Note: Descriptions are shown in the official language in which they were submitted.
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CONFIGURABLE DYNAMIC THREE DIMENSIONAL ARRAY
BACKGROUND OF THE INVENTION
Throughout this application various publications are referenced within
parentheses.
The disclosures of these publications in their entireties are hereby
incorporated by reference
in this application in order to more fully describe the state of the art to
which this invention
pertains.
1. Field of the Inyention
The present invention relates generally to arrays of probes. In particular,
the
invention relates to a system and method using a plurality of optical traps to
form a
configurable dynamic array of probes which may or may not be substrate bound.
2. Discussion of the Related Arts
Arrays of potentially reactive probes have a long history of use in assays and
other
chemical and biological tests and experiments. For example, arrays are often
used in the
fields of genetics, biochemistry, and biology to assay a sample for biological
or chemical
material (known as a target). Often the sample being assayed is only available
in relatively
small quantities. This limited availability of some materials led to the
development of
microarrays useful to present a relatively high density of probes, in a small
array, to assay for
targets in a small quantity of a sample.
Microarrays used in the testing of biological material are often referred to
as bio-
chips. Two principal applications of bio-chips are: extraction of sequence
information about
a specific nucleic acid, i.e., whether that nucleic acid corresponds to an
organism's entire
genome, a single gene, or a portion of a single gene (LJ.S. Patent No.
6,025,136); and
evaluation of gene expression. (See Sche~a, M. et al. "Quantitative monitoring
of gene
expression patterns with a complimentary DNA microarray," Science 270
(5235):467-70
(Oct. 20, 1995); I~.J. ahd Winzeler, E.A., "Genomics, gene expressiona and DNA
arrays,"
Nature 405(6788):827-836 (2000) and Ekins, R. arcd Chu, F. W , "Microarrays:
their origins
and applications," Trends in Biotechnology 17:217-18 (1999).)
Conventional microaxrays are comprised of either a linear or a two-dimensional
configuration of oligonucleotide probes, attached to the planar surface of a
solid support
(substrate). Different types of oligonucleotides are affixed to the substrate
at predetermined
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locations. Consequently, once the microarray is formed, the location of the
probes and hence
the location of any targets that react with the probes is always known. The
attachment of the
probe is achieved by either direct synthesis of the oligonucleotide onto the
substrate through a
process known as in situ photolithography synthesis (U.S. Patent Nos.
5,837,832 and
5,143,854), or attachment of the oligonucleotide after it has been
synthesized.
One drawback of such microarrays is that their linear or two dimensional
configuration provides a limited surface area to which probes can be attached,
thereby setting
a limit on the density of the probes to assay for the targets. In the case of
DNA hybridization
between targets (DNA or DNA fragments) and probes (immobilized
oligonucleotides) the
rate of hybridization is controlled by the rate at which the targets are able
to pass into contact
with the probes. Accordingly, the higher the density of probes, the greater
the rate of
hybridization.
A second drawback of such microarrays stems from the method of their
configuration.
Once a microarray is fabricated, the type and quantity of the probes become
fixed.
In an alternative approach to assaying for targets in a small quantity of a
sample,
probes are affixed to the surface of small bead-like substrates. (WO 00/61198
pending for
Kamba~a & Mitsuhashi.) Each bead containing a different probe is marked with a
distinct
label, thus permitting the identification of each probe and bound target by
discerning which
bead has what label after completion of the assay (See WO 00171243).
The identity of the bead and probe is maintained by physically transferring
the bead
with probe attached into a guide, capillary tube, groove, or holes within a
sheet, then washing
the beads with targets. While the non-flat nature of the beads does provide
greater surface
area for the targets to interact than does a microarray probe, the beads must
still be held in
some pre-determined order throughout the assay to maintain a record of the
identity of what
bead is supporting which probe or the bead probes must be collected and each
bead probe
examined after the assay to determine its identity.
An additional drawback of both the microarray and the bead assays is the
required
physical attachment of the probe to a substrate. In some instances the
attachment will in and
of itself alter the probe, or affect the process that the probe is being used
to assay. In other
instances, during or after the initial assay, information may be obtained that
would make for
desirable alterations of the quality or quantity of the probes, if the
identity of the probes was
both known throughout the assay and the configuration of the array could be
easily altered.
However, such alterations are not possible with either the microarray or bead
assays.
