Patent Application
20120267549
Fluorescent microscopy and fluorescent labeling have become powerful measurement and diagnostic techniques in the biological sciences. Genes, cells, molecules, small molecules, peptides, viruses, and oligonucleotides can be labeled with fluorescent tags tailored to provide diagnostic information about characteristics of the labeled species when the species is subjected to varied biological conditions. In laboratory studies, fluorescent emission is typically monitored from the labeled species to determine the effect of the varying biological conditions, e.g., the effect of a pharmaceutical agent applied to the species. Conventional fluorescence detection utilizes large microscopes adapted for fluorescent imaging applications or refractive optical imaging or detection systems also adapted for fluorescence detection. Conventional fluorescence detection apparatuses are typically large pieces of equipment, which are complex in design and require high-quality (expensive) optical components.
The inventors have appreciated that conventional fluorescence detection apparatuses are not suited for, or readily adapted for, high-throughput, parallel fluorescence detection, in which a plurality of fluorescing objects may be inspected simultaneously. To this end, the inventors have recognized and appreciated that microfabricated optical elements can be designed and integrated with microstructures (for holding/carrying samples/objects for inspection) in a system adapted for high-throughput, parallel fluorescence detection.
In view of the foregoing, the present disclosure is directed generally to inventive methods and apparatus for fluorescence sensing using one or more microfabricated optical elements. In various embodiments, the microfabricated optical elements may be diffractive optical elements; for example, in one implementation the optical elements may be Fresnel zone plates.
In some embodiments, a single microfabricated optical element (e.g., a single zone plate) or an array of optical elements may be used with a single low-numerical-aperture relay optic in a high-throughput fluorescence detection system. Also, in some embodiments one or more optical elements may be integrated with a microfluidic structure having an array of microfluidic channels. The microfluidic channels can be configured to support a stream of fluid that includes fluorescently labeled objects (e.g., microparticles and/or biological species), which objects are conveyed in the stream to respective optical elements in the array of optical elements. In one exemplary implementation, the stream of objects may be provided from a droplet generator that provides fluorescently-labeled droplets dispersed in the microfluidic stream. The inventors have appreciated that exemplary microfabricated, integrated optical element array/microfluidic apparatus (also referred to as “micro-optofluidic” apparatus) are useful in high-throughput drug screening applications.
According to one aspect of the technology described herein, an apparatus for detecting fluorescence from small objects comprises at least one microfluidic channel, at least one microfabricated optical element coupled to the at least one microfluidic channel, and a relay optic. The optical element may be disposed with respect to the microfluidic channel so that an optical axis of the microfabricated optical element passes through the microfluidic channel. The relay optic may be configured to operate as a confocal pinhole aperture and relay at least a portion of radiation from the at least one microfabricated optical element to a location, e.g., to a location at which a detector is mounted. The microfabricated optical element may be a microlens or a microfabricated Fresnel zone plate.
According to another aspect, a multilayer apparatus for detecting fluorescence comprises a first layer comprising a first substrate, and a second layer comprising a second substrate. In various embodiments, the second layer is in releasable contact with the first layer. At least one Fresnel zone plate may be disposed on the first substrate, and at least one microfluidic channel may be disposed on the second substrate. In various implementations, the Fresnel zone plate is configured to have a first focal region for excitation radiation, the first focal region substantially outside the microfluidic channel, and a second focal region for fluorescent emission excited by the excitation radiation, the second focal region substantially within the microfluidic channel.
In yet an additional aspect of the technology, an apparatus for detecting fluorescence from small objects comprises an array of microfabricated optical elements, and a microfluidic drop generator configured to generate microfluidic drops, wherein the array of optical elements and the microfluidic drop generator are integrated on a same chip. The array of optical elements may be an array of microfabricated Fresnel zone plates. A single relay optic may be configured to relay at least a portion of fluorescent emission collected by any one of the optical elements in the array to an imaging detector. High-throughput fluorescence detection at rates of nearly 200,000 samples per second can be achieved with an integrated optical element array, a single relay optic, and a high-speed imaging detector.
A further aspect of the technology is directed to a fluorescence sensing method that comprises steps of irradiating at least one microfabricated diffractive element with excitation radiation, the at least one microfabricated diffractive element being configured to focus the excitation radiation onto at least one microfluidic channel. The sensing method further comprises providing a flow of at least one object in the microfluidic channel. In various embodiments, the object generates a fluorescent emission when irradiated by the excitation radiation, and at least a portion of the fluorescent emission passes through the microfabricated diffractive element. The method may further comprise relaying at least some of the portion of the fluorescent emission passing through the microfabricated diffractive element to a location with a relay optic. The relay optic may be configured to exclude radiation emitted a selected distance from the object that generates the fluorescent emission. For example, the relay optic may be configured to operate as a confocal aperture and exclude fluorescent emission and other radiation originating from locations outside of a focal region, the focal region associated with the microfabricated diffractive element.
It should be appreciated that all combinations of the foregoing concepts and additional concepts discussed in greater detail below (provided such concepts are not mutually inconsistent) are contemplated as being part of the inventive subject matter disclosed herein. In particular, all combinations of claimed subject matter appearing at the end of this disclosure are contemplated as being part of the inventive subject matter disclosed herein. It should also be appreciated that terminology explicitly employed herein that also may appear in any disclosure incorporated by reference should be accorded a meaning most consistent with the particular concepts disclosed herein.
The foregoing and other aspects, embodiments, and features of the present teachings can be more fully understood from the following description in conjunction with the accompanying drawings.
The skilled artisan will understand that the figures, described herein, are for illustration purposes only. It is to be understood that in some instances various aspects of the invention may be shown exaggerated or enlarged to facilitate an understanding of the invention. In the drawings, like reference characters generally refer to like features, functionally similar and/or structurally similar elements throughout the various figures. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the teachings. The drawings are not intended to limit the scope of the present teachings in any way.
