1.52 .mu.m. The
illumination source for the example was a red LED (625 nm
wavelength) with 1 mW output power (measured after the objective
lens), which is sufficient to actuate 40,000 pixels. Tight focusing
is not required for OET, and the optical manipulation area can be
magnified by choosing appropriate objective lens. Using a 10.times.
objective, the manipulation area (1.3 mm.times.1.0 mm) is 500 times
larger than that of optical tweezers.
The patterning of high-resolution virtual electrodes is critical
for achieving single particle manipulation. OET has higher
resolution than the optically-induced electrophoretic methods
reported previously. The minimum size of the virtual electrode is
limited by the lateral diffusion length of the photogenerated
carriers in the photoconductor as well as the optical diffraction
of the objective lens. The large number of electronic defect states
in undoped a-Si:H results in a short ambipolar electron diffusion
length of less than 115 nm. The ultimate virtual electrode
resolution is thus determined by the optical diffraction limit. In
addition, the induced OET force is proportional to the gradient of
the square of the electric field, making it well confined to the
local area of the virtual electrodes, which is also a key property
for single particle manipulation.
It should be appreciated that the OET of FIG. 1 may be implemented
with a number of variations according to the present invention, the
following being provided by way of example. The OET device is
provided with a first surface and second surface separated by
chamber walls and configured for retaining a liquid which contains
particles, or cells, being manipulated. It is preferred that
electrodes are provided on the first and second surfaces of the OET
upon which a biasing current and/or field can be applied through
and/or across the retained liquid. FIG. 1 is shown with a single
photoconductive surface. However, the present system may be
implemented having at least one photosensitive/photoresponsive
surface which induces a local electric field on the surface of the
material in response to received light, therein creating virtual
electrodes for manipulating particles, cells, and the like at low
optical power levels. It should be understood that the OET
according to the invention may be created with a single or
double-sided photosensitive surface, in various combinations with a
conductive surface, non-conductive surface, or no opposing surface
(open structure).
The first and second surfaces of the OET are preferably formed as a
continuous film, wherein lithographic patterning of the surface is
not necessary for practicing the invention. However, it should be
appreciated that the OET of the present invention can be
implemented in combination with conventional DEP structures or
continuous optical electrowetting techniques (COEW) toward specific
application areas.
The OET of FIG. 1 can be configured having surfaces of amorphous
and/or micro/nano-crystalline semiconductor materials, amorphous
Si, or organic photoconductor materials, used with or without
dielectric layers, such as silicon nitride, silicon dioxide, and so
forth. When used, the thin dielectric layers of the OET, having an
impedance that is much less than the impedance across the liquid
retained in the OET device.
FIG. 2A through FIG. 2D illustrate results from an embodiment of
the device which provides massively parallel manipulations of
single particles across 15,000 particle traps created across a 1.3
mm.times.1.0 mm area. The 4.5 .mu.m diameter polystyrene beads
experiencing negative DEP forces are trapped in the dark area.
FIG. 2A depicts a portion of the array, with each trap in this
particular embodiment having a diameter of 4.5 .mu.m to fit a
single particle.
FIG. 2B illustrates by way of example parallel transporting of
single particles with three snapshots from the captured video
showing the particle motion within this section of the manipulation
area. The trapped particles in two adjacent columns move in
opposite direction as seen in FIG. 2C and FIG. 2D. The induced
negative DEP forces push the beads into the non-illuminated
regions, where the electric field is weaker. The size of each trap
is optimized to capture a single 4.5 .mu.m diameter polystyrene
bead.
By programming the projected images, these trapped particles can be
individually moved in parallel as shown in FIG. 2B. Compared with
the programmable CMOS DEP chip, the particle trap density of the
OET (11,500 sites/mm.sup.2) is 30 times higher in response to the
high-resolution addressing ability. Using direct imaging,
sophisticated virtual electrodes can be easily patterned and
reconfigured to create dynamic electric field distributions for
continuous particle manipulation without the assistance of fluidic
flow.
FIG. 3A through FIG. 3D illustrate an embodiment by way of example
of an integrated virtual optical machine in which the motion of
different components is synchronized. In FIG. 3A, the image
illustrates the structure which integrates a number of virtual
components including an optical sorter path, conveyers, joints and
a wedge. It should be appreciated that the OET device of the
present invention can be configured for implementing a large
variety of traps, combs, sorters, concentrators, loops, conveyers,
joints, particle channels, wedges, sweepers, which can be
implemented separately, in arrays of manipulation elements, and
combinations and sequences of manipulation elements and so forth,
without departing from the teachings of the present invention.
In FIGS. 3B-3C, two polystyrene particles with sizes of 10 .mu.m
and 24 .mu.m pass through the sorter path and are fractionated in
the z-direction due to the asymmetry optical patterns. The particle
traces can be switched at the end of the sorter path by
reconfiguring the tip position of the optical wedge. The
trajectories of particle movement are highly repeatable and
accurately defined, as can be seen in FIG. 3B and FIG. 3C in which
the optical sorting repeatability is represented by the dark and
light tracks. The light and dark loops in FIG. 3B represent the
particle traces after 43 cycles. The trace broadening at the
checking bar has a standard deviation of 0.5 .mu.m for the 10 .mu.m
bead and 0.15 .mu.m for the 24 .mu.m bead.
It should be appreciated that particles are transported through
different functional areas and recycled in this light-patterned
circuit, traveling through different paths depending on the
position of the wedge divider. Particles with different sizes are
fractionated in the lateral z direction as they pass through the
sorter path due to the asymmetric shape of the light-patterned
electric fields. At the end of the sorter path, an optical wedge
divides and guides the particles into the two conveyors. The looped
optical conveyors recycle the particles back to the sorter input to
repeat the process.
FIG. 3D shows a distribution of particle position in the middle of
the sorter (marked by a white bar) after the particles have passed
through the sorter 43 times. The standard variations of trace
broadening are 0.5 .mu.m for the 10 .mu.m bead, and 0.15 .mu.m for
the 24 .mu.m bead. The magnitude of the DEP force is proportional
to the particle volume. The larger particles exhibit tighter
confinement in the optically patterned DEP cages during
transport.
FIG. 4A through FIG. 4D illustrate by way of example the sorting of
biological cells according to their characteristics. According to
this example embodiment, the living cells are subject to positive
OET, trapping them in the bright areas, and pulling the live cells
into the center of the pattern. The dead cells (i.e., stained with
Trypan Blue dye) leak out through the dark gaps and are not
collected. By exploiting the dielectric differences between
different particles or cells, the DEP techniques described herein
have been used to discriminate and sort biological cells with
differences in membrane properties (e.g., permeability,
capacitance, conductivity, and so forth), internal conductivity,
and cell sizes. It should be appreciated that these techniques can
be extended to other particle or cell characteristics. The OET
technique not only inherits these DEP advantages but also provides
the capability of addressing each individual cell.
The selective concentration of live human B cells is demonstrated
from a mixture of live and dead cells in FIG. 4A through FIG. 4D.
The cells are suspended in an isotonic buffer medium of 8.5%
sucrose and 0.3% dextrose, mixed with a solution of 0.4% Trypan
Blue dye to check the cell viability, resulting in a conductivity
of 10 mS/m. The applied AC signal is 14V.sub.PP at a frequency of
120 kHz. The cell membranes of live cells are selectively permeable
and can maintain an ion concentration differential between the
intracellular and extracellular environments. By contrast the dead
cells are unable to maintain this differential ionic concentration
difference. So, then dead cells are suspended in a medium with a
low ion concentration, the ions inside the cell membrane are
diluted through ion diffusion, which results in a difference
between the dielectric properties of live and dead cells. Live
cells experience positive OET, and are collected in the center of
the shrinking optical ring pattern by attraction to the illuminated
region, while dead cells experience negative OET and are not
collected.
Single cell analysis is an important technique to comprehend many
biological mechanisms since it is capable of determining the
response spectrum of each individual cell under stimulation. A new
single cell and particle manipulation technique has been
demonstrated according to the invention which has enabled
manipulating a large number of single cells and particles in
parallel using direct incoherent optical images. By programming the
projected optical patterns, multi-step diagnostic protocols can be
achieved by combining multiple functions such as transporting,
sorting, recycling, and separating on a planar amorphous
silicon-coated glass slide. In addition to biological applications,
the high resolution electric field patterned on an OET surface can
also serve as a dynamic template to guide the crystallization of
colloidal structures.
2. Optoelectronic Tweezers.
The optoelectronic tweezers (OET) of the present invention are
designed for cell and microparticle manipulation using optical
control to permit functions such as cell trapping, collecting,
transporting, and sorting of cells and microparticles by using
sequentially projected images controlled by a spatial light
modulator (microdisplay or DMD mirrors). Since the optical
actuating power is as low as 1 mW, our invention permits the
optical manipulation using a lightly focused incoherent light
source and a direct image projection system. The system provides
substantially increased optical manipulating area and allows the
creation of complex optical patterns and thus more optical
manipulation functions.
FIG. 5 is a schematic example of the structure of an OET device 50
according to another embodiment of the invention. The example OET
shown includes spacers 44, 46 defining the horizontal extent of the
liquid cell structure and separating opposing surfaces 16, 18. An
AC bias may be applied across the top and bottom layers while
optical patterns are imaged on a photosensitive surface within the
lower layer 18. In a preferred embodiment the device comprises two
opposing surfaces with a top surface having a transparent layer 16
with a thin conductive layer 44 (i.e., aluminum) on an interior
surface and a bottom layer 18 with photosensitive surface 24. When
the light is illuminated on the photosensitive surface, such as
comprising a layer of amorphous silicon, it creates a light defined
virtual electrode 52 which generates a non-uniform electric field
in the liquid layer 12. The cells or particles nearby the virtual
electrode are manipulated by the electrostatic force.
FIG. 6 illustrates the OET of FIG. 5 within a cell manipulation
system 70. A programmable spatial light modulator 72, such as a
programmable DMD, is used to generate the required optical image
controlled by a processing means 74, such as a PC. The optical
image is projected onto the OET device, for example by reflecting
light generated by a light source, such as halogen lamp 76, to
create virtual electrodes within the structure for cell
manipulation.
The OET devices can be operated with or without pumps or channels
depending on the desired applications. Optional liquid pump 78 and
output ports 80 can be utilized for moving a liquid along with
particles or cells through the OET, or to change the conditions of
the OET.
The motion of the cells is captured by an imaging means 88, such as
a camera (i.e., charge coupled device (CCD)) with microscopic
focus, which provides a feedback signal for further processing. In
one mode of the invention a magnification lens system 82 is coupled
through a combination of color filters and beamsplitter 84 and a
light source 86 (i.e., mercury lamp) to allow images to be captured
by computer 74.
When an optical image is projected onto the photosensitive surface,
it creates a light patterned virtual electrode as shown in FIG. 5.
This virtual electrode generates an electric field around it for
manipulating the cells and particles through electrostatic force.
