WO2006125176A1 - Microsystem spectroscopy - Google Patents

Microsystem spectroscopy Download PDF

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Publication number
WO2006125176A1
WO2006125176A1 PCT/US2006/019533 US2006019533W WO2006125176A1 WO 2006125176 A1 WO2006125176 A1 WO 2006125176A1 US 2006019533 W US2006019533 W US 2006019533W WO 2006125176 A1 WO2006125176 A1 WO 2006125176A1
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WO
WIPO (PCT)
Prior art keywords
method
plurality
samples
microlenses
light beams
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PCT/US2006/019533
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French (fr)
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WO2006125176A8 (en
Inventor
Nicolae Damean
Samuel K. Sia
Vincent Linder
Max Narovlyansky
George M. Whitesides
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President And Fellows Of Harvard College
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Priority to US68245905P priority Critical
Priority to US60/682,459 priority
Application filed by President And Fellows Of Harvard College filed Critical President And Fellows Of Harvard College
Publication of WO2006125176A1 publication Critical patent/WO2006125176A1/en
Publication of WO2006125176A8 publication Critical patent/WO2006125176A8/en

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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using infra-red, visible or ultra-violet light
    • G01N21/17Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/255Details, e.g. use of specially adapted sources, lighting or optical systems
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01JMEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRA-RED, VISIBLE OR ULTRA-VIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/02Details
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01JMEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRA-RED, VISIBLE OR ULTRA-VIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/02Details
    • G01J3/0205Optical elements not provided otherwise, e.g. optical manifolds, diffusers, windows
    • G01J3/0208Optical elements not provided otherwise, e.g. optical manifolds, diffusers, windows using focussing or collimating elements, e.g. lenses or mirrors; performing aberration correction
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01JMEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRA-RED, VISIBLE OR ULTRA-VIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/02Details
    • G01J3/0256Compact construction
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using infra-red, visible or ultra-violet light
    • G01N21/01Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03Cuvette constructions
    • G01N2021/0346Capillary cells; Microcells
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using infra-red, visible or ultra-violet light
    • G01N21/01Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03Cuvette constructions
    • G01N21/05Flow-through cuvettes
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2201/00Features of devices classified in G01N21/00
    • G01N2201/06Illumination; Optics
    • G01N2201/063Illuminating optical parts
    • G01N2201/0638Refractive parts
    • GPHYSICS
    • G02OPTICS
    • G02BOPTICAL ELEMENTS, SYSTEMS, OR APPARATUS
    • G02B21/00Microscopes
    • G02B21/0004Microscopes specially adapted for specific applications
    • G02B21/002Scanning microscopes
    • G02B21/0024Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0032Optical details of illumination, e.g. light-sources, pinholes, beam splitters, slits, fibers

Abstract

The present invention generally relates to systems and methods for acquiring multiple spectra from a sample, for example, a microfluidic sample. In some aspects of the invention, spectra from a series of locations within a sample are recorded and analyzed, and optionally correlated with an image of the sample. The spectra can be obtained by passing a plurality of light beams through a sample, e.g., using an array of microlenses, diffracting the light beams passing through the sample with a transmission grating, before or after passing the light through the sample, and detecting the resulting light, e.g., as an image. The light beams may pass through the sample before or after passing through the transmission grating. The image can appear as an array of discrete spectra, where each spectrum is associated with a region of the sample where a light beam passed through the sample. The spectra can be analyzed to determine information about the region where the light beam passed through the sample. In some cases, the array of microlenses may be movable and/or replaceable with a second array of microlenses. Also, in some instances, the transmission grating may be movable and/or replaceable with a second transmission grating.

Description

MICROSYSTEM SPECTROSCOPY

FEDERALLY SPONSORED RESEARCH

Research leading to various aspects of the present invention were sponsored, at least in part, by DARPA and the NIH, Grant No. GM065364. The United States Government may have certain rights in the invention.

FIELD OF INVENTION

The present invention generally relates to spectroscopic techniques and, in particular, to systems and methods for acquiring multiple spectra from a sample, such as a microfluidic sample.

BACKGROUND

Microsystems are now ubiquitous in chemistry and biology. Applications of microsystems include the analysis of chemical reactions, sorting of cells, and high- throughput screening. Because these systems often require separation, mixing, or reactions of various components, they would benefit from methods that allow the components to be optically characterized. Advances in the miniaturization of spectrophotometric systems have produced a number of useful devices. However, these devices typically work at a single wavelength at any one time, or perform measurements at a single spatial location, and most cannot be easily interfaced with a microsystem. Thus, improvements in spectrophotometric design are needed.

