.-,,
`
`A PROVISIONAL APPLICATION
`
`SUBMITTED IN THE US. PATENT AND TRADEMARK
`OFFICE
`
`ENTITLED: SYSTEM AND METHOD FOR CELL SORTING
`USING HOLOGRAPHIC OPTICAL TRAPPING
`
`Inventors:
`
`Kenneth Bradley
`
`Lewis Gruber
`
`Ward Lopes
`
`Robert Lancelot
`
`and
`
`David G. Grier
`
`

`

`SYSTEM AND METHOD OF CELL SORTING USING HOLOGRAPHIC
`
`OPTICAL TRAPPING
`
`The present invention relates to a system and method of cell sorting using
`holographic optical trapping.
`
`BACKGROUND OF THE INVENTION
`
`In United States industry, there is a large number of unmet sorting and
`separation needs involving material made up of particles or units smaller than 50
`microns. These needs range across industries from particle sizing and sample
`preparation in the specialty chemicals and materials fields, to protein selection and
`purification in the pharmaceutical and biotechnology industries. Other examples
`include cell sorting and selection, in the medical, diagnostic and agriculture sectors.
`
`The importance of these needs can be seen by exploring the annual
`expenditures in areas where specialized or partial solutions have been developed, as
`well as by estimating the market value of sorted/separated/purified output in areas
`where there is currently not even a partial solution. As an example of the former, the
`biotechnology and pharmaceutical industries annually spend in excess of a billion
`dollars on equipment and supplies for protein purification.
`
`As an example of the latter, in the agricultural sector, there is currently no way
`to efficiently select the gender of offspring in farm animals; however, it is estimated
`that in the cattle area alone, over a billion dollars in value would be added by
`enabling such sperm selection as a part of the current artificial insemination process
`widely used in the industry.
`
`Outside of the animal husbandry market, the purification process of islet cells
`from human pancreases is currently a large concern of medical scientists developing
`new
`treatment methods for Type I diabetes.
`Significant progress
`in
`islet
`transplantation methods has been made, but the purification problem is one of the
`remaining stumbling blocks. Traditional methods for purifying islet cells are
`inefficient and result in damage to the cells.
`
`Islet cell transplantation is important because, in the Type I form of diabetes,
`the existing islet cells in the patient's pancreas have become damaged and no longer
`produce the insulin which is required for human survival. The current treatment for
`In spite of the
`Type I diabetes involves injection of insulin 1 to 5 times per day.
`treatment, the disease often leads to complications including blindness, blood flow
`problems requiring amputation, renal failure, and death. Greater purity and reduced
`contaminants for islet cells used in transplantation is expected to reduce the
`occurrence of these complications.
`
`Of the approximately 1 million current sufferers of Type I diabetes in the
`United States, at least 50,000 sufferers per year would submit to islet cell
`
`

`

`Islet cell purification, at $1000 to $3000 per
`transplantation if it were available.
`person, could result in $50 to $150 million in annual income per year. Upon large(cid:173)
`scale acceptance of islet cell transplantation as an effective therapy, these numbers
`would be expected to jump substantially. The jump would be driven by the difficulty
`of using today's treatment method (frequent injections) and the severe consequences
`even when the current treatment is adequately administered.
`
`Thus, islet purification is but one important problem reqmnng the highly
`selective sorting ofhuman cells in a non-damaging, non-invasive way.
`
`Another problem that needs to be addressed is the purification of normal cells
`from cancer cells in the bone marrow of persons undergoing whole-body radiation
`treatment for cancer.
`
`Still another is the selection of stem cells for research into the causes of, and
`therapies for, diseases such as Parkinson's disease.
`
`Yet another concern is developing new ways to automatically interrogate large
`numbers of human cells and select ones having characteristics not amenable to
`fluorescent tagging, which would enormously widen the scope and power of medical
`diagnoses.
`
`One conventional technique in manipulating microscopic objects is optical
`trapping. An accepted description of the effect of optical trapping is that tightly
`focused light, such as light focused by a high numerical aperture microscope lens, has
`a steep intensity gradient. Optical traps use the gradient forces of a beam of light to
`trap a particle based on its dielectric constant. "Particle" refers to a biological or
`other chemical material
`including, but not
`limited
`to, oligonucleotides,
`polynucleotides, chemical compounds, proteins, lipids, polysaccharides, ligands,
`cells, antibodies, antigens, cellular organelles, lipids, blastomeres, aggregations of
`cells, microorganisms, peptides, eDNA, RNA and the like.
`
`To minimize its energy, a particle having a dielectric constant higher than the
`surrounding medium will move to the region of an optical trap where the electric field
`is the highest. Particles with at least a slight dielectric constant differential with their
`surroundings are sensitive to this gradient and are either attracted to or repelled from
`the point of highest light intensity, that is, to or from the light beam's focal point. In
`constructing an optical trap, optical gradient forces from a single beam of light are
`employed to manipulate the position of a dielectric particle immersed in a fluid
`medium with a refractive index smaller than that of the particle, but reflecting,
`absorbing and low dielectric constant particles may also be manipulated.
`
`2
`
`