The features and advantages of the present invention will become more apparent from the detailed description set forth below when taken in conjunction with the drawings.
The inventors have appreciated that fluorescence detection is amongst the primary methods for conventional analysis of microscale objects disposed in microfluidic devices. At the same time, the inventors have recognized several drawbacks associated with conventional techniques for detecting fluorescence from microscale objects. In most microfluidic assays, fluorescence is observed using, for example, a conventional fluorescence microscope that suffers from a limited field of view and is difficult to parallelize. In order to achieve a high fluorescence signal collection efficiency, significantly large-valued numerical aperture (NA) microscope objective lenses often are used, which in turn limits the field of view (e.g., a conventional 40× microscope objective having a numerical aperture of 0.65 has a field of view less than 500 microns). The fluorescence detection region within the field of view is therefore usually confined to a single location along a channel, which limits the measurement of any dynamic process. To increase the field of view, signal collection efficiency (and signal quality) is sacrificed. Because the conventional microscope is large (and expensive), parallelization can be impractical.
Optical detection in biological or biochemical assays is typically performed conventionally by quantification of a fluorescence signal in a discrete volume or container. With respect to small sample volumes, recent research in microfluidics systems, and in particular two phase microfluidic technology, has demonstrated the ability to produce picoliter sized containers of fluid, called drops, at rates of several kilohertz per device. (See G. M. Whitesides, Nature, 2006, 442, 368-373; and S. L. Anna, N. Bontoux, and H. A. Stone, Applied Physics Letters, 2003, 82, 364:366). Based on the foregoing, the inventors have recognized and appreciated that droplet-based microfluidics is well-suited for carrying out high-throughput screening (and other applications that require a large number of reactions to be executed) in small volume samples.
The inventors also have appreciated that microfluidic devices can be parallelized on a single chip using lithographic techniques, further increasing the rate at which samples can be produced and manipulated. However, as the size of parallelized devices scale up, conventional optical detection becomes increasingly difficult, because of the tradeoff between field of view and collection efficiency for conventional fluorescence detection systems, e.g., conventional fluorescence microscopes.
To overcome these challenges, however, the inventors have recognized that optical imaging systems requiring a wide field of view and a small form factor can benefit from the use of a lens array. (See A. W. Lohmann, Applied Optics, 1989, 28, 4996-4998; R. Volkel, M. Eisner, and K. J. Weible, Microelectronic Engineering, 2003, 67-68, 461-472; and L. P. Lee and R. Szema, Science, 310, 2005, 1148-1150.)
In view of the foregoing, the inventors have developed fluorescence sensing apparatus and methods based on the integration of microfabricated optical lens elements (e.g., small diffractive elements, such as Fresnel zone plates), examples of which can be used readily in conjunction with (i.e., integrated with) massively parallel microfluidic devices in high-throughput sensing applications. Accordingly, various embodiments of the present invention are directed to using arrays of high numerical aperture (NA) microfabricated lenses, e.g., Fresnel zone plates, for both fluorescence excitation and collection associated with fluorescence detection of microscale objects. Both the microfluidics and the lens array may be integrated into a multilayer chip in various implementations, as discussed in detail below.
Embodiments of the lens array and detection apparatus according to the present invention facilitate simultaneous detection from a plurality of regions on a microfluidic chip, which may be separated by several times the field of view of traditional high-NA objective lenses used in conventional microscopes. The inventors have recognized that multiple detection at large sample spacing also makes possible temporal resolution of dynamic processes such as mixing or reaction kinetics.
In overview, methods and apparatus for high-throughput fluorescence detection using microfabricated optical element arrays integrated with microfluidic channels are described. Exemplary embodiments of the microfabricated optical element arrays may comprise one or more microfabricated Fresnel zone plates, which may be configured to collect light from microscale objects flowing in the microfluidic channels. In one exemplary implementation, multiple samples may be inspected in parallel at rates of about 200,000 samples per second for a single array having 64 lenses.
A microfluidic chip and fluorescence detection apparatus, according to some embodiments of the present invention, may be implemented as an integrated structure constituting a “chip.” In various aspects, such a chip may comprise a two or three layer structure within (or on) which is integrated a high-NA microfabricated optical element or microfabricated optical element array. Additional detection optics may comprise a single relay optic, a bandpass filter, and a photodetector or an imaging detector, according to certain embodiments. In exemplary implementations, the single relay optic combined with high numerical aperture integrated optical elements can provide signal enhancement on the order of 200 times or greater compared to that provided by conventional fluorescence detection methods.
As noted above, the optical elements of the array (also referred to herein as a “lens array”) may comprise microfabricated lenses such as Fresnel zone plates, or any other suitable optical elements, one or more of which may have a large numerical aperture, e.g., greater than about 0.5. The optical elements may be integrated onto a microfluidic chip and configured to provide high light collection efficiency, e.g., in some embodiments comparable to the collection efficiency of high performance objective lenses of conventional microscopes. The integration of a plurality of microfabricated lenses onto a microfluidic chip facilitates observation of a large number of sample areas with high light collection efficiency for each area. The lens array may be used to perform measurements in parallel, for example by using each of the microfabricated optical elements of the array to collect light (e.g., fluorescent emission from a respective microscale sample) for a separate measurement. Thus, large amounts of data may be collected simultaneously.
Further details of the apparatus and methods for fluorescence sensing according to various embodiments of the present invention are described in the following sections.
Apparatus
Referring to
For purposes of the following descriptions, the microfabricated optical element located near the object 103 providing an optical focusing characteristic is identified as a Fresnel zone plate 120 (i.e., a microfabricated diffractive lens). However, it should be appreciated that the optical element may be any suitable microfabricated diffractive optical element. For example, in other implementations contemplated by the present invention, the optical element may alternatively comprise a refractive microlens or may comprise a combination of a refractive and diffractive microfabricated optical element (e.g., a microfabricated binary optical element that may be produced by gray-scale dose exposure of a polymeric resist, by micromolding, or by imprint lithography). In various aspects, the microfabricated optical element provides at least one focusing characteristic and comprises a microfabricated optical lens. The optical element may provide additional focusing characteristics (e.g., multiple focal spots), as described in international patent application PCT/US2008/011033 to common inventors, filed Sep. 23, 2008, and now published under publication number WO2009/088399 (which publication is hereby incorporated by reference herein in its entirety).