SUMMARY OF THE INVENTION

The present invention generally relates to systems and methods for acquiring multiple spectra from a sample. The subject matter of the present invention involves, in some cases, interrelated products, alternative solutions to a particular problem, and/or a plurality of different uses of one or more systems and/or articles.

One aspect of the invention is generally directed to a method including acts of acquiring an image of one or more samples, acquiring diffraction spectra of a plurality of discrete locations of the one or more samples, and correlating the diffraction spectra and the image. The invention, in another aspect, includes acts of directing a plurality of isolated light beams through at least first and second distinct regions of one or more samples, and diffracting at least a portion of the isolated light beams passed through the one or more samples using a single transmission grating. Yet another aspect of the invention includes acts of passing a plurality of isolated light beams through at least first and second distinct regions of one or more samples, and diffracting at least a portion of the isolated light beams passed through one or more samples using a non-linear transmission grating. In still another aspect, the invention is directed to a method comprising acts of passing a first plurality of isolated light beams through at least first and second distinct regions of one or more samples, diffracting the isolated light beams using a first transmission grating, replacing the first transmission grating with a second transmission grating, passing a second plurality of isolated light beams through the one or more samples, and diffracting the isolated light beams using the second transmission grating. The method, according to yet another aspect, is directed to a method including acts of passing a first plurality of isolated light beams through at least first and second distinct regions of one or more samples, diffracting the isolated light beams using a transmission grating, moving the transmission grating, passing a second plurality of isolated light beams through the one or more samples, and diffracting the isolated light beams using the transmission grating.

The invention, according to another aspect, is directed to a method comprising acts of passing light through a first plurality of microlenses to produce a first plurality of isolated light beams, passing the first plurality of isolated light beams through at least first and second distinct regions of one or more samples, diffracting the first plurality of isolated light beams using a transmission grating, replacing the first plurality of microlenses with a second plurality of microlenses, passing light through the second plurality of microlenses to produce a second plurality of isolated light beams, passing the second plurality of isolated light beams through the one or more samples, and diffracting the second plurality of isolated light beams using the transmission grating. Still another aspect of the present invention includes an apparatus comprising a microscope comprising a plurality of microlenses and a diffraction grating, where the position of the diffraction grating, relative to the microlenses, is adjustable.

In another aspect, the present invention is directed to a method of making one or more of the embodiments described herein. In yet another aspect, the present invention is directed to a method of using one or more of the embodiments described herein. In still another aspect, the present invention is directed to a method of promoting one or more of the embodiments described herein.

Other advantages and novel features of the present invention will become apparent from the following detailed description of various non-limiting embodiments of the invention when considered in conjunction with the accompanying figures. In cases where the present specification and a document incorporated by reference include conflicting and/or inconsistent disclosure, the present specification shall control. If two or more documents incorporated by reference include conflicting and/or inconsistent disclosure with respect to each other, then the document having the later effective date shall control.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention. In the figures: Figs. 1 A-ID illustrate one embodiment of the invention;

Figs. 2A-2F are various diffraction patterns generated using certain embodiments of the invention;

Figs. 3A-3B are certain quantitative spectral data, generated using an embodiment of the invention; Figs. 4A-4C illustrate dyes used in another embodiment of the invention;

Figs. 5 A-5L illustrate the monitoring of dynamic events using another embodiment of the invention; Figs. 6A-6C illustrate data relating to another embodiment of the invention;

Figs. 7A-7D illustrate the calibration of yet another embodiment of the invention;

Figs. 8A-8B illustrate the determination of wavelength in another embodiment of the invention; Figs. 9A-9F illustrate an array of spectra, produced using a movable transmission grating, in one embodiment of the invention;

Figs. 1 OA- 1OC illustrate spectra producing using various transmission gratings, according to another embodiment of the invention;

Figs. 1 IA-11C illustrate various spectra producing using an array of microlenses, according to yet another embodiment of the invention;

Figs. 12A-12G illustrate still another embodiment of the invention that includes a non-linear transmission grating; and

Figs. 13A-13B are block diagrams illustrating examples of computer systems on which some embodiments of the invention may be implemented. DETAILED DESCRIPTION

The present invention generally relates to systems and methods for acquiring multiple spectra from a sample, for example, a microfluidic sample. In some aspects of the invention, spectra from a series of locations within a sample are recorded and analyzed, and optionally correlated with an image of the sample. The spectra can be obtained by passing a plurality of light beams through a sample, e.g., using an array of microlenses, diffracting the light beams passing through the sample with a transmission grating, before or after passing the light through the sample, and detecting the resulting light, e.g., as an image. The light beams may pass through the sample before or after passing through the transmission grating. The image can appear as an array of discrete spectra, where each spectrum is associated with a region of the sample where a light beam passed through the sample. The spectra can be analyzed to determine information about the region where the light beam passed through the sample. In some cases, the array of microlenses may be movable and/or replaceable with a second array of microlenses. Also, in some instances, the transmission grating may be movable and/or replaceable with a second transmission grating.