`

`The optical gradient force in an optical trap competes with radiation pressure
`which tends to displace the trapped particle along the beam axis. An optical trap may
`be placed anywhere within the focal volume of an objective lens by appropriately
`selecting the input beam's propagation direction and degree of collimation. A
`collimated beam entering the back aperture of an objective lens comes to a focus in
`the center of the lens' focal plane while another beam entering at an angle comes to a
`focus off-center. A slightly diverging beam focuses downstream of the focal plane
`while a converging beam focuses upstream. Multiple beams entering the input pupil
`of the lens simultaneously each form an optical trap in the focal volume at a location
`determined by its angle of incidence. The holographic optical trapping technique
`uses a phase modifying diffractive optical element to impose the phase pattern for
`multiple beams onto the wavefront of a single input beam, thereby transforming the
`single beam into multiple traps.
`
`Phase modulation of an input beam is preferred for creating optical traps
`because trapping relies on the intensities of beams and not on their relative phases.
`Amplitude modulations may divert light away from traps and diminish their
`effectiveness.
`
`When a particle is optically trapped, optical gradient forces exerted by the trap
`exceed other radiation pressures arising from scattering and absorption. For a
`Gaussian TEM00 input laser beam, this generally means that the beam diameter
`should substantially coincide with the diameter of the entrance pupil. A preferred
`minimum numerical aperture to form a trap is about 0.9 to about 1.0.
`
`One difficulty in implementing optical trapping technology is that each trap to
`be generated generally requires its own focused beam of light. Many systems of
`interest require multiple optical traps, and several methods have been developed to
`achieve multiple trap configurations. One method uses a single light beam that is
`redirected between multiple trap locations to "time-share" the beam between various
`traps. However, as the number of traps increases, the intervals during which each
`trap is in its "off' state can become long for particles to diffuse away from the trap
`location before
`the
`trap
`is re-energized.
`All
`these concerns have
`limited
`implementations of this method to less than about 10 traps per system.
`
`relies on
`traditional method of creating multi-trap systems
`Another
`simultaneously passing multiple beams of light through a single high numerical
`aperture lens. This is done by either using multiple lasers or by using one or more
`beam splitters in the beam of a single laser. One problem with this technique is that,
`as the number of traps increases, the optical system becomes progressively more and
`more complex. Because of these problems, the known implementations of this
`method are limited to less than about 5 traps per system.
`
`3
`
`

`

`In a third approach for achieving a multi-trap system, a diffractive optical
`element (DOE) (e.g., a phase shifting hologram utilizing either a transmission or a
`reflection geometry) is used to alter a single laser beam's wavefront. This invention is
`disclosed in U.S. Patent No. 6,055,106 to Grier et al. The wavefront is altered so that
`the downstream laser beam essentially becomes a large number of individual laser
`beams with relative positions and directions of travel fixed by the exact nature of the
`diffractive optical element. In effect, the Fourier transform of the DOE produces a set
`of intensity peaks each of which act as an individual trap or "tweezer."
`
`Some implementations of the third approach have used a fixed transmission
`hologram to create between 16 and 400 individual trapping centers.
`
`A fixed hologram was used to demonstrate the principle of holographic
`optical trapping but using a liquid crystal grating as the hologram permitted
`'manufacture' of a separate hologram for each new distribution of traps. The
`intensity distribution of the liquid crystal grating may be easily controlled in real time
`by a computer, thus permitting a variety of dynamic manipulations.
`
`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 trap. 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
`vortex 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. (See J. Arlt and M.J. 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 light cage (Neal 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 traps
`to surround a particle too large or reflective to be trapped with dielectric constants
`lower than the surrounding medium. However, unlike a vortex, no-zero electric field
`
`4
`
`