The electric field patterns generated by the optical patterned
electrode can have any kind of shape depending on the image
projected. It can form an electric field cage to capture a single
cell, or cell groups, or form an electric field channel to guide
cells. Since those virtual electrodes are all optically patterned,
they are fully programmable and reconfigurable. Optical
manipulation functions such as cell transport, cell collecting and
cell sorting can be achieved simultaneously on a single chip.
The exemplary OET device is particularly well-suited for
applications in cell manipulation at both multi-cell and single
cell levels. Liquid containing cells (or particles) are first
sandwiched between the two surfaces of the OET. The optical images
to be used are generated in the computer and then loaded for the
spatial light modulator, which is illuminated with either a
coherent or incoherent light source as shown in FIG. 6. The image
is then preferably projected onto the OET device through an
objective lens to create the optical patterned virtual electrodes.
The response of the cells or the particles can be captured by an
imaging means (i.e., camera) as a feedback signal for the computer
to generate a new optical pattern required for the optical
manipulation.
The microscopic imaging means coupled to the OET is preferably
configured with recognition algorithms which provide information
that allows image patterns to be created based upon the number,
characteristics, and position of the particles, and/or cells,
retained within the OET. For example this recognition algorithm can
be utilized for determining particles and/or cells of specific size
ranges, of specific colors and textures, or other directly
detectable characteristics such as colors, shape, texture,
conductivity, permeability, capacitance, motility, and so forth.
The imaging system is also preferably configured for detecting
indirect characteristics such as can be inferred from registering
the response of particles, and/or cells, to environmental changes
(e.g., aqueous solution changes, irradiation, temperature, pH, and
so forth), or to interaction between the particle, and/or cell, and
other particles, cells, and/or structures within the OET.
It will be appreciated that characteristics of particles, or more
typically cells, can be determined by microscopically detecting
response to changes in the environment, such as a shift in color,
shape, texture, and so forth of a particle or cell in response to a
temperature change, irradiation change, chemical characteristics
change of the surrounding liquid, interaction with other particles
and/or cells, and so forth. The microscopic imaging means can be
configured to store information for each particle, or cell, in its
field of view and to classify characteristics in response to
correlating detected changes in response to changes in the
environment. The microscopic imaging means can retain the
information about each particle, or cell, despite its movement
within the OET. In this way the present invention can be
implemented to perform a wide range of particle and cell sorting,
separation, classification, concentration, assembly, and other
desired objects of manipulation.
The OET device can also be integrated with pumps and channel
structures to provide for continuous optical manipulation.
Furthermore, the OET device can be combined with continuous optical
electrowetting (COEW) or conventional fixed electrode DEP
techniques to address specific applications suited to a hybrid
approach.
It should be appreciated that the present invention provides major
improvements to the art with respect to the OET structure and in
the utilization of photoconductive material. The OET device of the
invention can be comprised of substantially featureless layers
which do not require photolithography masking for fabrication,
wherein fabrication cost factors are substantially reduced. In
addition, it should be appreciated that low cost amorphous silicon
is preferably utilized as the photoconductive layer, while also
providing the benefits of low dark conductivity, high
photosensitivity and short electron diffusion lengths. It should be
appreciated that aside from amorphous silicon, other materials with
similar electrical and optical properties can also be utilized. In
addition, alternate embodiments can be providing by using other
mechanisms and forms of photoresponsivity, such as using a
phototransistor in place of the photoconductor structure.
The properties of the OET invention provide for optical
manipulation at very low optical power levels (i.e., on the order
of 1 mW) and sub-micron resolution of virtual electrode and also
permits optical manipulation with the OET using an incoherent light
source. It will be appreciated that conventional OET devices rely
on the use of coherent light sources, as dictated by their
structures.
The OET device of the present invention has been described and can
be used for a variety of applications, such as particle trapping,
collecting, multi-addressing, sorting on both microscopic particles
and live cells. The OET device of the invention allows for parallel
optical manipulation of cells on both single and multi-cell levels
using reconfigurable optical patterns from a direct image
projection system. No pumps, no microchannels and no valves are
required to handle cells in microfluidic environment. It is
contemplated that the use of the inventive OET device and methods
described herein will provide a significant step forward in the
field of particle manipulation, and in particular the manipulation
of cellular particles.
3. Dynamic DMD-Driven Optoelectronics Tweezers for Particle
Manipulation.
The ability to move and sort single cells is highly sought after in
the biomedical and biological community. Optical tweezers, and
dynamic holographic optical tweezers (HOT) arrays have provided a
means of performing individual cell manipulation, but require high
optical power levels (approximately 1 .mu.W-100 .mu.W) and have a
small trap area (<1 .mu.m). Optoelectronics tweezers (OET)
provides a method of cell manipulation which overcomes the
shortcomings of optical tweezers. It requires very low optical
power (i.e., on the order of microwatts), which opens up the
possibility of using incoherent light source and direct optical
imaging to pattern the traps.
Previously, we had demonstrated OET manipulation of microscopic
latex spheres and live E. coli cells using a single laser beam. A
spatial light modulator can be used to generate multiple OET traps
and novel patterns such as line and ring cages. In this paper, we
report on novel particle cages capable of trapping and moving
micro-particles by using a digital micromirror device (DMD) to
project dynamic images onto our OET device, via a standard
multimedia projector. It will be appreciated that this aspect of
the invention demonstrates microscopic particle manipulation using
a non-coherent light source, which should provide numerous benefits
within a number of applications.
FIG. 7A illustrates an example embodiment 90 of an optoelectronic
tweezers according to the invention which is based on the principle
of optically-induced dielectrophoresis. A buffer solution 12,
sandwiched between the nitride layer and the indium-tin-oxide (ITO)
top layer, contains the particles 14 of interest. In operation a
light source is focused onto photoresponsive layer 24, such as
comprising an AC-biased amorphous silicon (a-Si) photoconductive
substrate layer of the OET device. In the dark, the a-Si is highly
resistive, however, as the photoconductive layer is illuminated,
the conductivity of the a-Si is greatly increased, due to
photogenerated charge carriers, to create a localized virtual
electrode, and generate a non-uniform electric field in the buffer
solution. Dielectrophoretic (DEP) forces result from the
nonuniformity of the electric field. These forces are either
positive (particles attracted to electric field maxima) or negative
(particles attracted to electric field minima), depending upon the
dielectric properties of the particle and the media and the bias
frequency. FIG. 7B illustrates an experimental setup for the OET
material shown in FIG. 7A.
FIG. 8 depicts the spatial electric field distribution resulting
from a ring pattern projected onto the OET surface which can be
configured to form a single-particle trap. Negative DEP forces hold
a particle in the center of the light ring, as this corresponds to
a local electric field minima. Particles outside the ring are
repelled by the same forces.
Considering in detail the experimental setup 100 shown in FIG. 7B,
shown by way of example, a computer 102 (i.e., a personal computer
(PC)) outputs image signals to an InFocus.RTM. LP335 DMD-based
projector 104 used as both the light source 114 (i.e., having a
120-W 1000-ANSI lumen high-pressure mercury lamp) and as the DMD
driver circuit interface 116. The DMD, such as comprising an array
of MEMS mirrors, forms an image corresponding to the output of the
external monitor port of the PC. Light at the output of the
projection lens was collected, collimated, and directed by way
example with optics 118, lenses 106, 108, mirror 110 into an
objective lens 112 (i.e., 10.times.) onto OET device 90. The
objective focused the beam into the buffer solution 12 with a
conductivity of 0.1 mS/m, sandwiched between the ITO top layer and
photoconductive bottom layer. The photoconductive layer was
situated on the stage of a Nikon.RTM. TE2000E inverted microscope.
Observations were made via a CCD camera coupled into the inverted
microscope.
FIGS. 9A-9F depict images of particle caging and trapping according
to aspects of the present invention. The images in these figures
were formed on the focal plane of the objective using an optical
projector, such as standard presentation software (Microsoft
PowerPoint) on a PC connected to the DMD projector. Negative DEP
forces were observed on the 25 .mu.m latex spheres in solution, at
an AC bias of 19.5V and a frequency of 100 kHz. A variety of
patterns were used to manipulate the particles, including dynamic
line cages (FIGS. 9A-9C) and ring traps (FIGS. 9D-9F). Particle
movement was observed to be approximately 40 .mu.m/sec.
It should be appreciated that this aspect of the invention
demonstrates manipulation of micron-sized particles using
optically-induced dielectrophoresis from a non-coherent light
source. Various dynamic light patterns were successfully used as
particle traps and manipulators, moving 25 .mu.m latex spheres at
approximately 40 .mu.m/sec in a 0.1 mS/m buffer solution.
4. Dynamic Array Manipulation of Particles Via Optoelectronic
Tweezers.
Cellular-scale manipulation is an important tool in biological
research, and technologies that have demonstrated the capability
for such microscopic manipulation include optical tweezers and
dielectrophoresis. Although optical tweezers afford very fine
control of microparticles, the technique suffers from high optical
power requirements. Dielectrophoresis has been demonstrated to trap
particles as small as 14 nm. However, dielectrophoresis requires a
static pattern of electrodes, and is not easily reconfigurable.
Accordingly, the present invention demonstrates another method of
manipulating micrometer-scale objects using a technique of
optically-induced dielectrophoresis, or optoelectronic tweezers.
Using a laser to induce dielectrophoretic forces, the controlled
movement of 25 .mu.m latex particles, and E. coli bacteria has been
demonstrated. This technique can be utilized at very low optical
power levels, enabling the manipulation of particles and cells with
an incoherent light source. The use of a spatial light modulator in
the described optical system also allows for dynamic
reconfiguration of particle traps, providing increased versatility
in particle manipulation over conventional dielectrophoresis. The
present invention describes novel manipulation aspects in which
dynamic array manipulation of microparticles is performed using
optoelectronic tweezers. The self-organization of particles into an
array, and the formation of single particle arrays, are
demonstrated and provide the capability to individually address
each particle.
Dielectrophoresis (DEP) refers to the forces induced upon a
particle in the presence of non-uniform electric fields, which are
typically generated by a variety of electrode configurations. A
particle within an electric field forms an induced dipole, which
will experience a force due to the field gradient. The direction of
the induced dielectrophoretic force is dependent upon the frequency
of the electric field and the permittivity and conductivity of the
particle and the surrounding medium. Positive DEP results in
particle attraction to electric field maxima. In contrast, negative
DEP causes particles to be repelled from field maxima. Applying an
AC electric field thus allows the tuning of the type of DEP force
induced on a particle, as well as negating any electrophoretic
effects, or particle movement due to its surface charge.
The optoelectronic tweezers (OET) device according to this aspect
of the invention enables optically induced dielectrophoresis.
Unlike conventional DEP, no electrode pattern is required to
introduce non-uniformities into an applied electric field; instead,
a photoconductive layer is used to form virtual electrodes.
Focusing incident light onto the photoconductor substantially
increases its conductivity as compared to the dark areas,
effectively creating an electrode in the illuminated area,
analogous to the patterned electrodes in conventional DEP. In
addition, the virtual electrodes used by OET are movable and
reconfigurable, unlike the static electrodes of conventional
DEP.