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In an unrelated art, it is known to optically trap particles with multiple
simultaneously
generated optical tweezers. (See generally U.S. Patent No. 6,055,106 issued to
G~ier &
Dufi°eshe.) Optical tweezers use the gradient forces of a beam of light
to trap particles based
on the dielectric constant of a particle. To minimize its energy, a particle
having a dielectric
constant higher than the surrounding medium will move to the region of an
optical tweezer
where the electric field is the highest.
Other types of traps that can be used to optically trap particles include, but
are not
limited to, optical vortices, optical bottles, optical rotators and light
cages. An optical vortex
produces a gradient surrounding an area of zero electric field which is useful
to manipulate
particles with dielectric constants lower than the surrounding medium or which
are reflective,
or other types of particles which are repelled by an optical tweezer. To
minimize its energy
such a particle will move to the region where the electric field is the
lowest, namely the zero
electric field area at the focal point of an appropriately shaped laser beam.
The optical vortex
provides an area of zero electric field much like the hole in a doughnut
(toroid). The optical
gradient is radial with the highest electric field at the circumference of the
doughnut. The
optical vortek detains a small particle within the hole of the doughnut. The
detention is
accomplished by slipping the vortex over the small particle along the line of
zero electric
field.
The optical bottle differs from an optical vortex in that it has a zero
electric field only
at the focus and a non-zero electric field in all other directions surrounding
the focus, at an
end of the vortex. An optical bottle may be useful in trapping atoms and
nanoclusters which
may be too small or too absorptive to trap with an optical vortex or optical
tweezers. (J. Arlt
and MJ. Padgett. "Generation of a beam with a~dark focus surrounded by regions
of higher
intensity: The optical bottle beam," Opt. Lett. 25, 191-193, 2000.)
The optical rotator is a recently described optical tool which provides a
pattern of
spiral arms which trap objects. Changing the pattern causes the trapped
objects to rotate. (L.
Patersoh, M.P. MacDohald, J. Alt, I~h Sibbett, P.E. Byyant, aid K. Dholakia,
"Controlled
rotation of optically trapped microscopic particles," Science 292, 912-914,
2001.) This class
of tool may be useful for manipulating non-spherical particles and driving
MEMs devices or
nano-machinery.
The light cage, (heal in U.S. Patent No. 5,939,716) is loosely, a macroscopic
cousin
of the optical vortex. A light cage forms a time-averaged ring of optical
tweezers to surround
a particle too large or reflective to be trapped with dielectric constants
lower than the
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surrounding medium. However, unlike a vo~~tex, no-zero electric field area is
created. An
optical vortex, although similar in use to an optical tweezer, operates on an
opposite
principle.
There exists a need for an assay method and system in which the interaction of
the
probes and targets can be evaluated absent attachment of the probe to a
substrate. There also
exists a need for a method and system of forming an array of probes which is
configurable
(and re-configurable), the method maintaining the identity of the probes
throughout the assay
irrespective of the location of the probe. The present invention satisfies
these and other
needs, and provides further related advantages.
SUMMARY OF THE INVENTION
The present invention provides a novel and improved method and system to
construct,
configure and use a three dimensional array of probes.
Within a vessel optical traps are generated. The optical traps are produced by
directing a beam of light such as a laser beam, at an optical element which
alters the beam by
patterning its phase to generate beamlets. The beamlets in turn are focused
through a lens
and produce the gradient conditions necessary fox optical trapping. Probes,
each with a
known characteristic, are then added to the vessel. The probes for a given
assay are chosen
and then each is selected by containing it within an optical trap.
The quantity and quality of probes forming the array are readily re-
configurable by
using the optical traps to add, discard, or replace probes. The arrangement of
the probes, in
the array, relative to one another is also dynamic because the spatial
relationship of the
probes to one another can be altered while maintaining the identity of the
selected probes
from which the array was configured. Accordingly, both the array and each of
its probes are
also movable in three dimensions and can be positioned, moved and re-
positioned as a whole,
or separately within the vessel.
While a probe remains contained within an optical trap, regardless of whether
it has
been repositioned within the vessel and regardless of any change in it spatial
position "order"
in the array, the identity of the probe can be maintained by virtue of knowing
the identity of
the optical trap by which the probe is contained. Additionally, one optical
trap can pass the
probe to another optical trap and so on, while tracking the chain of optical
trap custody of the
probe thereby maintaining the identity of what probe is contained by which
optical trap.
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Other features and advantages of the present invention will be set forth, in
part, in the
descriptions which follow and the accompanying drawings, wherein the preferred
embodiments of the present invention are described and shown, and, in part,
will become
apparent to those skilled in the art upon examination of the following
detailed description
5 taken in conjunction with the accompanying drawings, or may be learned by
practice of the
present invention. The advantages of the present invention may be realized and
attained by
means of the instrumentalities and combinations particularly pointed out in
the appendant
claims.
DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates a partial cut-away side view of a system forming an array
of
configurable probes.
FIG. 2 illustrates a view of a free-probe contained within an optical trap.
FIG. 3 illustrates an overview of a system for forming an array of probes.
FIG. 4 illustrates a beam altering element with multiple static regions.
FIG. 5A illustrates a first operative movement of probes.
FIG. 5B illustrates a second operative movement of probes.
FIG. 6A illustrates a component view of a compact system to form optical
traps.
FIG. 6B illustrates an inverted microscope to which the compact system of Fig.
6A
attaches.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Particular embodiments of the invention are described below in considerable
detail for
the purpose of illustrating its principles and operation. However, various
modifications may
be made, and the scope of the invention is not limited to the exemplary
embodiments
described below. For example, while specific reference is made to biological
systems and
assays for gene sequencing and DNA hybridization, it can be appreciated that
the method and
system is of equal utility in such areas as optical circuit manufacturing and
testing,
nanocomposite material construction and testing, fabrication of opto-
electronics, electronic
components testing, assembly and testing of holographic data storage matrices,
chemical
assays, genomic assays, proteomics assays, facilitation of combinatorial
chemistry,
promotion of colloidal self assembly, and probing non-biological materials.
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Certain terminology will be used in the following specification, for
convenience and
reference and not as a limitation. Brief definitions are provided below:
A. "Beamlet" refers to a sub-beam of light or other source of energy that is
generated
by directing a beam of light or other source of energy, such as that produced
by a laser or
collimated output from a light emitting diode, through a medium which
diffracts it into two or
more sub-beams. An example of a beamlet would be a higher order laser beam
diffracted off
of a grating.
B. "Phase profile " refers to the phase of light or other source of energy in
a cross-
section of a beam or a beamlet.
C. "Phase patterning" refers to a patterned phase shift imparted to a beam of
light, or
a beamlet which alters its phase profile, including, but not limited to,
diffracting, phase
modulation, mode forming, splitting, converging, diverging, shaping and
otherwise steering a
beam or a beamlet.
D. "Probe" refers to a biological or other chemical material that selectively
binds to,
or reacts with, a target. Probes include, but are not limited to,
oligonucleotides,
polynucleotides, chemical compounds, proteins, peptides, lipids,
polysaccharides, ligands,
cells, antibodies, antigens, cellular organelles, lipids, blastomeres,
aggregations of cells,
microorganisms, cDNA, RNA and the like.
E. "Target" refers to a biological or other chemical material whose presence
or
absence in a sample is detected by binding the target to or reacting the
target with a probe.
For example, the presence of a target formed of genetic material is detected
by a reaction,
such as a hybridization reaction, of the genetic material of the target with
genetic material of
a probe, which possesses the particular characteristic, i.e., the
complimentary structure,
necessary for hybridization. Target materials also include, but are not
limited to,
oligonucleotides, polynucleotides, chemical compounds, proteins, lipids,
polysaccharides,
ligands, cells, antibodies, antigens, cellular organelles, lipids,
blastomeres, aggregations of
cells, microorganisms, peptides, cDNA, RNA and the like.
As shown in FIG.1, the probes 500-504 may be bound to or reacted with, any
suitable
substrate, through any suitable binding process or protocol. An important
characteristic of a
suitable substrate is that it be a material, which can be contained by, and
manipulated with, an
optical trap. Representative dielectric substrates include beads, irregular
small particles, or
other regular small particles. Suitable substrates are constructed of
materials, which include,
but are not limited to, control pore glass, ceramics, silica, titanium
dioxide, latex, plastics,
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such as polystyrene, methylstyrene, polymethyl methacrylate, paramagnetic
materials,
thoriosol, graphite, Teflon, cross-linked dextrans, such as sepharose,
cellulose, nylon, cross-
linked micelles, liposomes, and vesicles.
As shown in the alternative embodiment illustrated in FIG. 2, the method of
the
instant invention also includes using one or using more optical traps 1005
(one shown) to
contain one or more probes 505 (one shown) that are unbound to a substrate. It
should be
understood that the configurable arrays may contain only bound probes, only
unbound
probes, or a combination of bound and unbound probes. Selection of what
mixture, if any, of
bound and unbound probes may in part be influenced by a probe's physical
properties.