`

`area is created. An optical vortex, although similar in use to an optical trap, operates
`on an opposite principle.
`
`When the laser beam is directed through or reflected from the phase patterning
`optical element, 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
`traps, optical vortices, optical bottles, optical rotators, light cages, and combinations
`of two or more of these forms.
`r
`
`Accordingly, a method of cell sorting using a technique which isolates
`valuable cells from other cells, tissues, and contaminants is needed. Further, a way of
`achieving a unique contribution of optical trapping to the major industrial needs of
`(cell) sorting and purification is required. Still further, there is a need to separate
`sperm cells in the animal husbandry market.
`
`SUMMARY OF THE INVENTION
`
`The present invention provides a way of implementing a parallel approach to
`cell sorting using holographic optical trapping.
`
`Optical trapping is used to address cell sorting and purification in three ways:
`1) as reviewed earlier, the forces exerted by optical traps on a material are sensitive
`to the exact distribution of the dielectric value in that material - the optical force
`therefore depends on the composition and shape of the object; 2) other forces on the
`object are sensitive to the hydrodynamic interaction between the object and the
`surrounding fluid - control of the fluid flow probes material shape, size and such
`features as surface rugosity; and, 3) localizing an object at a known position allows
`additional methods of automated interrogation such as high speed imaging and
`particle-specific scattering measurements.
`
`In an embodiment, the present invention performs cell sorting of X and Y
`sperm for animal husbandry.
`
`In the beef cattle industry, the ability to change the male/female ratio of the
`offspring from the current 50%:50% mix to an 85%: 15% mix would increase the
`value of the annual offspring by $700 million. A similar, though smaller, increase in
`value would occur in the dairy industry.
`
`5
`
`

`

`In one embodiment, the present invention includes using optical trapping,
`which is a technology which has been used as a tool for manipulating microscopic
`objects. An accepted description of the effect is that tightly focused light, such as
`light focused by a high numerical aperture microscope lens, has a steep intensity
`gradient. Optical traps use the gradient forces of a beam of light to trap a particles
`based on its dielectric constant To minimize its energy, a particle having a dielectric
`constant higher than the surrounding medium will move to the region of an optical
`trap where the electric field is the highest.
`
`In one embodiment ofthe present invention, in achieving a multi-trap system,
`a diffractive optical element (DOE, e.g. a phase shifting hologram utilizing either a
`transmission or a reflection geometry) is used to alter a single laser beam's wavefront.
`The wavefront is altered so that the downstream laser beam essentially becomes a
`large number of individual laser beams with relative positions and directions of travel
`fixed by the exact nature of the diffractive optical element.
`
`In one embodiment of the present invention, spectroscopy of a sample of
`biological material can be accomplished with an imaging illumination source 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. A computer can be used to analyze the
`spectral data and to identify cells bearing either an X or Y chromosome, or a
`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 identified based on the reaction or binding of the
`contained cells with chemicals
`
`In one embodiment of the present invention, the 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 computer controlling the
`selection of cells and generation of optical traps.
`
`In other embodiments, the movement of cells 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 cell contained in each optical trap.
`
`In one embodiment of the present invention, the optical data stream can then
`be viewed, converted to a video signal, monitored, or analyzed by visual inspection of
`an operator, spectroscopically, and/or video monitoring. The optical data stream 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.
`
`6
`
`

`

`In one embodiment of the present invention, once a cell has interacted with a
`trap, spectral methods can be used to investigate the cell. The spectrum of those cells
`which had positive results (i.e., those cells which reacted with or bonded with a label)
`can be obtained by using imaging illumination such as that suitable for either inelastic
`spectroscopy or polarized light back scattering. A computer can analyze the spectral
`data to identify the desired targets and direct the phase patterning optical element to
`segregate those desired targets. Upon completion of the assay, selection can be made,
`via computer and/or operator, of which cells to discard and which to collect.
`
`There has thus been outlined, rather broadly, some features consistent with the
`present invention in order that the detailed description thereof that follows may be
`better understood, and in order that the present contribution to the art may be better
`appreciated. There are, of course, additional features consistent with the present
`invention that will be described below and which will form the subject matter of the
`claims appended hereto.
`
`In this respect, before explaining at least one embodiment consistent with the
`present invention in detail, it is to be understood that the invention is not limited in its
`application to the details of construction and to the arrangements of the components
`set forth in the following description or illustrated in the drawings. Methods and
`apparatuses consistent with the present invention are capable of other embodiments
`and of being practiced and carried out in various ways. Also, it is to be understood
`that the phraseology and terminology employed herein, as well as the abstract
`included below, are for the purpose of description and should not be regarded as
`limiting.
`
`As such, those skilled in the art will appreciate that the conception upon which
`this disclosure is based may readily be utilized as a basis for the designing of other
`structures, methods and systems for carrying out the several purposes of the present
`invention.
`It is important, therefore, that the claims be regarded as including such
`equivalent constructions insofar as they do not depart from the spirit and scope of the
`methods and apparatuses consistent with the present invention.
`
`7
`
`