FIGS. 10A-10B illustrate aspects of an OET trap according to the
present invention. In FIG. 10A, a single particle OET trap is shown
with a 45 .mu.m polystyrene sphere contained by optically-induced
negative DEP. In FIG. 10B distribution of the square of the
electric field is shown for the single particle trap in along the
cross-section A-A' as shown in FIG. 10A. DEP force is proportional
to the gradient of this distribution.
Shown by way of example, and not limitation, the single-particle
rectangular trap of FIG. 10A has inner dimensions of approximately
70 .mu.m by 50 .mu.m. A sphere with a diameter of 45 .mu.m is shown
"captured" by the surrounding light "walls" which are approximately
25 .mu.m wide. The corresponding cross-sectional distribution of
the square of the electric field shows that width of the trap as
experienced by the particle is approximately 50 .mu.m, as DEP force
depends on the gradient of this distribution as shown in FIG. 10B.
If negative DEP forces are induced by the trap pattern, all
particles outside the trap area will be repelled by the electric
field maxima forming the trap perimeter. Any particle within the
enclosed trap area will feel similar repulsive forces, however,
these forces balance and trap the particle. Once a particle is
contained within the rectangular pattern, the trap can be moved,
transporting the particle to a desired location. Furthermore,
multiple traps can be used as building blocks to form arrays of
trapped particles, which can be arbitrarily arranged, and
dynamically reconfigured.
The optical power required to induce DEP forces in the OET is much
lower than that required when implementing optical tweezers, as the
light energy provided for OET does not directly trap the particles.
Early experiments using OET showed movement of 25 .mu.m particles
at 4.5 .mu.m/sec with an optical power of 1 .mu.W, corresponding to
an incident power density of 440 mW/cm.sup.2. In comparison, a 1
.mu.m diameter optical tweezers trap, at a minimum trapping power
of 1 mW, has an optical power density of 32 kW/cm.sup.2.
The low optical power requirements of OET provide a number of
system design advantages. Inexpensive incoherent light sources can
be employed instead of lasers to provide the illumination necessary
for OET. In addition, light patterns can be produced by imaging
techniques (i.e., raster or vector based) rather than scanning
techniques. Furthermore, with no need to focus all optical energy,
a simple spatial light modulator can be utilized to pattern images,
rather than utilizing the holographic techniques employed by
optical tweezers arrays.
FIG. 11 illustrates an embodiment 90 of the optoelectronic tweezers
device with a liquid buffer containing the particles of interest
between the upper ITO glass layer and the lower photoconductive
layer. To separate the top and bottom layers, 100 .mu.m thick
spacers (not shown) are utilized.
In demonstrating the OET device and methods herein the digital
micromirror device (DMD) in a light projector was used to image the
virtual electrodes. An embodiment of the optoelectronic tweezers
device was formed by evaporating a 10 nm thick aluminum film onto a
glass substrate to provide electrical contact. A 1 .mu.m thick
undoped amorphous silicon (a-Si) photoconductive layer was then
deposited, for example by utilizing plasma-enhanced chemical vapor
deposition. It should be appreciated that detailed fine-pitched
features need not be created on the first and second retention
layers, wherein detailed lithographic steps are not necessary. To
protect the photoconductive film, a 20 nm thick silicon nitride
layer is preferably deposited over the a-Si in this embodiment. It
should be appreciated, however, that for some applications the
device can be formed without a dielectric. The liquid buffer layer
containing the particles of interest is sandwiched between this
photoconductive device and the opposing surface, such as comprising
indium-tin oxide (ITO) glass. An applied AC bias across the ITO and
a-Si produces the electric field.
Amorphous silicon has a dark conductivity of about 0.01 .mu.S/m to
1 .mu.S/m. Thus, in the dark, the a-Si has a much lower
conductivity than the liquid buffer (which has a conductivity of 10
mS/m), causing the majority of the voltage to drop across the
silicon layer. Incident light focused onto the photoconductive
layer substantially increases conductivity and creates a
non-uniform electric field surrounding the illuminated area, as the
majority of the voltage now drops across the liquid buffer layer.
In this manner, the light incident on the OET device can pattern
virtual electrodes for dielectrophoresis.
FIG. 12 illustrates by way of example an experimental setup 100 for
OET 90. In this example embodiment, the image from a projector 104,
such as an InFocus LP335, having mercury lamp 114, DMD 116, and
focusing optics 118 is focused via optical elements 106, 108 and
110 into a 10.times. objective lens 112, and projected down onto
OET device 90. Particle movement is observed on a microscopic
imaging means 120, for example a Nikon TE2000U inverted microscope,
coupled to computer 102.
A DMD-based projector (InFocus LP335) is shown used to display
images drawn on a PC, via Microsoft PowerPoint software. The
projector provides both the optical source (a 120 W, 1000-ANSI
lumen high-pressure mercury lamp) and the DMD-to-PC interface. The
output of the projector is collected, collimated, and directed into
an objective lens (i.e., Olympus MSPlan10 10X with NA=0.30),
projecting an image onto the OET device. The power at the projector
output was measured to be approximately 600 mW.
Approximately 7% of this power is collected by the objective lens
and focused onto the OET device. Therefore, the power of the light
incident on the OET is 42 mW, corresponding to an intensity of 12
W/cm.sup.2. The buffer solution comprises deionized water and KCL
salt, mixed to obtain a conductivity of 10 mS/m. Polystyrene
microspheres (45 .mu.m and 20 .mu.m) are mixed into the buffer
solution, and sandwiched into the OET device.
FIG. 12 illustrates, by way of example, a optical setup embodiment
100 for this OET demonstration. Observation of the particles under
test is performed preferably utilizing a microscopic imaging system
120, for example a Nikon TE2000U inverted microscope. A CCD camera
attached to the observation port of the microscope recorded images
and video of these demonstrations and tests. To produce the
electric field necessary for DEP, an AC voltage of approximately
10V.sub.PP at 100 kHz (i.e., Agilent.RTM. 33120A) was applied
across the top ITO surface and the bottom photoconductive surface
of the OET device.
FIGS. 13A-13B illustrate self-organization of 45 .mu.m polystyrene
spheres into an array configuration. After the initial grid
illumination shown in FIG. 13A, the randomly arranged particles
move towards the dark areas via negative DEP. After five seconds,
all particles are contained within the array cells as shown in FIG.
13B.
The self-organization of randomly distributed 45 kHz polystyrene
spheres into an array (FIGS. 13A-13B) is demonstrated by directing
a simple grid pattern of orthogonal horizontal and vertical lines,
such as drawn in PowerPoint, which are projected onto the OET
device. The pattern activates the optically-induced DEP, repelling
particles from the illuminated areas due to negative DEP forces.
This mechanism causes the self-organization of the particles once
the grid pattern is illuminated; wherein particles are pushed into
the non-illuminated cells. After a settling period, the particles
are trapped within the array of cells.
Due to a large trap relative to the particle size, the initial
self-organization may result in more than one particle per array
cell as shown in FIG. 13B. In this array, the largest cells are 80
.mu.m by 100 .mu.m. It may be possible to form self-organizing
arrays with a single particle per array cell by optimizing the
dimensions of a single array cell trap, such that only one particle
may fit into the potential well of the trap at any time.
FIGS. 14A-14B illustrate examples of single-particle manipulation
within the array. A particle in the lower-left side of the array is
made to change its array by combining cells in FIG. 14A, the
re-splitting the cell, moving the particle to the adjacent array
position shown in FIG. 14B.
In addition, it was found that certain particles within the self
organized array are able to escape when the array is moved around
the image plane. This phenomenon occurs for the array cells that
contain multiple particles. This occurrence, along with subsequent
manipulation of the self-organized array in FIG. 13B, allowed us to
obtain an array with a single particle per cell as shown in FIG.
14A. It should be noted that we were able to move the resulting
array of single particles around the image plane at approximately
25 .mu.m/sec.
Particles can be moved individually between adjacent cells, as
illustrated in FIGS. 14A-14B. The adjacent cells are merged by
first removing the dividing wall, and then re-separating the cells.
All movement of the trap walls are controlled in real-time by the
operator. To improve on the speed of this technique, a moving light
wall can be used to facilitate the transportation of the particle
between cells. This enables a single particle to be transferred to
any cell of the array, using repeated transfers between adjacent
cells.
FIGS. 15A-15D illustrate examples of flushing an array row to
remove undesired particles from the array. First, the walls of the
cells in the row to be flushed are removed as shown in the top row
of FIG. 15A. The particles are no longer bounded in the lateral
direction as shown in FIG. 15B. An operator-controlled light bar is
then used to push the particles out of the array as shown in FIG.
15C and FIG. 15D.
Since the patterns for manipulating the particles in the array are
created dynamically by optical illumination, a wide variety of
operations can be performed by simple software programming. For
example, to flush the particles in a single row of the array, we
remove the dividing walls of that row and use a moving wall to
sweep out the particles (FIGS. 15A-15D).
In addition to self-organizing behavior, arrays can be formed from
multiple single-particle traps. Each randomly positioned particle
is first contained within a square trap. This is performed by
drawing a rectangle around each particle in PowerPoint. The
multiple traps can then be positioned to form an array of
individually addressable cells.
FIG. 16 illustrates an example of an array of single particles,
formed from multiple single particle square traps. Each particle is
individually addressable. The time required to form this array of
20 particles was 3 minutes. Using this technique, we are able to
form a 4.times.5 array of single particle traps as shown in FIG.
16. Though the operation was performed manually, it can potentially
be automated by combining OET with a vision system. Biological
applications of such an array include studies on single-cell
behavior and interaction. Since each cell of the array is an
independent single particle trap, the array has the capability of
being dynamically rearranged.
FIGS. 17A-17D illustrate an example of dynamic rearrangement of an
array containing both 45 .mu.m and 20 .mu.m particles. An array is
rearranged by moving individual cells into a desired configuration.
Total rearrangement time for the embodiments was three minutes. The
spheres are reorganized under operator control in the images shown
in FIGS. 17A-17D, which demonstrates the addressability of each
particle trap, as well as the dynamic nature of the OET
patterns.
Movement of a single 45 .mu.m sphere in response to negative DEP
provides a maximum velocity of approximately 35 .mu.m/sec. This
corresponds to an estimated force of 15 pN, based upon Stoke's Law.
The maximum velocity of a 20-particle array is limited to
approximately 25 .mu.m/sec. Thus, the minimum holding force of each
individual array cell is 10 pN. This force is less than that
experienced by a single 45 .mu.m particle, probably due to slight
nonuniformities in image sharpness over the entire array area. The
more defocused areas will have less of an electric field gradient,
and a correspondingly lower DEP force. Thus, this 10 pN force
reflects the minimum trapping force of all of the array cells.