In
An embodiment of a Fresnel zone plate 120 is comprised of concentric rings 210 of varying width, as depicted in the plan views of
r
m=√{square root over (mλf+m2λ2/4)} EQ. 1
where m is an integer, f is the focal length for the optical wavelength λ as measured in the medium in which the zone plate focuses radiation. If the zone plate is immersed in a medium having a refractive index n, then λ takes on the effective value of the wavelength in the medium λ/n.
A cross-sectional view of a zone plate, according to one embodiment of the present invention, is shown in the atomic force microscopy trace of
The rings 210 of the zone plate provide the diffractive and optical focusing characteristic of the zone plate. The focusing characteristic of the zone plate 120 may be characterized, or parameterized, by a focal length f. Generally, the focal length f may be regarded as the “free space” focal length for the zone plate. For example, when collimated light impinges on and passes through the zone plate into air, it will come to a focus on an optical axis of the zone plate at a distance f from the zone plate. In
The focal length f of the zone plate 120 may be determined by the pattern of the rings 210. In general, ring patterns that have a larger radial periodicity provide a longer focal length, and ring patterns of shorter periodicity yield shorter focal lengths. Thus, one can design the pattern of the zone plate to provide virtually any desired focal length. The zone plate diameter dzp and focal length are related to the numerical aperture (NA) of the zone plate in free space according to the relation:
NA=sin[tan−1(dzp/2f)]. EQ. 2
A zone plate may also be characterized by its NA value. Generally, a larger NA value denotes a shorter focal length.
The inventors have recognized that employing zone plates 120 with significantly short focal lengths, or large NA values, can eliminate the need for a pinhole aperture in zone-plate-based confocal microscopy apparatus, similar to the apparatus as illustrated in
There is a subtlety in the operation of a zone plate when a material 110 and/or substrate 107 adjacent the zone plate 120 has an index of refraction n2 and/or n3 which differs from the index of refraction for air. In
In various embodiments, the zone plate 120 is disposed on a substrate 105, which may have a thickness ts. The substrate will transmit at least a portion of the radiation that is emitted from the object 103 and that passes through the zone plate 120. The substrate may have an index of refraction n1 that differs in value from 1.0. In exemplary embodiments, the thickness of the substrate 105 may be between about 5 microns and about 20 mm, or more particularly between 5 microns and 20 mm. In an alternative embodiment to
According to some embodiments of the present invention, a Fresnel zone plate 120 is designed to provide two focal lengths, a first focal length associated excitation or illumination radiation, and a second focal length associated with fluorescent emission from the object 103. An example of a dual-focus zone plate configuration is shown in
Excitation radiation 223′ that is not incident on the microfabricated optical element, such as zone plate 120, is not focused as can be seen in
An object 103 to be inspected or imaged may be illuminated in any suitable manner. For one embodiment, the object may be illuminated by illumination or excitation radiation 180 generally from a side opposite the zone plate 120, as illustrated in
The zone plate 120, or arrays of zone plates, may be patterned and fabricated using any of a variety of microfabrication techniques. Patterning techniques include, but are not limited to, electron beam lithography, ion beam lithography, contact photolithography, optical projection lithography, x-ray lithography, zone-plate array lithography, imprint lithography, interference lithography, micromolding lithography, and soft lithography. Fabrication techniques include, but are not limited to, lift-off techniques, reactive ion etching, wet chemical etching, material deposition, chemical-mechanical polishing. Any combination of these techniques may be used to fabricate a zone plate 120, a linear array of zone plates, or a two-dimensional array of zone plates.
For the embodiment depicted in
Referring again to
The layer of material 110 can serve several functions. First, the layer of material can protect the zone plate from damage or picking up particles that, may interfere with the optical properties of the zone plate. Any particles that may be picked up by the zone-plate structure may be readily cleaned from the surface of the material 110 without risk of damaging the zone plate 120. The layer of material 110 may be thin in some embodiments (e.g., less than 1 micron, less than 500 nm, less than 200 nm, and yet less than 100 nm in certain implementations). The layer of material 110 may also function as a “solid immersion” material, i.e., a material with a higher index of refraction than air that effectively shortens the focal length of the zone plate and increases the zone plate's NA. Additionally, the layer of material 110 may provide a boundary proximal the zone plate's focal region that can assure that an object will be located at the effective focal distance from the zone plate. This can facilitate positioning of the object in the zone plate's focal region.
The object 103 to be inspected may be located approximately at the focal region 251 of the zone plate 120, as illustrated in
The relay optic 140 may comprise any optic that relays an image of the zone plate 120 onto the detector 150. The image may be in focus, or out of focus at the detector. As one example, the relay optic may comprise a single refractive optical lens positioned between the zone plate 120 and the detector 150. As a further example, the relay optic may comprise a pair of refractive optical lenses. The relay optic may comprise a unity magnification telescope. In some implementations, the relay optic 140 may comprise a diffractive optical element and/or a binary optical element, or a combination of either or both of these optical elements and one or more refractive lenses. The relay optic 140 may function essentially as a pinhole aperture for the imaging system, and may have a suitable size (e.g., a one-inch diameter, a one-half-inch diameter, a two-inch diameter, or any other suitable size) selected so that the relay optic operates essentially as a pinhole aperture. To operate as a pinhole aperture, the relay optic 140 excludes unwanted radiation, travelling from the object 103, from the detector 150.