Specifically, the properties of certain probes, such as skin cells, may be
altered absent
adhesion to a substrate. Conversely, the action of other probes, such as
proteins, may be
better served by maintaining the tertiary structure of the probe/protein by
eliminating the
substrate.
FIG. 1 illustrates a configurable array 8 of substrate-bound probes 500-504
for
assaying a biological material. The probes are configured within a subject
cell 10 using
movable optical traps 1000-1004 constructed from focused beamlets 2000-2004.
The subject
cell 10 is a vessel constructed of a substantially transparent material, which
allows the
beamlets to pass through and which does not interfere with the formation of
the optical traps.
Illustrated in FIG. 3 is an overview of a system to generate and alter the
position of
the configurable array of probes, generally designated as 20. Movable optical
traps 1000-
1004 (FIG. 1) are generated within the vessel 10 by passing a collimated
light, preferably a
laser beam 100, produced by a laser 102 to area A' on a beam splitter 30. One
of the light
beams, beam 31, originates from the laser 102 and is redirected so that it
proceeds from the
area A' on the beam sputter 30 to area A or~ the phase patterning optical
element 22. Each
beamlet created by the phase patterning optical element 22 then passes through
area B at the
back aperture 28 of the focusing lens 12. Beamlets are converged by the
focusing lens 12
The resulting focused beamlets form the optical traps 1000-1004 by producing
the gradient
conditions necessary to contain and manipulate the probes in three dimensions.
For clarity,
only five sets of probes, beamlets, and optical traps are shown in FIG. 1, but
it should be
understood that a greater or lesser number can be provided depending on the
nature, scope,
and other parameters of the assay and the capabilities of the system
generating the optical
traps.
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Any suitable laser can be used as the source of the laser beam 100. Useful
lasers
include solid state lasers, diode pumped lasers, gas lasers, dye lasers,
alexanderite lasers, free
electron lasers, VCSEL lasers, diode lasers, Ti- Sapphire lasers, doped YAG
lasers, doped
YLF lasers, diode pumped YAG lasers, and flash lamp-pumped YAG lasers. Diode-
pumped
Nd:YAG lasers operating between 10 mW and 5 W are preferred. The preferred
wavelengths
of the laser beam 100 used to form arrays for investigating biological
material include the
infrared, near infrared, visible red, green, and visible blue wavelengths,
with wavelengths
from about 400 nm to about 1060 nm being most preferred.
The beam splitter 30 is constructed of a dichroic mirror, photonic band gap
mirror,
omni directional mirror, or other similar device. The beam splitter 30
selectively reflects the
wavelength of light used to form the optical traps and transmits other
wavelengths. The
portion of light reflected from area A' of the beam sputter is then passed
through an area A of
an encoded phase patterning optical element 22 disposed substantially in a
plane 24 conjugate
to a planar back aperture 28 of a focusing lens 12.
When the laser beam 100 is directed through the phase patterning optical
element 22,
the phase patterning optical element produces a plurality of beamlets having
an altered phase
profile. Depending on the number and type of optical traps desired, the
alteration may
include difFraction, wavefront shaping, phase shifting, steering, diverging
and converging.
Based upon the phase profile chosen the phase patterning optical element can
be used to
generate optical traps in the form of optical tweezers, optical vortices,
optical bottles, optical
rotators, light cages, and combinations of two or more of these forms.
In those embodiments in which the phase profile of the beamlets is less
intense at the
periphery and more intense at regions inward from the periphery, overfilling
the back
aperture 28 by less than about 15 percent is useful to form optical traps with
greater intensity
at the periphery of the optical traps than optical traps formed without
overfilling the back
aperture 28.
Suitable phase patterning optical elements are characterized as transmissive
or
reflective depending on how they direct the focused beam of light or other
source of energy.
Transmissive diffractive optical elements transmit the beam of light or other
source of
energy, while reflective diffractive optical elements reflect the beam.
The phase patterning optical element can also be categorized as having a
static or a
dynamic surface. Examples of suitable static phase patterning optical elements
include those
with one or more fixed surface regions, such as gratings, including
diffraction gratings,
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reflective gratings, and transmissive gratings, holograms, including
polychromatic
holograms, stencils, light shaping holographic filters, polychromatic
holograms, lenses,
mirrors, prisms, waveplates and the like. The static, transmissive phase
patterning optical
element 40, as shown in FIG. 4, is characterized by a fixed surface 41.
However, in some
embodiments, the phase patterning optical element itself is movable, thereby
allowing for the
selection of one more of the fixed surface regions 42-46 by moving the phase
patterning
optical element relative to the laser beam to select the appropriate region.