The forces attained in these experiments, using an optical power
density of 12 W/cm.sup.2, are in rough agreement with our earlier
results using a 632 nm laser light source. Our earlier data
suggests that the optical power density necessary to achieve a
force of 15 pN is 6.6 W/cm.sup.2. The difference between this
predicted power density requirement and our experimental findings
can be attributed to losses through the additional optics needed
for our current experiment.
These results compare favorably to other microparticle manipulation
techniques. Conventional dielectrophoresis uses static electrode
patterns, and is thus not reconfigurable. In addition, our device
is less expensive to produce, as no photolithographic steps are
needed. Addressable DEP arrays have also been demonstrated using
CMOS technology, but these devices are expensive to produce, and
the minimum electrode size is limited by the required CMOS
circuits. Both conventional DEP and optical tweezers are capable of
manipulating particles a few nanometers in diameter. The minimum
size of the virtual electrode in OET is limited by the 115 nm
ambipolar diffusion length of the a-Si. The OET can operate over a
large area (i.e., approximately 1.times.1 mm), which is much
greater than the 20 .mu.m.times.20 .mu.m area for optical
tweezers.
Though holographic tweezers can generate multiple traps, direct
imaging using a DMD is more versatile. It can generate any
arbitrary pattern with high contrast ratio. No computation is
required to generate the desired pattern. Furthermore, OET can
induce repulsive forces on transparent dielectric particles such as
biological cells, and form cell cages, which is not possible with
optical tweezers. On the other hand, optical tweezers traps are
three-dimensional, whereas our trap patterns are limited to two
dimensions. Utilization of the present invention generally requires
being more selective in the choice of buffer solutions, because the
conductivity of the solution plays an important role in the DEP
phenomenon.
The self-organizing of 45 .mu.m polystyrene particles into an
array, and the creation of an array from multiple single particle
traps utilizing optoelectronics tweezers have been demonstrated
with the present invention. Single particle movement within the
array has been demonstrated, showing the ability to address
individual array cells. Movement of single 45 .mu.m polystyrene
spheres was measured to be 35 .mu.m/sec (a force of 15 pN).
Movement of a 20-particle array was performed at 25 .mu.m/sec (a
force of 10 pN). Such particle manipulation techniques have many
applications to experiments with biological cells and
microparticles.
5. Microvision-Activated Automatic Optical Manipulator for
Microscopic Particles.
An embodiment of the present invention includes an automatic
optical manipulator that integrates microvision-based pattern
recognition and optoelectronic tweezers (OET) for processing
microscopic particles. This system automatically recognizes the
positions and sizes of randomly distributed particles and creates
direct image patterns to trap and transport the selected particles
to form a predetermined pattern. By integrating the OET with a
programmable digital micromirror device display (DMD), we are able
to generate 0.8 million pixels of virtual electrodes over an
effective area of 1.3 mm.times.1 mm. Each virtual electrode is
individually controllable for parallel manipulation of a large
number of microscopic particles. Combining the automatic
microvision analysis technology with the powerful optical
manipulator, this system significantly increases functionality and
reduces processing time for microparticle manipulation.
Tools for manipulating microscopic particles are important in the
fields of cell biology and colloidal science. Optical tweezers and
dielectrophoresis are two of the most widely used mechanisms for
manipulating microparticles. Optical tweezers use direct optical
forces to deflect the motion of microscopic particles. Optical
tweezers are noninvasive and have high positioning accuracy. The
use of holographic optical tweezers further extend the benefits to
allow manipulating multiple particles. However, these techniques
require very high optical power levels, and provides limited
working area (<100 .mu.m.times.100 .mu.m) due to the need of
tight focusing with high numerical aperture (N.A.) lenses. These
factors limit the use of these forms of optical tweezers in
large-scale parallel manipulation applications.
In contrast, dielectrophoresis (DEP) controls particle motion by
subjecting particles to non-uniform electric fields. The technique
provides high throughput and large working area, but requires a
fixed electrode pattern for a given function. Programmable DEP cage
array consisting of two-dimensional electrodes with integrated
driving circuits has been demonstrated on a CMOS (complementary
metal-oxide-semiconductor) chip. However, the resolution is limited
by the pitch of the electrode and the driving circuits of the unit
cell, and the cost may prohibit its use as disposable devices.
FIGS. 18A-18B illustrate aspects of an example embodiment of an
optical manipulation system. In FIG. 18A a schematic diagram is
shown of an example embodiment 130 of a microvision-based automatic
optical manipulation system. In FIG. 18B the structure of the OET
device is shown.
According to the present invention a novel optoelectronic tweezers
(OET) has been developed to address DEP forces on a photoconductive
surface using optical beams. OET enables virtual electrode patterns
to be created optically. The electrode size can be varied
continuously by the optical spot size down to the diffraction limit
of the objective lens. Because of the optoelectronic gain in the
photoconductor, the required optical power density is five orders
of magnitude lower than that of optical tweezers. This enables our
method to use a digital optical project with incoherent light
source to manipulate microparticles. The present invention
describes the use of "light walls" to confine microparticles in
virtual microfluidic channels and switch them by light pistons.
Interactive manipulation of virtual DEP cage arrays has also been
demonstrated by manually changing the optical patterns.
In this aspect of the invention automatic optical manipulator use
is described by integrating the OET with a microvision-based
analysis system. The microvision system automatically recognizes
the particle positions and sizes, generates the desired trapping
patterns, and calculates the moving paths of the particles. It
enables close-loop control of trapping, transporting, and
assembling a large number of particles in parallel.
The microvision based optical manipulation system 130 of FIG. 18A
is constructed with OET device incorporating a microscopic imaging
means and a mechanism for registering particle/cell characteristics
and position.
Particle or cell movement is controlled by projecting light
generated from source 114 reflected from DMD 116 through objective
112 onto OET 90. The light patterns are generated in response to
the positioning and characteristics of the particles or cells as
registered by a microscopic imaging means in combination with image
analysis and pattern recognition algorithms. In this example the
images are collected through lens 132 onto a CCD imager 134, and
the data is processed to control the patterns of light being
generated. The microscopic images, or image stream, is analyzed
within an image analysis circuit and/or routine 136. The image data
is then processed using pattern recognition circuits and/or
routines 138. The recognition of actual patterns is performed in
relation to the desired goal of the application, wherefrom
subsequent patterns are generated by pattern generator circuit
and/or routine 140, which is then converted by DMD circuit and/or
routine 142 to control the operation of the programmable DMD device
116. It should be appreciated that this may be implemented in a
number of alternative ways without departing from the teachings of
the present invention, such as using various imaging sources,
microscopic imaging, and different techniques for detecting,
analyzing, and generating subsequent optical images onto the
OET.
By way of example, this embodiment incorporates a Nikon inverted
microscope 132, 134. A 150 W halogen lamp 114 illuminates on a
programmable digital micromirror device (DMD) microdisplay 116. The
DMD pattern is imaged onto the OET device through a 10.times.
objective lens 112. The structure of OET device 150 is shown in
FIG. 18B.
FIG. 18B illustrates another example OET embodiment comprising a
top and bottom surface 16, 28, comprising such as indium-tin-oxide
(ITO) glass and photosensitive layer 24, such as amorphous silicon,
on the surface of the top and/or bottom layers. The liquid medium
12 containing the particles 14 are sandwiched between these two
surfaces. The OET is biased by a single AC voltage source 30.
Without light illumination, most of the voltage drops across the
amorphous silicon layer 24 because its impedance is substantially
higher than liquid layer 12. Under optical illumination, the
conductivity of the amorphous Si 24 increases in the areas upon
which the illumination is impinging by several orders of magnitude,
shifting the voltage drop to the liquid layer. This light-induced
virtual electrode thereby creates a non-uniform electric field 152,
and the resulting DEP forces drive the particles of interest. The
light-induced DEP force can be positive or negative, controlled by
the frequency of the applied AC signal. Negative DEP force repels
particles away from the high field region, and is preferable for
single particle cage, which can be easily formed by a light wall
around the particle. Positive DEP tends to attracts multiple
particles. We have employed negative DEP force in our automatic
optical manipulator experiments. The image on the OET device is
captured by a CCD camera through the inverted microscope and sent
to a computer for image processing.
Software according to the present invention analyzes the real time
video frames and generates the corresponding optical patterns for
trapping and moving the particles. These patterns are then
transferred to the DMD, and our test setup allows direct control of
individual pixels. The resolution of the projected optical image on
the OET device is 1.3 .mu.m, defined by the pixel size of the
mirror (13 .mu.m). The effective optical manipulation area on the
OET is 1.3 mm.times.1 mm. By combining the DMD mirrors with the OET
device, the silicon-coated glass is turned into a million-pixel
optical manipulator.
FIGS. 19A-19D illustrate the process of automatically recognizing
and arranging randomly distributed particles into a predetermined
pattern. First, the images of the particles are captured and
analyzed by the microvision system as in FIG. 19A, which identifies
the positions and the sizes of all particles as in FIG. 19B. The
software then generates a ring trap around each particle as in FIG.
19C. It also calculates the trajectories of the particles to reach
their final positions as in FIG. 19D.
FIGS. 20A-20D illustrate by way of example test images for a
particle recognition system. Polystyrene particles with three
different sizes, 10 .mu.m, 16 .mu.m, and 20 .mu.m, are mixed and
randomly distributed in the liquid medium. In FIG. 20B, the
microvision system recognizes the position of each particle and
projects a ring mark on each particle. In FIG. 20C, the histogram
showing the number of particles versus the number of recognized
dark pixels in this test image. In FIG. 20D the largest particles
are selectively picked up by setting a threshold for the dark
pixels.
Particle recognition is achieved by using a dark-pixel recognition
algorithm to scan through each pixel of the captured image. The
brightness value and the position of each pixel are then recorded
and calculated to determine the size of each particle and its
center position. The brightness value of the pixels at the particle
edge is smaller than that of the background and the color is darker
too. By setting a threshold brightness value between the background
and the particle edge, we can recognize the edge pixels of each
particle. Averaging the x and y position data of the edge pixels of
each particle, we can determine its position.
FIG. 20B shows the recognized particles marked by a white ring
pattern generated by the microvision analysis system. The same
algorithm also determines the size of each particle by counting the
number of the recognized dark pixels.
FIG. 20C is a histogram of data showing the number of particles and
the number of the dark pixels recognized for each particle on this
image. As larger particles have more dark pixels than smaller ones,
a threshold number can be set for the recognized pixels, as
indicated by the dash line in the histogram figure, wherein the
system can selectively register particles with certain sizes.
FIG. 20D depicts the seven largest beads (20 .mu.m), by way of
example, that are selected by setting a threshold number equal to
180. This recognition algorithm is used specifically for
determining spherical particles with different sizes. Other
algorithms can be developed to recognize particles with different
colors, shapes, or textures.
FIGS. 21A-21B illustrate, by way of example, the electric field
distribution induced by a single optical ring pattern. In static
state shown in FIG. 21A the particle is trapped in the electric
field minimum in the center. During moving as shown in FIG. 21B the
particle is displaced from the center as a result of the balance
between the DEP and the viscous forces.