`

`DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
`
`illumina1ion
`
`input
`beam
`
`imagin9 __ m
`J] l
`w
`
`optical
`1rap(s)
`
`:objective
`
`8
`
`op·
`
`I
`
`lmm
`
`FIG. 1 Schematic representation of a BioRyx™-type system. The
`laser hght powenng the system is incident from the left. The system IS
`controlled by a dynamic hologram shown below on the left. The trapped
`particles under system control are shown below on the right. "Ll," "L2,"
`and "objective" indicate lenses. "Dichroic" indicates a type of mirror. A
`phase pattern on the lower left produces the traps shown in the lower right
`filled With I p.m dtameter silica spheres suspended in water.
`
`The present invention will now be described with reference to various Figures.
`FIG 1 is a schematic of a system of the present invention for forming holographic
`optical traps.. The phase patterning optical element DOE is preferably a dynamic
`optical 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 phase patterned optical
`elements are computer controlled to generate the beamlets by a hologram encoded in
`the medium which can be varied to generate the beamlets and select the form of the
`beamlets.
`
`8
`
`

`

`The beam splitter in FIG. 1 is constructed of a dichroic mirror, photonic band
`gap mirror, omni directional mirror, or other similar device. The beam splitter
`selectively reflects the wavelength of light used to form the optical traps and
`transmits other wavelengths. The portion of light reflected from the area of the beam
`splitter is then passed through an area of an encoded phase patterning optical element
`disposed substantially in a plane conjugate to a planar back aperture of a focusing
`lens.
`
`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, 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 is characterized by a fixed
`surface .
`
`. 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
`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 and
`rotated with a controlled electric motor. The static phase patterning optical element
`has a fixed surface and discrete regions.
`In other embodiments of static phase
`patterning optical elements, either transmissive or reflective, the fixed surface has a
`non-homogeneous surface containing substantially continuously varying regions, or a
`combination of discrete 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
`
`9
`
`

`

`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.
`
`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 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.
`
`In some embodiments, the form of an optical trap can be changed from its
`original form to that of a point optical trap, an optical 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 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(cid:173)
`Laguerre mode while another beamlet may be formed in a Gaussian mode.
`
`1.
`
`Imaging system
`
`The current instrument design uses a high resolution CCD camera for the
`primary imaging system. The main advantage of the CCD camera is the favorable
`cost/performance ratio since this technology is a mature one. Another advantage of
`CCD cameras is their wide dynamic range and the ease of generating digital output.
`
`The images are viewed on a computer screen to provide both a frame of
`reference for selecting the location of the traps as well as to minimize the possibility
`of inadvertent exposure ofthe operator to the laser.
`
`2.
`
`User Interface
`
`a. Object Display
`
`The user interface consists of a computer screen which displays the field of
`view acquired by the CCD camera. The user designates the loci of the traps with a
`mouse. There also an option to delete a location.
`
`As described in greater detail below, the user is also able to specify the power
`per trap so as to be able to avoid specimen damage. In addition it is desirable to be
`able to vary trap power because trapping depends upon the difference between the
`index of refraction of the specimen and the suspending medium which can be
`expected to vary from specimen to specimen.
`10
`
`