The static phase
patterning optical element may be attached to a spindle 47 and rotated with a
controlled
electric motor (not shown). The static phase patterning optical element in the
embodiment
shown in FIG. 4 has a fixed surface 41 and discreet regions 42-46. In other
embodiments of
static phase patterning optical elements, either transmissive or reflective,
the fixed surface 41
has a non-homogeneous surface containing substantially continuously varying
regions, or a
combination of discreet regions, and substantially continuously varying
regions.
Examples of suitable dynamic phase patterning optical elements having a time
dependent aspect to their function include computer generated diffractive
patterns, phase
shifting materials, liquid crystal phase shifting arrays, micro-mirror arrays,
including piston
mode micro-mirror arrays, spatial light modulators, electro-optic deflectors,
accousto-optic
modulators, deformable mirrors, reflective MEMS arrays and the like. With a
dynamic phase
patterning optical element, the medium which comprises the phase patterning
optical element
encodes a hologram which can be altered, to impart a patterned phase shift to
the focused
beam of light which results in a corresponding change in the phase profile of
the focused
beam of light, such as diffraction, or convergence. Additionally, the medium
can be altered
to produce a change in the location of the optical traps. It is an advantage
of dynamic phase
patterning optical elements, that the medium can be altered to independently
move each
optical trap.
Preferred dynamic optical elements include phase-only spatial light modulators
such
as the "PAL-SLM series X7665", manufactured by Hamamatsu, of Japan or the "SLM
512N15' and SLM 512SA7," both manufactured by Boulder Nonlinear Systems of
Layafette
Colorado. These phase patterning optical elements are computer controlled to
generate the
beamlets 2000-2004 (FIG. 1) by a hologram encoded in the medium which can be
varied to
generate the beamlets and select the form of the beamlets.
In some embodiments, the form of the optical traps and/or the locations of
optical
traps used to form the array are altered and hence configured and re-
configured. The form
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can be changed from its original form to that of an optical tweezer, an
optical, a vortex, an
optical bottle, an optical rotator or a light cage The optical trap can be
moved in two or three
dimensions.
The phase patterning optical element is also useful to impart a particular
topological
5 mode to the laser light, for example, by converting a Gaussian into a Gauss-
Laguerre mode.
Accordingly, one beamlet may be formed into a Gauss-Laguerre mode while
another beamlet
may be formed in a Gaussian mode.
The probes are configured within a vessel 10. The vessel 10 is a subject cell
constructed of a substantially transparent material, which allows the beamlets
to pass through
10 and which does not interfere with the formation of the optical traps. In
those embodiments,
where the substrate is labeled with a wavelength specific dye, the subject
cell should be
transparent to the specific wavelength. Furthermore, the subject cell should
be constructed of
a material that is inert to the substrate. For example, biological substrates
such as cells,
proteins, and DNA should not stick to the surface of the subject cell and must
not be changed
or destroyed by the material.
Probes which possess the particular characteristics necessary for binding
andlor
reacting with the target of interest axe selected for addition to the vessel
and inclusion in the
configurable array. In some of the embodiments, where the probe is bound to a
substrate, the
substrate is labeled with a marker (such as a wavelength specific dye) to
facilitate selection of
the probe. In preferred embodiments, all the substrate bound probes that have
the same
binding or reactivity characteristic are labeled with the same type of
markers. When the
substrate is labeled with a wavelength specific marker, the selection of
probes 500-504 can be
accomplished by adding the probes bound to the labeled substrate to the vessel
10. Then, as
illustrated in FIG. 3, spectral measurement of the probe's labeled substrate
can be used to
select (or not to select) a probe for inclusion in the array. In some
embodiments (FIG. 2) the
probe may be unbound to a substrate and may also be labeled.
In embodiments where unlabeled probes are chosen to form all or part of the
array,
the probes can be added to the vessel 10 in a sequential order. In such a case
the identity of a
probe is known by its load order or a probe's identify can be known on the
basis of the time
that the probe is added. Alternatively, the probes having binding or
reactivity characteristics
that differ from one another, can be segregated to different predetermined
locations, based on
the difference in properties. The probes are then selected on the basis of
their location within
the vessel.
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As seen in FIG. 3, spectroscopy of a sample of biological material can be
accomplished with an imaging illumination source 39 suitable for either
spectroscopy or
polarized light back scattering, the former being useful for assessing
chemical identity, and
the later being suited for measuring dimensions of internal structures such as
the nucleus size.