Particle trapped by an optical ring pattern trapping of a single
particle is achieved by operating OET in the negative DEP regime.
We create an optical ring pattern to form a virtual DEP cage that
allows only one single particle to be trapped inside the ring, as
shown in FIG. 21A.
In static state, the trapped particle will be focused at the center
or the ring pattern where the minimum electric field strength
occurs. When the optical ring moves, the trapped particle also move
in the same direction but with a position deviated from the ring
center so that the DEP force pushes the particle in the direction
toward the center. This deviation distance depends on how fast the
particle moves. When the optical ring moves too fast, the particle
will escape the optical ring because the DEP force is not strong
enough to hold it. The escaping speed of a 20 .mu.m particle is 40
.mu.m/sec in our current system. To trap a particle with a smaller
size, a smaller optical ring would be required to ensure a single
particle in the ring.
FIGS. 22A-22C each illustrate multi-frame example sequences of
microvision-based automatic optical manipulation of microscopic
particles. In FIG. 22A randomly distributed particles are shown
being arranged into a hexagonal shape. In FIG. 22B the hexagonal
pattern of FIG. 22A is shown being transformed into a line. In FIG.
22C the line pattern of FIG. 22B is then transformed into a
triangle shape. In each case the unwanted particles are swept away
by a scanning line.
Once the particle positions are recognized, the software is
configured to generate the corresponding ring-shaped traps and
calculates the transport trace for each particle. These optical
patterns are stored as image files and are batch loaded to the DMD
control software to create dynamic optical patterns to trap and
transport particles. These processes are shown in FIG. 22A. The
image of the randomly distributed particles was scanned vertically
from left to right. The first six particles were identified and
trapped by the OET by the 0 second frame. The trapped particles
were transported by moving the ring traps, and reached the
hexagonal configuration in 12 seconds.
FIGS. 22B and 22C show the video sequences of rearranging the
particles into linear and triangular shapes and the unwanted
particles were swept away by a scanning line pattern.
An automatic optical manipulator has been demonstrated that
provides a feedback control through a microvision analysis system.
This system can automatically recognize particles with specific
size from a mixture of particles with different sizes and generate
optical manipulating patterns to trap and move these selected
particles to form a predetermined pattern. The large optical
manipulation area (>1 mm.times.1 mm) of our OET device permits
parallel manipulation of a large number of microscopic particles.
The automatic parallel optical manipulation system greatly reduces
the time for sorting and patterning microscopic particles. With
further optimization, the system will be able to sort particles
with different colors, shapes, or textures. More sophisticated
optical manipulation functions can also be performed. The automatic
optical manipulator has many potential applications in biological
cell analysis and colloid science fields.
6. Manipulation of Live Red and White Blood Cells.
Optoelectronic tweezers (OET) provides a new tool for single-cell
manipulation for biological research applications. Current
cell-manipulation technologies, such as optical tweezers and
dielectrophoresis, have limitations that can be overcome by
OET.
Optical tweezers are a widely used tool for the manipulation of
cells and microparticles in the micro-scale and nanoscale regimes.
By integrating holographic imaging techniques with optical
tweezers, multiple particle traps can be created from a single
laser source. However, optical tweezers requires expensive,
high-power lasers, and is limited in its effective manipulation
area.
Dielectrophoresis (DEP) describes induced particle motion along an
electric field gradient due to the interaction of the induced
dipole in the particles and the applied electric field. This
technique has been used to perform many biological experiments,
including cell and DNA trapping and cell sorting. A limitation of
conventional DEP devices is the difficulty of reconfiguring the
devices for different experiments, as they rely on patterned metal
electrodes to create the required non-uniform electric fields. By
using CMOS technology to create DEP traps, real-time reconfigurable
DEP devices can be achieved. However, these CMOS-based devices have
a limited resolution, due to the area of the circuitry.
Optoelectronic tweezers offer low-power optically-controlled
actuation of cells and microparticles via light-patterned virtual
electrodes on a photoconductive surface. Since OET is directly
controlled by optical images, it is easy to reconfigure in real
time. In addition, high-resolution cell manipulation is achieved
over a large area. As one of the major potential applications of
OET is biological analysis, a number of experiments were performed
using live cells. The first demonstration of OET on living cells
was performed by Chiou et al., on E. coli bacteria, proving the low
optical power of OET is capable of manipulation of live single
cells without causing photodamage. In this aspect of the invention
we present a description and demonstration of OET manipulation of
live mammalian cells.
Optical tweezers work by directly converting photon momentum to a
mechanical force on a microparticle or nanoparticle. This requires
a highly-focused, intense laser beam. In contrast, optoelectronic
tweezers creates an optically-patterned electric field, which in
turn produces a dielectrophoretic force on particles. Due to the
photoconductive gain, the required optical power is on the order of
100,000 times less than that required of a typical optical
tweezers. As a result, an incoherent light source, such as an LED
or halogen lamp, is sufficient for OET actuation. Furthermore, the
optical pattern does not need to be highly focused, allowing OET to
be effective over a larger area (currently 1.3 mm.times.1.0 mm)
than optical tweezers traps.
FIG. 23 illustrates an example embodiment 170 of the structure of
an OET device according to the invention. The OET consists of an
upper planar electrode 16 of ITO-glass and a lower photoconductive
layer 24, between which are sandwiched a layer of liquid solution
12 containing the cells or microparticles 14 of interest. The upper
planar electrode in this embodiment consists of a conductor such as
indium-tin-oxide (ITO) which itself is transparent over a
transparent glass slide, while the lower photoconductive layer is
preferably formed with hydrogenated amorphous silicon (a-Si:H)
deposited onto a ITO-coated glass slide via plasma-enhanced
chemical vapor deposition (PECVD). Projecting light patterns 172,
174 modifies the electric field profile, creating a
dielectrophoretic force. An AC bias 30 is placed across the upper
electrode and the lower photoconductive layer.
In the dark regions, the applied AC voltage is dropped primarily
across the highly-resistive (R.sub.S) a-Si:H layer, which has a
much higher impedance than (R.sub.L) of the liquid 12, resulting in
a low electric field in the liquid solution. However, in the
illuminated regions, the projected light creates virtual
electrodes, by locally increasing the conductivity of the a-Si:H.
The photoconductor is now less resistive than the liquid, creating
a high electric field region in the liquid above the virtual
electrode. This creates non-uniform electric fields, which in turn
creates a dielectrophoretic force to drive the cells.
Dielectrophoretic force is AC frequency-dependent. Thus, by varying
the frequency of the applied AC bias, the force can be adjusted
from an attractive force to a repulsive force, or vice-versa. Since
OET uses optically-induced DEP, the OET force is also tunable in
the same manner. As a result, there are two operating modes for
OET: positive OET, in which cells and microparticles are attracted
to the illuminated areas, and negative OET, in which cells and
microparticles are repelled by the illuminated areas.
FIG. 24 illustrates an example embodiment 190 configured for
manipulation of bovine red blood cells utilizing optoelectronic
tweezers according to the present invention. For this
demonstration, the optical source 114 consisted of a 0.8 mW He--Ne
laser (.lamda.=633 nm). A spatial light modulator 116 is not
necessary if a laser is used as the light source, as the laser can
be directed into the objective either directly or through a mirror.
A 10.times. objective lens 112 was used to reduce the laser beam
size to about 20 .mu.m in diameter. A personal computer (PC) 102 is
shown coupled to control the light imaging via laser output control
or a spatial modulator as well as to control the signals generated
at OET device 170, such as bias voltage, from function generator
192 and for collecting data from a microscopic imager 88. The
prepared cell solutions consisted of red blood cells (RBCs) from
bovine serum, suspended in an isotonic solution (8.5% sucrose, 0.3%
dextrose) at concentrations ranging from approximately 1 to 10% by
volume. Approximately 5 .mu.L of this solution was introduced into
the OET device.
FIGS. 25A-25D depict resultant concentrations of blood cells using
the OET of FIG. 24. A strong positive OET response was observed at
an applied AC bias of 3 V.sub.PP at 200 kHz, attracting the red
blood cells towards the laser spot shown in FIG. 25A. Initially,
the laser is on in FIG. 25A, but no electric field is applied. An
AC bias is then applied to the OET device, producing OET force,
which attracts the blood cells to the illuminated area as shown in
FIG. 25B. It was also observed that the cells align vertically
along the electric field lines as in FIG. 25B. When the laser is
turned off, the concentrated cells remain in the area that the
laser spot was focused as shown in FIG. 25C. As the applied voltage
is switched off, the concentrated red blood cells began to slowly
pulsate, migrating away from the central area as shown in FIG. 25D,
implying that they remain alive and viable.
The use of an incoherent light source and direct image patterning
techniques increases the flexibility and functionalities of OET. A
spatial light modulator can pattern any arbitrary image to be
projected onto the photoconductive surface, creating the
corresponding virtual electrodes on the OET device. Complex,
reconfigurable manipulation patterns can thus be created by simple
software programming. This powerful technique is demonstrated in
the arrangement of human B-lymphocytes into a complex pattern.
A 100 W halogen lamp was used as the incoherent optical source. The
spatial light modulator consisted of the Texas Instruments digital
micromirror device (DMD). The DMD is a 1024.times.768 array of
individually-addressable micromirrors, each of which is 13.68
.mu.m.times.13.68 .mu.m. The images displayed on the DMD are
controlled via a computer. A 10.times. objective lens was used to
increase the resolution of each DMD mirror to approximately 1.4
.mu.m. The prepared cell solutions consisted of human white blood
cells (B-lymphocytes), suspended in an isotonic solution.
Approximately 5 .mu.L of this solution was introduced into the OET
device.
FIGS. 26A and 26B illustrate how B-lymphocytes can be manipulated
into an arbitrary pattern. In this example we chose to assemble the
cells into the shape of a "U" (FIG. 26A) and a "C" (FIG. 26B)
character (for Univ. of California). At an applied bias of
14V.sub.PP at a frequency of 100 kHz, the white blood cells exhibit
positive OET behavior. A shrinking concentric ring pattern is used
to concentrate the cells towards the character image. Cells are
attracted to each concentric ring. As the rings shrink, the cells
are transported towards the center of the concentric rings, where
the character image is projected. The cells then become trapped by
the static character pattern.
Optoelectronic tweezers provides a powerful tool for single-cell
manipulation. The use of direct imaging and incoherent light
sources provides OET with more flexibility than conventional DEP.
The OET technique also uses considerably less optical power than
optical tweezers, while still providing a larger effective
manipulation area. Concentration and manipulation of cells,
specifically red blood cells, has been demonstrated by using the
OET and methods of the present inventive aspect. It should be
appreciated that these manipulation functions can be easily
tailored to a specific biological experiment.
7. Novel Optoelectronic Tweezers Using Light Induced
Dielectrophoresis.