In certain aspects of the invention, one or more light beams are directed at a sample. As used herein, a "light beam" is defined as a substantially unidirectional stream of photons emitting from a light source, i.e., in the light beam, the photons define a cone of light. The cone of light may have an opening angle (defined as the angle between the center line of the cone and the outer edge) of less than about 45°, less than about 30°, less than about 25°, less than about 20°, less than about 15°, less than about 10°, less than about 5°, or less than about 2° (i.e., the cone appears to be nearly cylindrical). In many cases, the light beams described herein are isolated light beams. An "isolated" light beam is a light beam that is readily identifiable, and can be distinguished from other light beams, although some "leakage" or "blurring" may be evident, i.e., the isolated light beam may not necessarily have a precisely-defined edge, although the light beam is nevertheless distinguishable from other light beams, e.g., due to differences in intensity.

The term "light," as used herein, generally refers to electromagnetic radiation, having any suitable wavelength (or equivalently, frequency) that can be manipulated using the systems and method of the present invention, for example, using an optical component such as a lens. For instance, in some embodiments, the light may include wavelengths in the optical or visual range (for example, having a wavelength of between about 400 nm and about 700 nm), infrared wavelengths (for example, having a wavelength of between about 300 micrometers and 700 nm), ultraviolet wavelengths (for example, having a wavelength of between about 400 nm and about 10 nm), or the like. The light may have a single wavelength, or include a plurality of different wavelengths. For instance, in some cases, the light may have a range of wavelengths between about 350 nm and about 1000 nm, between about 300 micrometers and about 500 nm, between about 500 nm and about 1 nm, between about 400 nm and about 700 nm, between about 600 nm and about 1000 nm, between about 500 nm and about 50 nm, etc. A plurality of light beams may be produced, for instance, using a plurality of optical components, which may be arranged as an array. As used herein, the term "optical component," includes passive elements or devices that do not produce light, but rather, diffract, reflect, and/or refract light incident on the component. Non-limiting examples of optical components include filters, prisms, mirrors, a diffractive lenses, refractive lenses, reflective lens, a spherically-shaped lenses, non-spherically-shaped lenses, plano-convex-shaped lenses, polygonal convex-shaped lenses, waveguides, or optical fibers. Additionally, as used herein, the term "optical component" also includes active elements or devices that produce light including, for example, incandescent lights, fluorescent lights, halogen lights, lasers, or light-emitting diodes.

In certain embodiments, the optical component includes one or more lenses, i.e., a transparent or at least partially transparent object which refracts or changes the direction of light beams incident on and passing through it. As used herein, a "partially transparent object" is an object that allows at least a portion of any visible light incident on the object to be transmitted therethrough, for example, at least about 50% of the instant light may be transmitted therethrough, in other cases, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or substantially all of the instant light may be transmitted therethrough. In some cases, the lens may be characterized by an index of refraction, or a distribution of refractive indices. In some instances, the lens may be capable of refracting, or otherwise changing the direction of light incident on the lens, such that the light converges on or near a predetermined plane, location, or region. The distance between the lens and the predetermined location can be referred to as the "image distance." The image distance depends on, inter alia, the index of refraction of the lens, the medium through which the electromagnetic radiation travels around the lens, the size and/or shape of the lens, the wavelength(s) of the incident light, etc., and the image distance of a lens can be determined by those of ordinary skill in the art. In some cases, the optical components include one, or a plurality of microscale optical components, such as a plurality of microlenses, for example, arranged in an array. The term "microscale optical component," as used herein, refers to an optical component having at least one dimension on the micrometer scale (e.g., less than about 1 millimeter), and includes, for example, microlenses, micromirrors, microprisms, etc, where the prefix "micro-" generally denotes a dimension on the micrometer scale of the corresponding component. Other non-limiting examples of potentially suitable microscale optical components include microfilters, microoptical fibers, microwaveguides, or the like. The plurality of microscale optical components may be arranged in any suitable configuration, for example, in a hexagonal close-packed configuration, in a square or rectangular close-packed configuration, randomly, etc. In some cases, the plurality of microscale optical components may be movable or adjustable (e.g., without breaking or dissassembling a component or mechanism that the component is attached to); for example, the distance between the plurality of microscale optical components and the sample may be adjusted, e.g., during use of the optical components, for example, to bring the sample into focus. The plurality of microscale optical components may include the same or different types of optical components, for example, the plurality of microscale optical components may have a variety of shapes and/or a variety of spatial and/or optical arrangements. For example, the microscale optical component may comprise, in some embodiments, at least one spherically-shaped optical component.