In various embodiments, the relay 140 optic has a numerical aperture with a value less than the numerical aperture of the zone plate 120. The NA of the relay optic may have a value between about 0.01 and about 0.5, more particularly between 0.01 and 0.5. In some embodiments, at least one lens of the relay optic has an NA value between about 0.01 and about 0.5, more particularly between 0.01 and 0.5. In various implementations, the relay optic 140 accepts a portion of radiation that is emitted from the object 103 and collected by the zone plate 120. It will be appreciated that selecting a relay optic 140 with a small NA value will exclude radiation travelling from the zone plate 120 at angles with respect to the optical axis 101 that exceed the acceptance angle of the small NA relay optic 140.
In various embodiments, the relay optic 140 is aligned substantially centrally with an optical axis of the zone plate 120. Referring to
An optical filter 145 may be disposed between the object 103 and the detector 150 and be any type and form of optical filter, e.g., an interference filter, an optical density filter, a polarizing beam splitter, a pellicle, etc. In some embodiments, more than one filter may be used. The optical filter 145 may be selected to block a portion of radiation directed toward the detector. For example, the filter may block radiation longer than a selected wavelength, shorter than a selected wavelength, or within a wavelength band. The blocked radiation may be radiation that would otherwise contribute to background signal or noise in the detector. As an example, the filter 145 may block ambient light. The filter 145 may be used in certain embodiments to block excitation radiation that is used to excite fluorescent radiation emitted from the object 103.
The filter 145 may be located at various positions in the apparatus 100. It may be located within the relay optic 140, before or after the relay optic, incorporated with the detector 150, or incorporated with the zone-plate structure, e.g., incorporated with the substrate 105 or material 110, in some embodiments. In some embodiments, the filter 145 may be incorporated in a lens or optical component of the relay optic 140. In certain implementations, the apparatus 100 may omit the filter, e.g., when illumination radiation scattered from the object 103 is being detected.
A detector 150 may be used with the sensing apparatus 100 to detect a level of radiation emitted from the object 103 and provided to the detector by the zone plate 120/relay optic 140 combination. The detector may be any type and form of detector that is responsive to radiation from the object 103, the radiation being desirable to detect. For example, the detector 150 may be responsive to fluorescent radiation emitted from the object 103. In some embodiments, the detector 150 may be responsive to illumination radiation emitted from the object 103, the illumination radiation scattered by the object. The detector may comprise a photodetector, an array of photodetectors, e.g., a CCD array. The detector 150 may comprise a photomultiplier, an avalanche photodiode, a CMOS photodetector, or an array of any of these types of detectors.
In one exemplary implementation, the sensing apparatus 100 may sense and record radiation emitted from an object 103 in the following way. Radiation emitted from the object 103 generally will radiate in all directions. The radiation emitted from the object may be fluorescent emission or may be illuminating radiation scattered by the object. In some implementations, it may be desirable to detect fluorescent emission, while in other implementations it may be desirable to detect scattered illuminating radiation. A portion of the emitted radiation will be collected by the zone plate 120 and directed generally towards the relay optic 140. Some of the radiation collected by the zone plate 120 will travel toward the relay optic at an angle with respect to the optical axis 101 that falls within an acceptance angle of the relay optic 140. The acceptance angle of the relay optic is determined by the NA of the relay optic. Radiation collected by the zone plate 120 and travelling toward the relay optic at an angle within the acceptance angle of the relay optic 140 will be relayed to and detected by the detector 150. All other radiation will be excluded from detection.
The inventors have recognized that by using a relay optic with a small NA value and a zone plate with a large NA value, off-axis and out-of-focus radiation emitted from the object 103 can be excluded from detection without using a confocal pinhole aperture in the apparatus 100. This aspect of operation is described in the co-pending application referred to above and entitled “Methods and Apparatus for Scanning Microscopy Using One or More Fresnel Zone Plates.” The inventors have recognized that the combination of NA values, for the zone plate 120 and for the relay optic 140, can be selected to limit detected radiation to be that which is emitted substantially from only within the focal region 251 of the zone plate 120. The apparatus 100 may then be used to detect radiation from a microscale region without the use of a pinhole aperture. The apparatus 100 may also be used for high-resolution imaging, by moving the object 103 with respect to the zone plate 120 and recording a sequence of detected light levels corresponding to each movement step of the object. High-resolution fluorescence imaging may be carried out in two dimensions, by moving the object 103 transverse to the optical axis 101 (i.e., in an X-Y plane), or alternatively in a direction along the optical axis (i.e., in a plane containing the Z axis), in a raster-scanning method. Three-dimensional imaging may also be obtained by systematically scanning the object in all three dimensions (e.g., recording a series of X-Y images at sequential Z positions).
The operation of the apparatus 100 may also be described as follows. By using a relay optic with a small NA value, the “field of view” of the zone plate/relay optic combination is restricted to a small area, e.g., in some embodiments, an area having a width on the order of the size of the focal region 251 of the zone plate. As used herein, “focal region” refers to a small volume at the effective focus of the zone plate that would be substantially filled by a diffraction limited focal spot. Expressed alternatively, the focal region refers to the spot focus that results when a uniform beam of monochromatic radiation impinges normally on the zone plate. The focal region may also be expressed as a surface which bounds a majority portion of the focused radiation (e.g., the full-width-half-maximum value of the focused radiation, the 1/e value of the focused radiation, the 1/e2 value of the focused radiation).