Optical tweezers have become an important tool in biological
research areas since they were first demonstrated. However, the
potential photodamage caused by the intense optical energy has
restricted its use. For example, a 100 mW optical tweezers has a
light intensity on the order of 10.sup.10 mW/cm.sup.2 when focused
to diffraction limit. Such an intense light energy may cause
damages due to local heating or two-photon absorption. To reduce
the photodamage, lasers with wavelengths in the near infrared
region are often chosen to avoid the absorption in water or
biological objects. However, the recent research shows the cell
metabolism may still be affected even using infrared lasers.
Recently, a light induced electrophoresis mechanism has been
proposed to optically address polymer beads by using DC electric
bias. The electrically charged particles are attracted to the
electrode with opposite polarity. In this paper, we present a light
induced dielectrophoresis mechanism that would allow the optical
addressing of electrically neutral micro-particles with .mu.W
optical energy, which is much lower than the approximately 1 mW to
100 mW of optical energy used by optical tweezers.
Dielectrophoresis (DEP) refers the motion of an electrically
neutral particle resulting from the interaction between the applied
electric field and the induced dipole. It has been used widely in
the manipulation of microparticles or sub-micro-particles and
biological cells. An analytical expression of DEP force is given by
the following expression.
.times..times..pi..times..times..times..epsilon..times..function..times..-
times..omega..times. .function. ##EQU00001##
.times..times..omega..times..times..times..sigma..omega..times..sigma..om-
ega. ##EQU00001.2##
According to the above equation E is field strength, a is particle
radius, .di-elect cons..sub.m and .di-elect cons..sub.p are the
permittivities of the surrounding medium and the particle,
respectively, .sigma..sub.m and .sigma..sub.p are the conductivity
of the medium and the particle, respectively, with .omega. the
angular frequency of the applied electric field.
FIGS. 27A-27B illustrate an OET embodiment 210 in FIG. 27A with an
Illustration of the light induced dielectrophoresis mechanism in
FIG. 27B.
The term Re[K*(.omega.)] can have any value between 1 to -1/2,
depending on the applied AC frequency and the polarizability of the
particle and the medium. If Re[K*(.omega.)]<0, it is called
negative DEP with the direction of the DEP force towards lower
electric field. Since the DEP force is proportional to the gradient
of the square of the applied electric field, a highly non-uniform
electric field is desired to achieve a higher trapping force. In
the following experiment a light induced negative DEP force is
demonstrated.
In FIG. 27A, the structure of the optoelectronic tweezers are shown
with a liquid solution 12 containing the particles sandwiched
between two surfaces separated by a gap spacing of 100 .mu.m. The
top surface 16 is a commercial ITO glass. The bottom surface is a
glass substrate 28 coated with three pattern-less layers: a 2000
.ANG.-thick aluminum layer 46, a 2 .mu.m-thick photoconductive
(amorphous silicon) layer 24, and a 200-.ANG.-thick silicon nitride
layer 26. An AC bias 30 is applied between the top (ITO) and the
bottom (aluminum) electrodes. In the dark state, the majority of
the voltage drops across the photoconductor due to its high
electrical impedance, which results in a very weak electric field
in the liquid layer. When the laser beam 40 is focused through
objective 38 on photoconductive layer 24, the local
photoconductivity at the site under light illumination is greatly
increased due to the photogenerated electron-hole pairs.
FIG. 27B depicts a light defined micro electrode turned-on locally
and creating a highly non-uniform field in liquid layer 12. The
laser spot creates a light defined electrode and a highly
non-uniform electric field in the liquid layer. The particles
inside the liquid are polarized by the non-uniform field and pushed
away from the illuminated site by the negative DEP force.
Since light is used to switch the AC voltage drop between the
photoconductive layer and the liquid layer, rather than to directly
trap the particles, the required optical power is orders of
magnitude lower than that of conventional optical tweezers.
FIG. 28 depicts experimental results for the OET of FIG. 27A with
the relationship between particle speed and optical power. In the
experiment, a 800 .mu.W laser with a beam width 0.24 mm and a
wavelength of 632 nm is used as the light source. The laser beam is
preferably steered by a pair of orthogonally scanning galvanometer
mirrors and then sent through a combination of a convex lens and a
40.times. objective lens. The optical spot size on the
photoconductive layer is around 17 .mu.m. Neutral density filters
are used to control the incident optical energy. A 100 kHz AC bias
is applied between the top and the bottom electrodes to drive 25
.mu.m latex particles. To measure the particle speed, the scanning
mirror is programmed to scan at a constant speed to push the
particle. The particle is pushed by the optical beam, until at
sufficiently high scan rate, the particle can no longer keep up
with the optical beam. The maximum speed at which the particle
responds to scanning optical beam is measured for various optical
powers and AC bias voltages. An optical beam with power as low as 1
.mu.W light is sufficient to transport the particle at a speed of
4.5 .mu.m/sec at 10 V AC bias. The maximum speed observed here is
397 .mu.m/sec, which corresponds to a force of 187 pN estimated by
Stokes' law.
FIG. 29A and FIG. 29B illustrate an example of multi-particle
focusing, in which the laser beam is programmed to scan in circular
patterns. The four particles are focused, or squeezed, to the
center of the shrinking circular pattern.
According to the present aspect of the invention a novel
optoelectronic tweezers is demonstrated which is successfully
applied to transport neutral micro particles. The required optical
power on the order of from (i.e., approximately 1 .mu.W-100 .mu.W)
is one to two orders of magnitudes lower that that of optical
tweezers. Particle transport speed of 397 .mu.m/sec and trapping
force of 187 pN are measured for 25 .mu.m latex particles with 100
.mu.W optical power and 10 V AC bias.
8. Optical Sorting Mechanism in Dynamic Electric Field.
Optoelectronic tweezers (OET) have been proposed herein as a
powerful tool for cell and microparticle manipulation, via direct
optical images. The optically-patterned electrical field generated
on the OET surface can be configured to trap and transport single
or multiple cells in parallel. Such a dynamic reconfigurable
electric field provides driving forces for sorting particles
without the need for pumps to introduce fluid flow, as presented in
most of the microfluidic sorters. It completely eliminated the need
for the fabrication and integration of complex microfluidic
components, adding lots of flexibility in the applications of cell
or particle manipulation. In the present invention an OET based
sorting mechanism is demonstrated using a dynamic moving light
beam. Particles on the OET surface can be sorted simply by scanning
a light beam across the OET surface. The sorted particles can be
transported to other areas by other dynamic optical patterns that
have been demonstrated in the industry. The following portion of
the present invention focuses on the fundamental mechanism of
OET-based optical sorting.
FIGS. 30A-30C illustrate dynamic electric fields induced by an OET
according to the present invention. In FIG. 30A a schematic diagram
is shown for the OET device, in which different sized particles 14,
14', 14'' are sandwiched between a top ITO glass 16 and a bottom
OET photoresponsive surface 24. In FIGS. 30B-30C randomly
distributed particles are sorted out when the line-shape laser beam
scans across the OET surface.
Optoelectronic tweezers are a novel mechanism that enables optical
patterns to induce highly non-uniform electric fields on a
photoconductive thin film material. The particles near the
non-uniform electric field experience a net force, resulting from
the interaction between the electric field and the induced electric
dipole of the particles. This force is called dielectrophoretic
(DEP) force, which can be expressed in the following relation.
.times..times..pi..times..times..times..epsilon..times..function..times..-
times..omega..times. .function. ##EQU00002##
According to the above equation E is field strength, r is particle
radius, .di-elect cons..sub.m and .di-elect cons..sub.p are the
permittivities of the surrounding medium and the particle,
respectively, .sigma..sub.m and .sigma..sub.p are the conductivity
of the medium and the particle, respectively, with .omega. the
angular frequency of the applied electric field and K*(.omega.) is
the Clausius-Mossotti (CM) factor, which has a value between 1 and
-0.5, representing the polarizability of the particle.
This force is very sensitive to the size of the particle a.sup.3
and the non-uniformity of the field ( VE.sup.2). If a particle is
less polarizable than the medium, its real part of the CM factor is
negative, and the particle will be pushed away from the high
electric field area. When a line-shaped laser beam scans across the
OET surface, it produces an electric field pattern that moves at
the same speed. This light-induced electric field will push
particles in the OET device. The relative distance between the
moving light beam and the particles is determined by the balance
between the DEP force and the viscous force. Using Stoke's Law to
estimate the viscous force for a moving particle, we obtain the
following relationship between the particle size and nonuniformity
of the field.
.times.
.function..times..times..eta..times..times..times..function..time-
s..times..omega. ##EQU00003##
In the above equation r is the particle radius and C is a constant
determined by the light scanning speed v, real part of the CM
factor, and also the viscosity .eta., and permittivity .di-elect
cons..sub.m, of the surrounding medium. Since the term VE.sup.2 is
a function of the relative distance between the particle and the
electric field maximum, particles with different sizes will have
different deterministic relative distances to the center of the
scanning laser beam. Based on this principle, particles with
different sizes will be sorted out when the laser beam scans across
the OET surface.
FIG. 31 illustrates an example embodiment 230 of an experimental
setup for optical sorting of microscopic particles. A single-mode
fiber pigtailed laser diode 114 with a wavelength of 635 nm is
coupled through a fiber collimator 232, producing a beam spot size
of 3 mm and an optical power of 120 .mu.W. A cylindrical lens 234
directed through a 2D scanning mirror 236 and dichroic mirror 238
and a 10.times. objective 38 lens are used to shape the circular
Gaussian beam into a line shaped pattern and focus it onto the OET
surface. A scanning mirror is programmed to steer the laser beam.
The OET device 210 is shown on a stage 240 and configured with a
microscopic imaging means for registering particle (or cell)
position and characteristics within the OET.
FIGS. 32A-32D depicts the result when the laser beam (120 .mu.W red
diode laser at wavelength=635 nm) scans across the OET surface of
FIG. 31 where the 10 .mu.m and 20 .mu.m diameter polystyrene beads
are randomly distributed. In FIG. 32A the particles are randomly
distributed on the surface. In FIG. 32B the optical beam scans
across the area of the 10 .mu.m beads and aligns them into a line
pattern. In FIG. 32C the 10 .mu.m and 20 .mu.m beads are aligned
and moving with different relative distances to the center of the
optical beam. In FIG. 32D the optical beam is programmed to "jump"
into the spacing between these two groups of particles and further
separate them.
After the line-shaped laser beam scans across the assortment of
beads, the 10 .mu.m and 20 .mu.m beads become aligned at different
distances relative to the center of the beam. The laser beam is
programmed to "jump" between these two groups of particles and
further separate them. This sorting process finished in 25
seconds.
FIGS. 33A-33B shows the sorting of particles of different sizes;
specifically 5 .mu.m, 10 .mu.m, and 20 .mu.m particles with
relative distances of 15 .mu.m, 20 .mu.m, and 40 .mu.m,
respectively, under the scan speed of 6 .mu.m/sec. The optical beam
scans at a constant rate from the left to the right. These three
sizes of particles are moving at the same speed as the light beam.