The microscale optical components, in certain embodiments, may be configured to create a plurality of light beams. For instance, if the microscale optical components include one or more microlenses, the microlenses may transform light incident thereon into a plurality of light beams. In some cases, the plurality of light beams may be parallel or substantially parallel. In certain instances, the plurality of light beams may be focused on a specific plane, location, or region for example, on a sample, on another optical component (for example, a lens), a transmission grating, or the like. Optionally, the plurality of light beams may be passed through one or more optical components (including microscale optical components), for example, one or more lenses, prisms, etc., which may be used, for instance, to focus or direct the plurality of light beams at a sample or at a transmission grating, etc. For example, the plurality of light beams may be passed through a common lens, redirected using a mirror, etc.

Techniques for making and using microscale optical components, such as microlenses, are discussed in more detail in U.S. Patent Application Serial No. 10/384,080, filed March 7, 2003, entitled "Microlens for Projection Lithography and Method of Preparation Thereof," by Wu, et at, published as U.S. Patent Application Publication No. 2004/0027675 on February 12, 2004, incorporated herein by reference. For example, the microscale optical component may be fabricated from an at least partially transparent material. Some examples of potentially suitable materials include, but are not limited to, polymers such as polystyrene or polydimethylsiloxane.

The plurality of light beams may be directed at and/or passed through at least a portion of a sample, or a plurality of samples. In some embodiments, the sample may be used at least partially transparent, i.e., the sample allows at least a portion of the light instant on the sample to pass therethrough. The sample may be, for example, a chemical sample, a biological sample, a biochemical sample, or the like. In certain embodiments where a microscope is used as part of a light detector (as discussed in more detail below), the sample may be any sample able to fit on the microscope stage. In some cases, a microfiuidic system may be used to contain a sample. The microfluidic system may contain, for example, one or more samples that are able to pass through one or more channels in the microfluidic system. For example, the microfluidic system can contain at least one channel carrying a fluid, and the sample may be dissolved and/or suspended in the fluid. Microfluidic systems are discussed in more detail below.

In some embodiments, the sample, or at least a portion of the sample, is fluorescent, i.e., the sample is able to emit light in response to a stimulus (e.g., a light stimulus, a chemical stimulus, etc.). For example, the sample may contain an entity attached to a fluorescent moiety, such as a fluorescein, rhodamine, Texas red, calcein, Lucifer yellow, etc. that is able to emit light when stimulated. In other cases, the sample, or at least a portion of the sample, is a dye, i.e., the sample is able to absorb light in at least a portion of the spectra. For instance, the sample may contain an entity attached to a dye. Non-limiting examples of potentially suitable dyes include bromophenol blue, methylene blue, methyl orange, Janus green, safranin O, orange green, xylene cyanole, pyronin, etc. In other embodiments, however, the sample is able to generate light (for example, the sample may be chemiluminescent, or contain a source of energy that is converted into light), and the stimulus (e.g., a light source able to produce a plurality of light beams) may be omitted.

In some embodiments, multiple locations within the sample may be analyzed with respect to spectra (e.g., wavelength) and/or spatial position, in some cases simultaneously, for example, using a plurality of light beams. As a particular non- limiting example, the spectra at each of a series of discrete locations in a sample can be simultaneously determined and/or recorded, e.g., as shown in the image of Fig. 2B. Similarly, the spectra at each point along a portion of microfluidic channel (shown in Fig. 2C) can be simultaneously determined and/or recorded, as shown in the image of Fig. 2D. In some cases, the spectra at each location may appear one or more line segments positioned proximate the location, where, within the light segment, the position is correlated with the wavelength of the spectra. Thus, with reference to Figs. 2B and 2D, the spectra indicated as "+1" and "-1" appear as spectra of colors ranging from red to blue.