Multiple different combinations of respective NA values for the relay optic 140 and the zone plate 120 are contemplated according to various embodiments of the present invention. For example, in some implementations, the inventors have found that the combination of NA values for the relay optic and the zone plate can be selected for some embodiments to yield a field of view for the apparatus 100 between about 200 nanometers and about 2 microns, more particularly between 200 nanometers and 2 microns. As a non-limiting example, if a zone plate has a diameter of about 50 microns and a NAzp of 0.9, and the relay optic has an NAro of 0.05, the field of view will be about 1 micron. In other implementations, the combination of NA values for the zone plate and relay optic may be selected to yield a field of view for the apparatus between about 100 nanometers and about 500 nanometers, more particularly between 100 nanometers and 500 nanometers. As a non-limiting example, if a zone plate has a diameter of about 40 microns and a NAzp of 0.95, and the relay optic has an NAro of 0.025, the field of view will be about 300 nanometers. Other combinations of NA values and zone plate design to yield different field of views FOV will be evident to those skilled in the art of optics from the following relation expressed in terms of zone plate focal length f and the numerical aperture of the relay optic NAro:
FOV≈2f×NAro. EQ. 3
or in terms of zone plate diameter dzp, zone plate numerical aperture NAzp and numerical aperture of the relay optic NAro:
For the expressions of EQ. 3 and EQ. 4, it is assumed that the focal length of the zone plate is much less than the diameter of the zone plate. Not all implementations described herein will employ a zone plate for which its focal length is much less than its diameter. In such implementations, expressions other than those above may be used to determine a suitable zone plate/relay optic combination.
From EQ. 3 and EQ. 4 it will be appreciated that a zone plate and relay optic combination can be designed to provide a field of view that has a size substantially equal to the width of the microfluidic channel 117 (wc in
As used herein, the term “glass” may be used to refer to any type and form of an inorganic composition generally transmitting excitation radiation and fluorescence emission. Examples of glass include, but are not limited to, plate glass, quartz, fused silica, BK-7, sapphire, indium tin oxide, borosilicate glass, to name a few.
In exemplary implementations, the substrate 107, having at least one microfluidic channel formed thereon, is in releasable contact with the substrate 105, on/in which the zone plate is fabricated, or with the intermediate substrate 109. Releasable contact can be implemented when one of the substrates comprises a soft polymer (e.g., polydimethylsiloxane PDMS) and another substrate, to which it contacts, comprises any suitable material to which PDMS adheres (e.g., SiOx, fused silica, quartz, glass, silicon, sapphire.) Releasable contact is advantageous in that the substrate 107 having the microfluidic channels formed thereon may be removed, subjected to cleaning, and reused. The substrate 105 also may be cleaned and reused. As an example, referring to
To assemble the integrated structures as shown in
The inventors have recognized and appreciated that parallel confocal fluorescence microscopy may be performed using apparatus similar to that shown in
There are several advantageous aspects of the present invention. Among these are advantages related to fluorescence sensing with micro-optofluidic chips utilizing a plurality of microfabricated optical lenses, as depicted in
With reference now to
In exemplary implementations, a droplet generator 505 provides a plurality of microscale drops 503 flowing in a plurality of microfluidic channels 517. The drops 503 may comprise a biochemical specimen and may contain at least one fluorescing component. The presence, signal level, and/or absence of a fluorescent emission from each drop may be indicative of a biochemical condition for specimen. In certain embodiments, the drops 503 may comprise microscale spheres within the fluid of the microfluidic channel.
As depicted in
The channel spacing dc will then be given by dc=Λcos(α).
It may be appreciated that the device shown in
Description of Methods
It should be appreciated that various inventive methods according to the concepts described herein may be carried out with some or all of the apparatuses described above. An exemplary method of zone-plate-based microscopy according to one embodiment of the present invention is depicted in the flow chart of
In a particular implementation, a method 700 for optically inspecting an object may comprise illuminating 710 at least one object, providing 720 a flow of at least one object in at least one microfluidic channel, and relaying 730 to a location at least a portion of fluorescent emission emitted from the at least one object. The exemplary method 700 may further comprise detecting 740 the at least a portion of fluorescent emission with a detector, which may be located at the location to which the portion of fluorescent emission is relayed. The method 700 may further comprise recording 750 at least one signal level associated with the detected fluorescent emission. As may be appreciated, the steps of relaying 730, detecting 740, and recording 750 may be iterated to repetitively detect and record signals for a plurality of microscale objects provided in the flow of objects (e.g., a flow of droplets through a device similar to that depicted in
The step of illumination 710 may comprise illumination a microscale object in a microfluidic channel with any suitable wavelength and intensity of radiation. The radiation may be in the infrared wavelength band, visible wavelength band, ultraviolet or deep ultraviolet wavelength band. The illumination radiation may be coherent radiation, e.g., radiation from a laser, partially coherent, or incoherent radiation. The step of illuminating may comprising providing polarized or unpolarized illumination radiation. Polarized radiation may be polarized in any manner, e.g., linear, elliptical, or circular.
The step of illuminating 710 may comprise exciting fluorescence in the object, whether a property of the object itself or a fluorescing component added to the object or bound to the object. The fluorescent radiation emitted from the object may be the radiation desired to be detected by detector 150 in some embodiments. In other embodiments, the step of illuminating 710 may comprise scattering illumination radiation from the object 103, wherein the scattered radiation is detected by the detector 150. In some embodiments, the step of illuminating 710 may comprise both exciting fluorescence and scattering illumination radiation. Both fluorescent and scattered radiation from the object may be detected using time-division multiplexing or wavelength-division multiplexing detection methods.
The step of illuminating 710 may or may not comprise focusing the illumination radiation onto the object 103. A separate focusing optic may be used to focus illumination radiation onto the object 103 in some embodiments, e.g., a lens located near the object but opposite the zone plate. (See, for example,
The act of providing a flow 720 of at least one object may comprise providing a flow of one or more microscale objects in at least one microfluidic channel. In some embodiments, a single channel may be used to provide a flow of objects to a plurality of microfabricated optical elements, whereas in other embodiments an array of channels may be used to provide a flow of objects to a plurality of microfabricated optical elements. In certain implementations, the objects generate fluorescence when illuminated by excitation radiation. When an object is excited by excitation radiation and at least a portion of the object is located within the field of view for a particular microfabricated optical element, at least a portion of the fluorescent emission emitted from the object passes through the particular microfabricated optical element.