Their deterministic relative distances remain constant during the
movement. The relative distance is scan speed dependent. At a high
scan speed, the particle experiences a larger viscous force. In
order to balance this force, the particle moves closer to the
scanning beam, where a stronger electric field gradient exists.
FIG. 34 depicts the relationship between the scan speed and the
relative distances of microparticles from the scanning beam center
as a function of scanning speed. Theoretical calculations are shown
in solid lines and experimental data is shown by the dots.
For the 20 .mu.m particle, the relative distance to the scanning
beam center decreases from 50 .mu.m to 25 .mu.m when the scan speed
increase from 20 .mu.m/s to 100 .mu.m/s. This trend is also
reflected in the data for 5 .mu.m and 10 .mu.m particles. Thus, low
scanning rates provide a larger spatial separation between
different sizes of particles. At a scanning speed of 17 .mu.m/sec,
the spacing between 5 .mu.m and 10 .mu.m particles is 7 .mu.m, and
the spacing between 10 .mu.m and 20 .mu.m particles is 28 .mu.m.
The maximum scanning rate for a 5 .mu.m particle is approximately
70 .mu.m/sec. If the scanning rate is increased beyond this limit,
the particle becomes levitated by the vertical non-uniformity of
the electric field, causing the particle to escape the lateral
"pushing force" of the scanning beam. The escape speed is higher
for bigger particles; for a 10 .mu.m bead, it is 90 .mu.m/sec.
The present invention provides a novel optical sorting mechanism
based on optoelectronic tweezers (OET). The light induced dynamic
electric fields sort out particles with diameters of 5 .mu.m, 10
.mu.m, and 20 .mu.m by simply scanning a light beam across the OET
surface. This technique completely eliminates the requirement of
extra pumps as a driving force for liquid flow, greatly simply the
fabrication and integration process of microfluidic system. The
deterministic relative distances from the beads to the beam center
are size-dependent. Particle sorting performed on 5 .mu.m and 10
.mu.m-diameter beads resulted in a spacing of 7 .mu.m between the
separated groups. The spacing between sorted 10 .mu.m and 20 .mu.m
diameter particles was 28 .mu.m.
9. Moving Toward an all Optical Lab-on-a-Chip System.
Miniaturization and integration of microfluidic systems could
reduce the cost as well as increase the speed of many analytical
biological and chemical processes. Multiple microfluidic functions
are integrated on a chip, referred to as "lab-on-a-chip", to
perform the biological analysis. These functions include microfluid
delivery mixing, cell trapping, concentrating, and sorting.
Conventional lab-on-a-chip systems consist of micro pumps, valves,
and fluidic channels.
The fluidic circuits, and therefore their functions, are usually
fixed by the specific structure which has been fabricated. By
contrast to the conventional "fixed" microfluidic system, the
optical manipulation approach taught herein offers several
advantages. The present invention is flexible and easily
re-configurable. Optical tweezers have been widely used to trap
cells and other bio-particles, and recently, holographic optical
tweezers have been proposed to perform multiparticle trapping,
optical sorting, particle spinning, three-dimensional manipulation,
and optical pumping of microfluid. These microfluidic functions
permit an all-optical-lab-on-a-microscope system that is
programmable and reconfigurable in response to light input.
However, the optical tweezers-based systems suffer from the
following limitations. First, control of microfluids using optical
force is not energy efficient. The optical energy is first
transferred to the kinetic energy of colloidal particles or beads.
The moving beads induce liquid flow through the viscous force. The
maximum force from optical trap is around 100 pN, which is not
large enough to drive liquids through microchannels effectively due
to a large pressure drop. Second, the optical power required by
optical tweezers is very high. It requires tightly focused laser
beams to provide optical gradient force for trapping or deflecting
the particle motion.
Typically, a single trap requires 1 mW of optical power, and
multiple traps require even higher power. Here, instead of using
optical force the present invention relies upon a light-induced
electrowetting mechanism, called optoelectrowetting (OEW), for
controlling microfluids and a light-induced dielectrophoresis
mechanism, called optoelectronic tweezers (OET), for manipulating
microscopic particles.
FIG. 35 illustrates the concept by a comparison of energy transfer
paths of different optical manipulation methods, wherein optical
energy is first converted to electrical energy, which in turn
drives the liquids or particles through electrowetting or DEP
processes, respectively. As a consequence of the optoelectronic
gain of the photoconductor, the required optical power is reduced
by four to five orders of magnitude. Optical tweezers transfer
energy from optical domain directly to mechanical domain, while
optoelectrowetting (OEW) and optoelectronic tweezers (OET) transfer
optical energy to electrical domain first and then trigger the
electrical force for the manipulation.
Surface tension is the dominant force for controlling liquids in
microscale. Several mechanisms have been proposed to control the
surface tension. Electrowetting is attractive because of its fast
response and low power consumption. It changes the contact angle of
a droplet on a solid surface by modulating the surface energy at
the liquid-solid interface with electrostatic energy.
Optoelectrowetting uses optical beams to control the amount of
electrostatic energy stored in that interface and thus the contact
angle.
FIGS. 36A-36B illustrate an embodiment 250 of optoelectrowetting in
which a water droplet 252 is placed on a glass substrate coated
with a transparent conductive glass 20, a photoconductive layer 24,
and a thin dielectric layer 16. FIG. 36A depicts the droplet
without light illumination, with the contact angle 254 being the
same as the initial angle without bias. In FIG. 36B illumination is
generated by source 212 and the contact angle of droplet 252'
decreases due to the electrowetting effect.
An AC electrical bias is applied between the bottom electrode and
the droplet. The photoconductor is configured with a high
electrical resistance in the dark, resulting in a RC charging time
much longer than the AC signal cycle. A very small amount of the
voltage drops across the capacitor between the droplet and the
photoconductor. The contact angle in the dark is the same as the
initial angle without bias, as shown in FIG. 36A. When light shines
on the photoconductor layer, it creates electron-hole pairs and
increases the photoconductivity by several orders of magnitude. The
RC time becomes much smaller than the cycle of the AC signal,
resulting in a fully charged capacitor, as shown in FIG. 36B. These
extra electrical charges stored in the capacitor change the surface
energy between the solid-liquid interface and thus the droplet
contact angel. The relation between the contact angel and the
voltage across the insulator can be expressed by the following.
.function..theta..function..function..theta..times..times..times..gamma..-
times. ##EQU00004##
In the equation above the value .theta..sub.0 is the initial
contact angle, .di-elect cons. is the permittivity of the
insulator, d is the thickness of the insulator, .gamma..sub.LV is
the surface tension of the liquid-vapor interface, V is the
root-mean-square voltage of the applied signal.
FIGS. 37A-37C illustrate induced movement by electrowetting. In
FIG. 37A, a schematic of an example embodiment is illustrated which
induces movement of a liquid droplet by an optical beam on the COEW
surface. In FIG. 37B an equivalent circuit of the COEW device of
FIG. 37A is shown. In FIG. 37C the layered structure of the COEW
surface is shown.
OEW allows optical tuning of the voltage across the insulator and
thus the contact angle. Our previous results have demonstrated that
using halogen lamp with an intensity of 65 mW/cm.sup.2 is
sufficient to reduce the contact angle of the droplet from
105.degree. to 75.degree., turning hydrophobic surface to
hydrophilic. Using OEW, we have demonstrated a droplet-based
microfluidic device that allows an optical beam to extract a
droplet from a liquid reservoir and transport it freely on a
two-dimensional surface. Separation of the droplet is achieved with
two optical beams moving in opposite directions. The droplet (100
nL volume) follows the scanning optical beam up to a speed of 70
mm/sec, demonstrating the effectiveness of OEW for optical
manipulation of microfluid. The minimum droplet size is limited by
the area of the OEW electrodes (>1 nL for an electrode area of
100 .mu.m.times.100 .mu.m).
For manipulating sub-nano-liter droplet the present invention
includes a continuous OEW (COEW) device that allows optical beams
to create virtual electrodes that can be continuously addressed on
a two-dimensional surface.
FIG. 37A shows the structure of the COEW device. It consists of two
surfaces, a top ITO glass and a bottom photosensitive COEW surface.
The top ITO glass is coated with a 0.2 .mu.m thick silicon dioxide
layer and a 20 nm thick Teflon layer, making the surface
hydrophobic; and the bottom COEW surface consists of multiple
featureless layers, including a 200 nm thick ITO, 10 nm thick
aluminum, 5 .mu.m thick undoped amorphous silicon, 200 nm thick
silicon dioxide, and a 20 nm thick Teflon layers, as shown in FIG.
37C. The liquid droplet is sandwiched between these two surfaces
with a 10 .mu.m gap defined by a photoresist spacer. Due to the
hydrophobic Teflon coating, the initial contact angle of the
liquid-solid interface is larger than 90.degree.. When a light beam
illuminates at one edge of the droplet, it creates a virtual
electrode at the photoconductive layer right underneath this edge.
The optoelectrowetting effect is turned on locally, reducing the
droplet contact angle at the illumination site.
This process can be understood from the equivalent circuit model
shown in FIG. 37B. The amorphous silicon layer has smaller
capacitance (higher AC impedance) than the silicon oxide layers in
the dark because it is ten times thicker. Very small amount of
voltage drops across the silicon oxide layer. Under light
illumination, the conductivity of amorphous silicon increases by
several orders of magnitude, thereby reducing the electrical
impedance to a much smaller value than that of the oxide layers.
The contact angle is reduced locally, creating an unbalanced
pressure on the droplet. The net capillary force pushes the droplet
to move toward the laser beam. By scanning the light beam, the
droplet is continuously addressed on the COEW surface. There are
two factors that may limit the resolution of the light-patterned
virtual electrodes: optical diffraction limit and ambipolar
electron-hole diffusion length. In the case of amorphous silicon,
the ambipolar diffusion length is less than 115 nm, resulting in a
electrode resolution only limited by optical diffraction.
The movement of a 100 .mu.L droplet moving on the COEW device is
captured by a video camera through a microscope. The movement is
directed by a 100 .mu.W HeNe laser with a wavelength of 632 nm. The
focused spot size is 20 .mu.m using a 10.times. objective. The
laser beam is steered by a pair of orthogonal galvanometer scanning
mirrors (Cambridge Inc.). A 100V AC bias with frequency of 10 kHz
is applied through the top ITO glass and the bottom COEW
surface.
FIGS. 38A-38D show a sequence of video snapshots showing the
droplet moving in a circular pattern with approximately a 100 .mu.m
radius. The speed of the droplet is 785 .mu.m/sec. These images
show microdroplet transport by COEW. The droplet has a volume of
100 .mu.L and moves in a circular pattern directed by a scanning
laser beam. The speed of the droplet is 785 .mu.m/sec.
The virtual electrode created by light illumination can also be
used to move microscopic particles in liquid through
dielectrophoretic (DEP) force. The DEP force is generated by the
interaction of the applied electric field and the induced electric
dipoles in neutral particles. An aspect of the invention provides
an optoelectronic tweezers (OET) device that exploits such
light-induced DEP to move microscopic particles with very low power
optical beams. The DEP force has been widely used to manipulate
microscale or nanoscale particles. The analytic expression of the
DEP force is given by the following equation.