Using such spectroscopic methods, in some embodiments, cells are interrogated
and the array
of probes created from selected interrogated cells. For instance, a computer
3~ can be used to
analyze the spectral data and to identify suspected cancerous, pre-cancerous
and/or non-
cancerous cell types. The computer then can apply the information to direct
optical traps to
contain selected cell types. The contained cells then may be used as the
probes in assays,
based on the reaction or binding of the contained cells with targets such as
other cells,
antibodies, antigens, and other biological material, or drugs and other
chemicals. Those
skilled in the art will recognize that the methodology used to interrogate and
concentrate cells
based on parameters specific to cancerous cells, may be altered, without
departing from the
scope of the invention, for use with interrogating and/or separating
blastomeres, cells, or
other material.
In other embodiments, labeled or unlabeled probes, such as unlabeled probes
having
differing binding or reactivity characteristics may be placed in a series of
sub-cells 16
disposed within the vessel 10. In FIG. 1, for clarity, only one sub-cell is
shown. However, it
should be understood that a plurality of such sub-cells can be provided. In
some
embodiments, the boundaries of a sub-cell are constructed with optical traps.
A number of
optical traps placed in the right orientation create an optical sub-cell which
can perform the
same functions as the physical sub-cell 16.
Placement of the probe in a sub-cell 16 is by any suitable means including
movement
by optical traps, through flow channels, through micro-capillaries or by other
equivalent
mechanism. In each sub-cell, one or more probes having the same binding or
reactivity
characteristics are placed. Selection of the probes for inclusion in the array
is then made on
the basis of the sub-cell in which the probe is contained.
The optical traps 1000-1004 are then used to trap the selected probes 500-504
by
containing the probes within the optical traps 1000-1004. A group °of
such contained probes
are thereby configured to form an array.
The inventive method and system lends itself to a semi-automated or automated
process for tracking the movement and contents of each optical trap. The
movement can be
monitored, via video camera, spectrum, or an optical data stream and which
provides a
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computer controlling the selection of probes and generation of optical traps
information
useful to adjusting the type of probes contained by the optical traps and the
composition of
the probes forming the array. In other embodiments, the movement is tracked
based on
predetermined movement of each optical trap caused by encoding the phase
patterning optical
element. Additionally, in some embodiments, a computer is used to maintain a
record of
each probe contained in each optical trap.
Returning to the beam splitter 30, the beam splitter 30 also provides a light
beam 32
originating from the imaging illumination source 39 which passes through the
subject cell 10
forming an optical data stream corresponding to the location of one or more of
the beamlets,
derived from the location and position of a probe contained by an optical
trap.
The optical data stream can then be viewed, converted to a video signal,
monitored, or
analyzed by visual inspection 34a of an operator 36, spectroscopically 34b,
and/or video
monitoring 34c. The optical data stream 32 may also be processed by a
photodectector to
monitor intensity, or any suitable device to convert the optical data stream
to a digital data
stream adapted for use by a computer 38.
To construct the array, the operator 36 andlor the computer 38 will adjust the
hologram encoded by the phase patteniing optical element 22 to direct the
movement of each
optical trap to acquire the selected probe and trap it. The plurality of
optical traps with
contained probes form the composition of the configured array that may be
reconfigured as to
the composition or position of the probes depending on the needs of the user.
Using the
optical data stream, the position of one or more of the trapped probes can be
identified and
their positions monitored. Based on such information, the surface of the phase
patterning
optical element can be altered, in some embodiments independently, to change
the form of
one or more of the optical traps containing the probes.
Additionally, the position of one or more of the trapped probes in the array
can be
tracked by monitoring the position of the optical trap which contains it. Then
using such
information, any given probe in the array may be independently re-positioned
within the
subject cell by altering the surface of the phase patterning optical element
and the identity of
each probe remains known by the optical trap in which it is contained
irrespective of where
the optical trap positions the probe.
In a preferred embodiment, the computer 38 controls the movement of the
optical
traps both before and after the probes are trapped. In other embodiments, the
optical data
stream is first converted to a video signal which is then used to produce an
image
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13
corresponding to the array and the operator views the image to control the
movement of at
least one of the optical traps based on the image.
Referring to both FIGS. l and 3, to perform an assay, a first batch of targets
T1-TS is
added to the subject cell 10, which also contains a fluid medium 3000, via an
inlet port 14.
The array of probes 500-504 is suspended in the medium 3000 via their
containment by the
optical traps 1000-1004. To increase the opportunity for interaction with the
targets T1-T5,
the probes may be moved about the subject cell corresponding to movement of
the optical
traps.