In some implementations, providing a flow 720 comprises providing a flow of a plurality of microfluidic drops, which may be produced by a droplet generator 505. The microfluidic drops may be provided in one microfluidic channel or an array of microfluidic channels. The droplet generator may or may not comprise one or more flow dividers. The drops may be generated in at least one microfluidic stream, which conveys the drops to one or more microfabricated optical elements.
In some implementations, providing a flow 720 comprises providing a flow of a plurality of microscale objects in at least one microfluidic stream. The microscale objects may dispersed in a solution which is provided to one or more microfluidic channels. Flow dividers may or may not be used in combination with one microfluidic channel or an array of microfluidic channels. In various embodiments, the microscale objects may comprise biological or biochemical specimens, or microparticles such as colloidal particles, beads, or agglomerations. Exemplary size ranges of the microscale particles are between about 50 nanometers and about 200 microns, more particularly between 50 nanometers and 200 microns.
Flow may be provided in any suitable manner. In some implementations, flow is provided using pressure to force a fluid through the microfluidic channels. In some cases, vacuum may be applied to the microfluidic channels to draw a fluid through the channels. In some embodiments, electrophoretic flow may be employed. Electrodes may be disposed on the microfluidic chip and configured to provide electric fields along the microfluidic channels when activated by an electrostatic potential. The electric fields within the microfluidic channels can convey charged particles along the channels.
The relaying 730 may comprise directing a first portion of the portion of radiation collected by the microfabricated optical element (e.g., a Fresnel zone plate) to a location (e.g., a location at which the detector 150 is positioned), and excluding a second portion of the portion of radiation collected by the microfabricated optical element from the location. The directing and excluding may be carried out by a low-NA relay optic. The step of relaying 730 may comprise selecting a relay optic having an NA value so that the relay optic will provide a selected field of view for the microfabricated optical element/relay optic combination. The step of relaying 730 may further comprise positioning the relay optic, e.g., aligning the optical axis of the relay optic to the optical axis of the microfabricated optical element, or aligning the optical axis of the microfabricated optical element to the optical axis of the relay optic.
The step of detecting fluorescence 740 may comprise detecting an electrical signal level representative of the amount of fluorescence emitted by a fluorescing microscale object 103, collected at least in part by a particular microfabricated optical element 120, and relayed at least in part by a relay optic 140 to a detector 150. The detector may comprise a single photodetector, or an array of photodetectors (e.g., a CCD linear or imaging array) as described above. Signals from the detector may be provided to a signal recording and/or signal display apparatus. The display apparatus may comprise and video camera in some implementations and/or an oscilloscope. The step of detecting 740 may further comprise filtering the optical signal (e.g., filtering the fluorescent emission signal with a wavelength and/or polarization filter), and filtering the electrical signal (e.g., filtering the signal from the detector with a low-pass, high-pass, or band-pass filter).
In certain embodiments, diffractive properties of the microfabricated diffractive optical elements 120 may be used to spectrally resolve fluorescent emission at different wavelengths, e.g., emitted from different fluorophores. According to one embodiment, the apparatus 100 described herein may be used for multi-color fluorescence applications. In such applications, optical filtering utilizes the spectral resolution in the lens itself, and may obviate any need for a spectral filter bank. As an example and referring to
The act of recording 750 may comprise recording a data signal representative of at least one light level from at least one microfabricated optical element detected by detector 150. The data signal may be recorded in computer-readable storage media. As an example, the detector 150 may be interfaced with a computer or processor (not shown in the figures) that repeatedly records data signals from the detector. The data signals may be plotted to show a time evolution of detected radiation from an object. When an object 103 moves with respect to a particular microfabricated optical element (e.g., moves along a microfluidic channel), the data signals may be recorded and plotted as a function of time to show evolution of the fluorescence. Accordingly, the step of recording may further comprise displaying at least one signal representative of the at least one light level detected by the detector 150.
It will be appreciated that when an array of microfabricated optical elements (e.g., zone plates 120) are used, the step of recording 750 may comprise recording a plurality of data signals representative of light levels from each of the zone plates in the array. In an embodiment employing an array of zone plates, a detector having an array of optical detectors may be used, e.g., a one- or two-dimensional CCD array, or an array of sensitive photodiodes or photomultipliers. In some embodiments, one pixel in the array of photodetectors may correspond to one zone plate in the array, e.g., receive radiation predominantly from the corresponding zone plate. In some embodiments, a group of pixels in the array of photodetectors may correspond to one zone plate in the array. A computer or processor may then record light data signals associated with each of the microfabricated optical elements and their corresponding microscale object 103 in the array. The step of recording 750 may further comprise recording separately, as well as displaying separately, data signals representative of light levels from each of a plurality of objects 103 and/or each of a plurality of optical elements in the array.
When parallel detecting 740 and recording 750 is carried out using computer processing methods, high-throughput fluorescence sensing can be achieved. For high-throughput sensing, the step of providing 720 a flow of objects may be carried out for an extended period of time, e.g., between about 10 seconds and about 10 hours, more particularly between 10 seconds and 10 hours. The steps of relaying 730, detecting 740, and recording 750, may be carried out in an automated or semi-automated process monitored or managed by a computer processor during the flow interval. Generally, the throughput rate Rt, or number of fluorescing objects that may be detected per second, will depend on several factors such as number of microfabricated optical elements in an array 520 that can be viewed simultaneously by an imaging detector 150, microfluidic flow rate, amount of fluorescent emission, on average, from the microscale objects, sensitivity of the detector 150, and noise levels. In one implementation, described below, the inventors have demonstrated that throughput rates R, on the order of 190,000 objects/second are obtainable with the inventive apparatus and methods described above.
In this example, the effect of longitudinal dispersion on the transit times of fluorescent spheres through 12 detection regions along a serpentine microfluidic channel is investigated. This example illustrates how temporal dynamics over a long microfluidic channel length can be investigated.