.times..times..pi..times..times..times..epsilon..times..function..times..-
times..omega..times. .function. ##EQU00005##
.times..times..omega..times..times..times..sigma..omega..times..sigma..om-
ega. ##EQU00005.2##
In the above equation E is field strength, a is particle radius.
.di-elect cons..sub.m and .di-elect cons..sub.p are the
permittivities of the surrounding medium and the particle,
respectively, .sigma..sub.m and .sigma..sub.p are the conductivity
of the medium and the particle, respectively, with .omega. the
angular frequency of the applied electric field. The value
K*(.omega.) is the Clausius-Mossotti factor and is a frequency
dependent complex number. The real part of K*(.omega.), or
Re[K*(.omega.)], has a value between 1 and -0.5, depending on the
polarizabilities of the medium and the particle and on the
frequency of the applied AC electric bias. If Re[K*(.omega.)]>0,
the particle will move towards higher electric field region and
this is called positive DEP. On the other hand, if
Re[K*(.omega.)]<0, the particle will move away from the high
field region and this is called negative DEP.
FIG. 39 illustrates an example embodiment of an OET structure in
which the liquid containing the cells or particles are sandwiched
between an ITO glass and a photoconductive surface. To achieve
light-induced DEP, we use a device structure that is very similar
to the OEW device but without the Teflon and silicon dioxide layers
on either the ITO or the photosensitive surfaces, as shown in the
figure. The 1 .mu.m thick amorphous silicon is coated with 20 nm
silicon nitride layer to prevent electrolysis. As in the OEW
device, the amorphous silicon layer has high resistance in the
dark, resulting a small voltage drop across the liquid layer. Under
light illumination, the virtual electrodes create a non-uniform
electric field in the liquid layer, producing a DEP force on the
particles nearby. The particles can be attracted or repelled by the
optical beam, depending on the sign of the Clausius-Mossotti
factor. The photoconductive gain of the amorphous silicon allows
OET to operate with very low optical power.
Our previous results showed a 1 .mu.W He--Ne laser with wavelength
at 633 nm is sufficient to transport a 25 .mu.m particle at 4.5
.mu.m/sec. Though the structures are similar, the OET and the OEW
are different in the following aspect: the OET switches the voltage
between the photoconductive layer and the liquid layer, while the
OEW switches the voltage between the insulating layer and the
photoconductive layer.
In this section, we present the applications of OET for collecting
and transporting biological cells. To accommodate the size of E.
coli cells, the gap spacing in OET is reduced to 15 .mu.m. FIG. 40A
shows the simulated electric field distribution in the liquid layer
for a 17 .mu.m virtual electrode generated by a focused laser beam
and a bias voltage of 10 V. The conductivity of the liquid is 1
mS/m. The conductivity of the amorphous silicon follows the
intensity distribution of the laser beam, and is assumed to have a
Gaussian shape with a peak conductivity of 10 mS/m at the center.
The three-dimensional electrical field distribution is calculated
using FEM-LAB.
FIG. 40A and FIG. 40B illustrate electric field properties for a
photoconductor layer. FIG. 40A illustrates electric field
distribution in the liquid layer when the photoconductor is
illuminated by a focused laser beam with 17 .mu.m spot size. FIG.
40B The electric field strength at three different heights above
the photoconductive layer. The electric field distribution at 4
.mu.m, 8 .mu.m, and 12 .mu.m above the photoconductive surface are
plotted in FIG. 40B. Since the DEP force is proportional to the
gradient of E2, the electric field distribution shows that the OET
can generate strong DEP force within a radius of approximately 20
.mu.m in the lateral direction. The vertical gradient attracts the
particles towards the photoconductive surface. Both the lateral and
the vertical gradients are strongest near the edge of the laser
spot, similar to those generated by a physical electrode.
FIG. 41 illustrates a demonstration setup for trapping biological
cells. The OET device is coupled to an microscopic imaging means,
such as placed on an inverted microscope (i.e., Nikon.RTM. TE2000E)
with the photosensitive side up. A 0.8 mW He--Ne laser
(wavelength=632 nm) is used to power the optoelectronic tweezers.
The incident power is controlled by neutral density filters. The
optical beam is delivered to the device through a 40.times.
objective lens with a numerical aperture (N.A.) of 0.5, thereby
producing a 17 .mu.m focused spot size. The fluorescent image of
the cells is captured by a CCD camera through the bottom objective
lens.
FIG. 42A is an image of fluorescent E. coli cells before OET is
turn on. FIG. 42B is the same image as FIG. 42A after the OET is
turned on for 14 seconds. It should be appreciated that the E. coli
cells are "focused" by the OET to the laser spot.
When the laser beam is focused on a fixed spot, the OET attracts
cells within the trapping area towards the center of the beam, as
shown in FIG. 42A and FIG. 42B. It functions as a cell
concentrator. In this experiment, we use the E. coli cells that can
express green fluorescent protein (GFP) for the convenience of
observation under fluorescent microscope. The liquid has a
conductivity of 1 mS/m. We apply a 100 kHz, 10V.sub.PP (volts
peak-to-peak) AC electric bias between the top and the bottom ITO
electrodes. The E. coli cells experience positive DEP force under
these conditions. The effective capturing distance is around 20
.mu.m from the focal point. Due to the electric field gradient in
the vertical direction, the cells are trapped right on top of the
photosensitive surface. When the laser beam is turned off, these
trapped E. coli cells swim away. No "opticution" is observed even
for light in the visible wavelength range, thanks to the low
optical intensity.
We have investigated the minimum optical power required to operate
this OET. Cell concentrating is observed for optical power as low
as 8 .mu.W. This optical power density is almost five orders of
magnitude lower than that of conventional optical tweezers with 1
mW laser focused to diffraction-limited spot size. The concentrated
cells can be transported to any arbitrary location by scanning the
laser beam. FIG. 43 shows the transport of multiple E. coli cells
using a single scanning laser beam.
To study the effective trapping area and the velocity of the
trapped cells, we recorded the trapping action and analyzed the
video images frame by frame. The recording microscope is focused on
the surface of the photoconductor to capture the trapped cells. We
have measured the velocities of cells trapped by lasers with
optical powers of .mu.m 8 .mu.W, 120 .mu.W, 400 .mu.W, and 800
.mu.W. The OET traps work at all power levels.
FIG. 44 shows the measured velocities of the E. Coli cells towards
the center of the focused light spot cells versus the radial
distance from the center of the trap. At 800 .mu.W, cells as far as
30 .mu.m away are attracted by the OET. Initially, they move at a
relatively low speed of 5 .mu.m/sec. The speed increases sharply
when they are within 20 .mu.m, eventually reaching a speed of 120
.mu.m/sec at about 15 .mu.m from the focal point. The transport
speed becomes smaller after the peak value. The cells are stopped
at 9 .mu.m from the center by the trapped cells. This result
matches very well with the simulated electric field distribution in
FIGS. 40A-40B. The maximum slope of the top curve (4 .mu.m above
photoconductor) happens at about 15 .mu.m from the center. The cell
velocity is a function of the optical power. The peak velocity
increases from 26 .mu.m/sec at 8 .mu.W to about 90 .mu.m/sec at 120
.mu.W. Above 120 .mu.W, the peak velocity increases more slowly,
and eventually saturates at about 200 .mu.W. This can be explained
by the following: when the optical power is lower than 120 .mu.W,
the photoconductor is not fully turned on, for example the
conductivity is lower than, but not negligible, compared to the
liquid. At about 200 .mu.W, the conductivity of liquid becomes
dominant in the electrical circuit, and most of the electric field
drops across the liquid layer.
Further increases of optical power do not change the peak electric
field. However, the electric field distribution becomes more
"square" like because of this saturation effect. The capturing area
increases slightly after the peak field saturates. It should be
pointed out that the minimum optical power required for OET depends
on the liquid conductivity.
The liquid conductivity used in our experiments is 1 mS/m. The
current laser power can attract cells in liquid with conductivities
up to 100 mS/m. The optical power can be further reduced by
shrinking the optical spot size. The current power level can be
reduced by 100 times by decreasing the spot size from 17 .mu.m to
1.7 .mu.m.
In this present aspect of the invention we have presented
optoelectrowetting (OEW) and optoelectronic tweezers (OET), for
manipulating microdroplets and microparticles by light. The OEW
uses light-induced electrowetting to control the surface tension,
the dominating force in microscale, and actuate microdroplets. Our
result shows that a 100 pL water droplet is transported at a speed
of 785 .mu.m/sec with an optical power of 100 .mu.W. The OET
exploits light-induced dielectrophoretic force for manipulating
microparticles. The optical power required by OET is as low as 8
.mu.W and the optical power density is five orders of magnitude
lower than that of optical tweezers. We have used OET to
concentrate and transport live E. coli cells without photodamage.
The low power requirement of OET opens up the possibility of
trapping microscopic particles using incoherent light sources.
Embodiments have been described for practicing the apparatus and
method of the invention by way of example. It should be appreciated
that the specific demonstration/experimental setups were provided
only by way of reference and that the invention can be implemented
in a wide variety of ways using various equipment as will be
recognized by one of ordinary skill in the art. It should be
recognized that although the present invention provides an OET
which can be implemented with unpatterned surfaces, the teachings
herein can be combined with patterned techniques to provide a
hybrid approach without departing from the teachings of the present
invention. Additionally, specific values for particle transport,
times, and other measured characteristics were provided to aid
those in understanding the approximate results which can be gleaned
from this technology; one of ordinary skill in the art will
appreciate that in many cases the results can be significantly
improved with more detailed implementations beyond these
demonstrations. Furthermore, it should be appreciated that the
aspects of the present invention can be practiced on the areas as
described, or in other areas which will be recognized by those of
ordinary skill in the art based on the teachings herein.
Although the description above contains many details, these should
not be construed as limiting the scope of the invention but as
merely providing illustrations of some of the presently preferred
embodiments of this invention. Therefore, it will be appreciated
that the scope of the present invention fully encompasses other
embodiments which may become obvious to those skilled in the art,
and that the scope of the present invention is accordingly to be
limited by nothing other than the appended claims, in which
reference to an element in the singular is not intended to mean
"one and only one" unless explicitly so stated, but rather "one or
more." All structural, chemical, and functional equivalents to the
elements of the above-described preferred embodiment that are known
to those of ordinary skill in the art are expressly incorporated
herein by reference and are intended to be encompassed by the
present claims. Moreover, it is not necessary for a device or
method to address each and every problem sought to be solved by the
present invention, for it to be encompassed by the present claims.
Furthermore, no element, component, or method step in the present
disclosure is intended to be dedicated to the public regardless of
whether the element, component, or method step is explicitly
recited in the claims. No claim element herein is to be construed
under the provisions of 35 U.S.C. 112, sixth paragraph, unless the
element is expressly recited using the phrase "means for".
* * * * *