`

`b. The Hologram
`
`The purpose of designating the loci of the traps is to provide input for the
`hologram calculation. The hologram is essentially a function whose Fourier
`transform produces the desired trap array. However in the case of the liquid crystal
`display this function must be a phase object (i.e., an object that changes the phase of
`the wavefront without absorbing any energy
`
`c. Methods for choosing the set of traps
`
`When a large number of traps are needed, the time to designate their location
`with a computer mouse can take an inordinate amount of time. Therefore, there are
`several options to reduce the time required.
`
`Often one wishes to use the traps to move an object in a particular direction.
`This can be accomplished by using the mouse to create a line (by dragging). The
`software interprets a line as calling for a series of traps to be deployed sequentially
`and sufficiently close together so as to move the target in small steps without losing
`the lock.
`
`The present invention also includes the capability of changing the height of
`the traps. If a laser beam is parallel to the optical axis of the objective lens, then a
`trap forms at the same height as the focal plane of the lens. Changing the height of a
`trap is accomplished by adjusting the hologram so that the beam of light forming a
`trap is slightly converging (or diverging) as it enters the objective lens of the
`microscope. Adjusting the height of a trap is possible using lenses but only HOT
`allows the height of each individual trap to be adjusted independently of any other
`trap. This is accomplished in software by adjusting the phase modulation caused by
`the liquid crystal hologram
`
`3.
`
`Sample Holder
`
`a. General
`
`The sample chamber of the present invention is inexpensive and disposable.
`Although the sample chamber of the present invention is described below, another
`object of the present invention is to create a flexible design that can be changed for
`differing applications.
`
`The sample chamber lies on the surface of a microscope slide. The sample
`chamber contains a series of channels for introducing specimens. The channels are
`connected to supply and collection reservoirs by thin tubing (commercially available).
`
`11
`
`

`

`Samples will be suspended in a liquid medium and will be introduced into the
`working area via the channels.
`
`b. The Sample Chamber
`
`In one embodiment of the present invention, a poly(dimethyl siloxane)
`(PDMS) resin is used to fabricate the chamber which consists of a series of channels.
`The process involves creating the desired pattern of channels on a computer using
`standard CAD/CAM methods and transferring the pattern to a photomask using
`conventional photoresist/etching techniques. The photomask is then used as a
`negative mask to create an inverse pattern of channels which are etched on a silicon
`wafer. The depth of the channels is controlled by the etch time. The silicon wafer is
`a negative replica of the actual sample chamber. The final step consists of creating
`the positive sample chamber by pouring PDMS onto the wafer and polymerizing.
`This results in a PDMS mold which is bonded to a glass slide and overlaid with a
`cover slip. The glass to PDMA bonding is effected with an oxygen etch which
`activates the exposed surfaces.
`
`A number of additional steps are necessary to ensure consistent quality. For
`instance the PDMS solutionlhardner is maintained under a vacuum in order to prevent
`bubble formation. The silicon wafer is silanized to prevent the PDMS from sticking
`to the wafer. There are a variety of steps involving cleaning the replicas and
`maintaining proper environmental controls. These represent standard technology.
`
`The channels are connected to microbore tubing using small syringe needles
`which are inserted through the PDMS mold into small circular wells which connect to
`each channel. Sample solutions are introduced into the cell using micropumps.
`
`FIG 2 shows a diagram of a typical arrangement for the introduction of a
`sample. FIG 3 presents a scanning electron micrograph of the diagram in FIG. 2 as
`actually created from the process described above. The channels are approximately
`50 microns wide and 50 microns deep. FIG 4 presents a scanning electron
`micrograph of the 'working' volume where manipulations of the specimen under
`study would occur. The micrographs clearly show that the channels are smooth and
`clean. (The imperfections evident on the images were caused by manipulation of the
`specimen during preparation for scanning electron microscopy). Although the
`channels are rectangular in cross section, other shapes can be devised as well. The
`channels are designed to allow samples to be flowed to a 'working area' whose shape
`can be custom designed for experimental requirements.
`
`12
`
`

`

`; CO"V"eT "l-ip
`
`A
`
`B
`
`FIG 2. Diagram showing how sample is introduced into sample holder.
`
`FIG 3. A scanning electron micrograph of the sample chamber fabricated as described in the text.
`
`FIG 4 An enlarged view of the 'working' area ofthe sample chamber.
`
`13
`
`

`

`c. Advantages
`
`Unlike scanned optical traps which address multiple trapping points in
`sequence, and thus are time-shared, holographic optical traps illuminate each of their
`traps continuously. For a scanned optical trap to achieve the same trapping force as a
`continuously illuminated trap, it must provide at least the same time-averaged
`intensity. This means that the scanned trap has to have a higher peak intensity by a
`factor proportional to at least the number of trapping regions. This higher peak
`intensity increases the opportunities for optically-induced damage in the trapped
`material. This damage can arise from at least three mechanisms: ( 1) single-photon
`absorption leading to
`local heating, (2) single-photon absorption
`leading to
`photochemical transformations, and (3) multiple-photon absorption leading to
`photochemical transformations. Events (1) and (2) can be mitigated by choosing a
`wavelength of light which is weakly absorbed by the trapping material and by the
`surrounding fluid medium. Event (3) is a more general problem and is mitigated in
`part by working with lo