For this example, a multilayer optofluidic device similar to the structure shown in
Using a serpentine microfluidic channel similar to that illustrate in
In order to simultaneously collect a fluorescence signal from each detection region, the zone plate array was imaged onto a fast CMOS camera. A telescope was used as the relay optic. The zone plate array was illuminated with an excitation laser beam that has a width of 2 mm, which overfilled the array width of 1 mm. Video movies were recorded of the zone plate array at rates of 2k to 75k frames per second (fps) using a sub-region (16×32 pixels) of the camera. Each zone plate subtends approximately twenty pixels, which are integrated to obtain the signal from each detection region. Fluorescent beads were loaded 5 μm into the device at a 0.2% concentration by mass.
In addition to measuring the average velocity of beads in the channel, the dispersion of velocities can be measured by analyzing the cross-correlation of the signals at different detection regions. The peak value of the cross-correlation signal gives the average time delay between the two measurements, and the width and shape of the peak give the distribution of bead velocities. Beads at different transverse locations in the channel travel at different velocities due to the parabolic velocity distribution in laminar flow.
In this example, the inventors demonstrate a zone-plate array optical detection system that is integrated into a massively parallel microfluidic device. The zone-plate array efficiently collects fluorescence signals at well defined regions spannihg a large area. Consequently, this multilayer micro-optofluidic platform enables parallelization not possible using a single conventional microscope objective lens.
The high throughput microfluidic device produces drops traveling through 64 parallel microfluidic channels. The drops are formed in a single flow-focus drop maker and then are subsequently split by six layers of drop splitters or flow dividers in a microfluidic device similar to that shown in
Although a microscope image of a substantial area of the device can be obtained with a conventional low magnification objective lens (10× magnification, 0.3 NA), the field of view would not be sufficient for imaging the entire device. It would be possible to use a conventional objective lens with an even lower magnification to increase the field of view, but such an objective lens would have an even smaller numerical aperture (NA) and consequently lower light collection efficiency.
Instead of using a single low NA objective, the optical detection system comprised an array of high NA zone plates, each confocally detecting from a localized region in a separate channel. Increasing the NA is significantly advantageous, because collection efficiency C scales approximately with the square of NA (C∂NA2). With the zone-plate array, fluorescence from a region in every microfluidic channel may measured simultaneously without scanning, in contrast to what is frequently done in other parallel detection systems. The lens array is illuminated by a laser providing excitation radiation, and is imaged onto a high speed CMOS camera using a unity magnification 0.13 NA telescope for the relay optic.
Since the zone plates are planar in contrast to lenses that use refraction across curved surfaces, they can readily be integrated into flat substrates in multilayer designs. Both the microfluidic device and the zone-plates are fabricated from PDMS using soft lithography. In addition to the advantages of being planar, zone plates are advantageous in lens arrays because their focal length can be defined with much higher accuracy and precision than refractive lens arrays. Zone plates can also provide significantly large NA values, and can be matched to the focusing medium, consequently minimizing effects of spherical aberration.
In various exemplary embodiments, each zone plate performs at least two functions. A first function is to produce a focused excitation spot for each microfluidic channel in the array. A second function is to collect the fluorescence emission over each detection region efficiently. For this example, the zone plates are designed to focus light through a 170 μm (tcover) thick No. 1 coverslip 109 that is bonded to the top surface of the microfluidic substrate 107, as depicted in
For the implementation of this example, as depicted in
In addition to a high collection efficiency, each zone plate provides for confocal filtering of the emitted fluorescence, meaning that the detection regions are well defined and do not overlap. Lower NA lens arrays have overlapping fields of view, which in applications like this example would produce cross-talk from neighboring channels. The detection region of each zone plate is confined because of vignetting in the relay optics. The condition for vignetting can be expressed as Δx=rrofZP/frelay, where rro is one-half the clear aperture diameter of the relay optic (selected to be 10 mm in this example), frelay is the relay lens focal length (selected to be 100 mm in this example), and fZP is the zone plate focal length (selected to be 180 μm in this example). Vignetting starts to occur in this system at a transverse distance of about 9 μm from the optical axis of the zone plate. Thus, for this implementation the detection region has a width of about 18 μm, slightly larger than the width of the microfluidic channels.
Excitation and collection characteristics for a micro-optofluidic device are measured in this example. For this study, the micro-optofluidic device of Example 2 above was used. By aligning a single zone plate to a dye filled microfluidic channel, separate measurements of enhancement due to increased excitation intensity and enhancement due to increased collection efficiency, relative to the low NA telescope, can be made. To characterize the excitation enhancement, the excitation laser is focused into the dye filled microfluidic channel using the zone plate. The emitted fluorescence is imaged onto a CCD camera using a microscope objective lens, and the image is recorded. The recorded image shows a bell-shaped distribution of detected fluorescence.
The size and the relative enhancement of the collected fluorescent emission using the zone plate are characterized by scanning a tightly focused fluorescence excitation spot, generated by a microscope objective lens, and quantifying the fluorescence captured by a zone plate and imaged onto a CCD camera. Only fluorescence emitted from inside the zone plate's detection region is collimated and captured by the relay optics, otherwise it is filtered out by the confocal combination of zone plate and relay optic.
Since the detection region for a zone plate is smaller than the channel-to-channel spacing in this embodiment, fluorescent emission that could arise from neighboring microfluidic channels will not corrupt the signal from the zone plate. In addition for this implementation, the detection region is also slightly larger than the microfluidic drop size, so little of the fluorescence emission from each drop should be filtered out when the drop is centered under the zone plate.