For example, in one embodiment, the probes 500-504 are trolled through the
medium
3000 containing the taxgets Tl-T5. By containing the probes optically, as
opposed to
physically, and moving the probes within the subject cell 10, the opportunity
for interaction
of a probe with each target is increased, thus improving the speed and
efficiency of the assay.
The movement of an array of probes 500-502 via the sequential creation of sets
of
optical traps is illustrated in FIGS. 5A and SB. In the embodiment illustrated
in FIG. 5A,
there is shown a simple linear movement of the array of probes, configured
along a line P 1
representing a first set of predetermined positions. Movement is accomplished
by
transferring the probes from a first set of optical traps to a second, third,
and then fourth set.
Referring additionally to FIG. 4, the first set of optical traps is generated
by directing a laser
beam at a first region 42 of the phase patterning optical element 40. When the
beamlets
emanating from the first region 42 pass through a focusing lens, they form the
first set of
optical traps at a first position P1 containing the probes 500-503.
To move the probes 500-502 from the first position Pl to a second position P2,
the
static phase patterning optical element 40 is rotated around a spindle 47 to
align the laser
beam with a second region 43 which generates the second set of optical traps
at a
corresponding second set of predetermined positions P2. By constructing the
second set of
optical traps in the appropriate proximity to the first position Pl, the
probes can be passed
from the first set of optical traps to the second set of optical traps. The
sequence may
continue passing the probes from the second set of predetermined positions P2
to a third set
of predetermined positions P3, from the third set of positions P3 to a fourth
set of
predetermined positions P4, and from the fourth set of predetermined positions
P4 to a fifth
set of predetermined positions PS by the rotation of the phase patterning
optical element to
align the appropriate region 42-46 corresponding to the desired position P1-
P5. The time
interval between the termination of one set of optical traps and the
generation of the next
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should be of a duration to ensure that the probes are transferred to the next
set of optical traps
before they drift away.
Such movement of the probes can be useful to troll the probes through the
medium
thereby enhancing the opportunity to have targets within the medium interact
with the probes.
This type of simple movement may also be useful in moving the probes from a
sub-cell 16
(FIG. 1) to another area of the subject cell 10, or segregating probes into a
sub-cell 16.
In the embodiment illustrated in FIG. 5B there is shown a staggered movement
of the
probes from a wide to narrow proximity. The staggered movement of the probes
occurs in a
similar fashion as described in reference to FIG. 5A. However, the first
region 42 now
produces staggered optical traps with two probes 500 and 502 configured along
a line P1,
while a third probe 501 is configured at P2, a position between the two
probes, but spaced
apart from the line P1. As the probes are passed from a first set of optical
traps to a second
set and moved to second and subsequent positions, the staggered arrangement of
the probes
allows the probes to be packed densely without placing a set of traps in too
close a proximity
to two probes at the same time wluch could cause the probes to be contained by
the wrong
optical trap
Once a target has interacted with a probe, spectral methods can be used to
investigate
the targets. The spectrum of those probes which had positive results (i.e.,
those probes which
reacting with or bonded with the targets) can be obtained by using imaging
illumination 39
such as that suitable for either inelastic spectroscopy or polarized light
back scattering. The
computer 38 can analyze the spectral data to identify the desired targets and
direct the phase
patterning optical element to segregate ,those desired targets. Those skilled
in the art will
recognize that the methodology used to segregate targets based on spectral
data may be
altered, without departing from the scope of the invention, to identify and/or
segregate targets
based on other information obtainable from the targets and/or the optical data
stream.
Upon completion of the assay, selection can be made, via computer 38 and/or
operator 36, of which probes to discard and which to collect. The
reconfigurable nature of
the array allows for selective movement of a given optical trap and contained
probe. In some
cases the medium 3000 and unbound targets will be removed or flushed from the
subject cell
10 through an outlet port 18 and the assay will be completed. In other cases,
at least some of
the probes still contained by optical traps, are reused with additional
targets to perform
further assays. This technique can be useful in the case of probes that tested
positive or
negative, depending on the parameters of the assay. In yet other cases,
because the array of
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probes is reconfigurable as to the quantity and characteristics of the probes
forming the array,
the optical traps can be used to discard unbound probes and acquire additional
probes for
further experimentation.
In some embodiments, it is not necessary to generate beamlets from each region
of the
5 static beam altering optical element 40, or move the beam altering optical
element 40 in a set
direction. Instead, changing the order of the regions will change the location
of the sets of
optical traps.