In this example, high-throughput detection of fluorescence from microscale drops flowing in an array of microfluidic channels is demonstrated. An 8×8 zone-plate array patterned on a PDMS substrate is aligned to a 64-channel-array microfluidic device using a mask aligner and adhered to an opposite coverslip surface with a reversible bonding step. The multilayer, integrated micro-optofluidic chip appears similar to the structure of
To form microscale drops in the microfluidic channels, water containing 2 mM resorufin dye and hydrocarbon oil (HFE-7500) with a surfactant of carboxylic acid of Krytox 157 FSL at 1.8% by weight is loaded into the droplet generator 505 as the inner and outer fluid phases respectively. For the selected implementation of this example, the inner and outer flow rates of the drop maker are 8 and 20 μL/min, respectively. This results in drops being produced at approximately 200 Hz. When a drop containing the fluorophore passes through the detection region of a zone plate, the collected fluorescence peaks due to the excitation and collection enhancement. Images of the device in operation are taken with a CMOS camera operating at 1 k frames per second (fps).
At similar flow rates, the zone-plate array may also be imaged onto an electron-multiplying CCD (EMCCD), which has much greater sensitivity and lower pixel noise but a maximum frame rate of approximately 300 fps. Drops with resorufin concentrations as low as 1 μM have been imaged with signal to noise ratios of 20, but at rates approaching 100 drops per second per channel.
In order to obtain high-throughput detection, high frame rates achievable with high-speed CMOS cameras are utilized in an exemplary embodiment. The zone-plate array is illuminated with about 200 mW of excitation radiation and a beam diameter that substantially matches the diagonal length of the square zone-plate array, resulting in approximately 1.44 mW of excitation power incident on each zone plate in the array. The CMOS camera is operated in sub-array mode where each frame is 128×128 pixels and each pixel comprises a 22 μm square area. Because of the unity magnification telescope, the sub-array dimensions correspond to a 2.8 mm square field size, which is slightly larger than the 2 mm square zone-plate array. In this configuration, there are about 100 pixels of the imaging detector associated with each zone plate of the array.
From the captured image stack, the fluorescence signal corresponding to each detection region is found by integrating the ˜100 pixels associated with each zone plate. This is carried out for 62 of the 64 zone plates. The remaining two zone plates are misaligned, and do not produce appreciable signals. A Fourier transform of the time signals from active channels reveals that the drop rate in each channel is about 2975 Hz when the droplet generator is operated with inner and outer flow rates of 120 and 300 μL/min, respectively. When all 64 output channels are considered, this corresponds to an aggregate production and detection of about 190,000 drops per second.
All literature and similar material cited in this application, including, but not limited to, patents, patent applications, articles, books, treatises, and web pages, regardless of the format of such literature and similar materials, are expressly incorporated by reference in their entirety. In the event that one or more of the incorporated literature and similar materials differs from or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way.
While the present teachings have been described in conjunction with various embodiments and examples, it is not intended that the present teachings be limited to such embodiments or examples. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art.
It should be appreciated that the various techniques described herein may be operated in different modes, and that the technology is not limited to being operated in any particular mode. For example, in one embodiment, an imaging system may be operated in epifluorescence mode (i.e., the entire sample is illuminated with fluorescence excitation). In some embodiments, the imaging system may be operated in a confocal mode. The differing modes may be chosen based on a particular application. For example, the confocal mode may provide higher resolution, and may allow for three dimensional inspection of a microscale object in addition to two dimensional sectioning.
While various inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.
The above-described embodiments of the invention can be implemented in any of numerous ways. For example, some embodiments may be implemented using hardware, software or a combination thereof. When any aspect of an embodiment is implemented at least in part in software, the software code can be executed on any suitable processor or collection of processors, whether provided in a single computer or distributed among multiple computers.
In this respect, various aspects of the invention, e.g., signal acquisition from the detector 150, flow rate control, and droplet generation, may be embodied at least in part as a computer readable storage medium (or multiple computer readable storage media) (e.g., a computer memory, one or more floppy discs, compact discs, optical discs, magnetic tapes, flash memories, circuit configurations in Field Programmable Gate Arrays or other semiconductor devices, or other tangible computer storage medium or non-transitory medium) encoded with one or more programs that, when executed on one or more computers or other processors, perform methods that implement the various embodiments of the technology discussed above. The computer readable medium or media can be transportable, such that the program or programs stored thereon can be loaded onto one or more different computers or other processors to implement various aspects of the present technology as discussed above.
The terms “program” or “software” are used herein in a generic sense to refer to any type of computer code or set of computer-executable instructions that can be employed to program a computer or other processor to implement various aspects of the present technology as discussed above. Additionally, it should be appreciated that according to one aspect of this embodiment, one or more computer programs that when executed perform methods of the present technology need not reside on a single computer or processor, but may be distributed in a modular fashion amongst a number of different computers or processors to implement various aspects of the present technology.
Computer-executable instructions may be in many forms, such as program modules, executed by one or more computers or other devices. Generally, program modules include routines, programs, objects, components, data structures, etc. that perform particular tasks or implement particular abstract data types. Typically the functionality of the program modules may be combined or distributed as desired in various embodiments.
Also, the technology described herein may be embodied as a method, of which at least one example has been provided. The acts performed as part of the method may be ordered in any suitable way. Accordingly, embodiments may be constructed in which acts are performed in an order different than illustrated, which may include performing some acts simultaneously, even though shown as sequential acts in illustrative embodiments.
All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.
The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”
The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.
As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently; “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.
The claims should not be read as limited to the described order or elements unless stated to that effect. It should be understood that various changes in form and detail may be made by one of ordinary skill in the art without departing from the spirit and scope of the appended claims. All embodiments that come within the spirit and scope of the following claims and equivalents thereto are claimed.
The present application claims priority to U.S. provisional patent application No. 61/176,361 filed on May 7, 2009, which is incorporated herein by reference.
This invention was made with government support under PHY-0646094, DBI-0649865, and DMR-0820484 awarded by the National Science Foundation and under HR0011-06-1-0044 awarded by the U.S. Department of Defense. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US10/01372 | 5/7/2010 | WO | 00 | 7/11/2012 |
| Number | Date | Country | |
|---|---|---|---|
| 61176361 | May 2009 | US |