Shown in FIG. 6A is an elevational view of a compact system for forming the
optical
traps, generally designated 50. The phase patterning optical element 51 is a
dynamic optical
10 element, with a reflective, dynamic surface, which is also a phase only
spatial light modulator
such as the "PAL-SLM series X7665," manufactured by Hamamatsu of Japan, the
"SLM
512SA7" or the "SLM 512SA15" both manufactured by Boulder Nonlinear Systems of
Lafayette, Colorado. These dynamic optical elements have an encodable
reflective surface in
which a computer controls a hologram formed therein.
15 FIG. 6A shows a compact system for forming the optical traps, the optical
element 51
is aligned with, or attached to, a housing 52 through which a first light
channel 53a is
provided. One end 53b of the first light channel is in close proximity to the
optical element
51, the other end 53c of the first light channel intersects with and
communicates with a
second light channel 53d formed perpendicular thereto. The second light
channel is formed
within a base 54a of a microscope lens mounting turret or "nosepiece" 54b. The
nosepiece
54b is adapted to fit into a Nixon TE 200 series microscope (not shown). The
second light
chamlel communicates with a third light channel SSa which is also
perpendicular to the
second light channel. The third light channel SSa traverses from the top
surface of the
nosepiece 54b through the base of the nosepiece 54a and is parallel to an
objective lens
focusing lens 56. The focusing lens has a top and a bottom forming a back
aperture 57.
Interposed in the third light channel between the second light channel and the
back aperture
57 of the focusing lens is a dichroic mirror beam splitter 58. Other
components within the
compact system for forming the optical traps 50 include a first mirror Ml,
which reflects the
beamlets emanating from the phase patterning optical element through the first
light channel,
a first set of transfer optics TO1 disposed within the first light channel,
aligned to receive the
beamlets reflected by the first mirror M1, a second set of transfer optics T02
disposed within
the first light channel, aligned to receive the beamlets passing through the
first set of transfer
lenses TO1, and a second mirror M2, .positioned at the intersection of the
first light channel
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and the second light channel, aligned to reflect beamlets passing through the
second set of
transfer optics TO2 and through the third light channel SSa.
To generate the optical traps, a laser beam (not shown) is directed through an
optical
150 out a collimator end 151 and reflected off the dynamic surface 59 of the
optical element
51. The beam of light (not shown) exiting the collimator end 151 of the
optical fiber 150 is
diffracted by the dynamic surface 59 of the optical element 51 into a
plurality of beamlets
(not shown). The number type and direction of each beamlet may be controlled
and varied
by altering the hologram encoded in the dynamic surface medium 59. The
beamlets then
reflect off the first mirror M1 through the first set of transfer optics TO1
down the first light
channel 53a through the second set of transfer optics T02 to the second mirror
M2; and are
directed at the dichroic mirror 58 up to the back aperture 57 of the objective
lens 56, are
converged through the objective lens 56, thereby producing the optical
gradient conditions
necessary to form the optical traps. That portion of the light which is split
through the
dichroic mirror 58, for imaging, passes through the lower portion of the third
light channel
SSb forming an optical data stream (not shown).
In those embodiments in which the phase profile of the beamlets is less
intense at the
periphery and more intense at regions inward from the periphery, overfilling
the back
aperture 57 by less than about 15 percent is useful to form optical traps with
greater intensity
at the periphery of optical traps than optical traps formed without
overfilling the back
aperture 57.
Shown in FIG. 6B is an elevational view of a Nixon TE 200 series microscope
into
which the compact system for forming the optical traps 50 has been mounted,
generally
designated 60. The nosepiece 54 with the attached a housing 52 fits directly
into the
microscope via the mount (not shown) for the nosepiece 54a and 54b. The
housing and its
contents and attached optical element 51 are secured to the nosepiece 54a and
54b require
few or no alterations or modifications to the remainder of the microscope. For
imaging, an
illumination source 61 may be provided above the objective lens 56.
The first and second set of transfer optics TO1 and T02 are shown containing
two
lens elements each. The lenses can be either convex or concave. Different and
varying types
and quantity of lenses such as symmetrical air spaced singlets, symmetrical
air spaced
doublets and/or additional lenses or groups of lenses, can be chosen to
achieve the image
transfer from the first mirror M1 to the second mirror M2. In some embodiments
the first and
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second set of transfer optics are symmetrical air spaced doublets, spaced at a
distance to act
in combination as a telephoto lens.
Since certain changes may be made in the above systems apparatus and methods
without departing from the scope of the invention herein involved, it is
intended that all
matter contained in the above description, as shown in the accompanying
drawings and
specification shall be interpreted in an illustrative, and not a limiting
sense.