In some embodiments of the invention, the spectra determined for at least some of the locations may be correlated with a visual image of the sample. For instance, the spectral data of the micro fluidic channel, as shown in Fig. 2D may be correlated with a visual image of the microfluidic channel, as shown in Fig. 2C. Additionally, in some cases, a series of spectra and/or visual images may be determined and/or recorded with respect to time, for example, using a video camera.

In certain embodiments of the present invention, the plurality of light beams are directed through a transmission grating, e.g., before or after passing through at least a portion of the sample. In some cases, the plurality of light beams are directed through the transmission grating prior to detection. As used herein, a transmission grating is an optical component comprising a series of grooves on a reflecting or transparent substrate. In one embodiment, the transmission grating is a diffraction grating. The transmission grating may be fabricated from an at least partially transparent material, for example, glass or plastic, and such transmission gratings are commercially available, or can be custom-made in some cases. The transmission grating is flexible in some embodiments. In some cases, the transmission grating may be movable or adjustable; for example, the distance between the transmission grating and the sample may be adjusted. Typically, the dimensions and spacing of the grooves of the transmission grating are on the same order of magnitude of the light incident to the grating. The grooves can cause diffractive and/or interference effects that are able to concentrate the incident light in discrete directions, called "orders" or "spectral orders," to form various diffraction patterns (zeroth order generally corresponds to light passing through the transmission grating without being diffracted). For instance, a light beam diffracted by a transmission grating may be diffracted into zeroth order light, a +1 order, and/or a -1 order. Of course, in some cases, even higher orders of diffracted light may be produced, for example, +2 or +3 spectral orders. The +1 and -1 orders of light may appear as lines proximate the zeroth order light, and the directionality of the +1 and -1 orders of light may be controlled, in some cases, by controlling the orientation of the transmission grating.

There may be hundreds or thousands of grooves per millimeter in a transmission grating in some cases. For example, the transmission grating may have groove spacings of about 100 micrometers (corresponding to about 10 grooves/mm), about 50 micrometers (about 20 grooves/mm), about 20 micrometers (about 50 grooves/mm), about 10 micrometers (about 100 grooves/mm), about 5 micrometers (about 200 grooves/mm), about 2 micrometers (about 500 grooves/mm), about 1 micrometer (about 1000 grooves/mm), about 500 nm (about 2000 grooves/mm), about 200 nm (about 5000 grooves/mm), about 100 nm (about 10,000 grooves/mm), about 50 nm (about 20,000 grooves/mm), etc. In some cases, the grooves are straight lines, and such a transmission grating is a "linear transmission grating." In other cases, however the transmission grating may include one or more grooves that are not straight lines, for example, a "non- linear transmission grating," or a holographic diffraction grating.

A non-limiting example of a non-linear transmission grating is now described. With reference to Fig. 12 A, a microfluidic channel 120 is illustrated, having a non-linear shape. At several discrete locations 125 within the non-linear microfluidic channel, spectra are to be recorded, as is illustrated in Fig. 12B. An array of spherical microlenses corresponding to these locations is fabricated, and combined with a linear transmission grating, illustrated in Fig. 12C. The spectra corresponding to each of these locations is then determined, as is shown schematically in Fig. 12D. However, in other embodiments, by using a non-linear transmission grating, the spectra of entire regions of the microfluidic channel, rather than discrete locations, may be recorded. In Fig. 12E, a microlens is fabricated having a shape substantially similar to that of the microfluidic channel in Fig. 12 A. Fig. 12F shows the corresponding non-linear transmission grating. When the microlens and the non-linear transmission grating are used together, using the methods of the invention as described herein, the spectra at each point along the nonlinear microfluidic channel may be simultaneously determined and/or recorded. The plurality of light beams, after passing through the transmission grating, may be determined using a detector, for example, an optical detector or a ultraviolet detector. As used herein, the term "determining," when used with respect to light, generally refers to the analysis of light (e.g., at a particular wavelength or frequency), for instance, quantitatively or qualitatively, and/or the detection of the presence or absence of the light. The detector may be any detector able to collect at least a portion of any light incident on the detector and convert at least some of that light into electricity, e.g. to determine the light. Examples of detectors include, but are not limited to, cameras such - l i as CCD cameras or video cameras, photosensors, photomultipliers, photocells, photodiodes, or the like. Commercially-available detectors may be used in certain embodiments of the invention.

In one set of embodiments, the detector includes an microscope, such as an optical microscope. The microscope may be a simple microscope or a compound microscope, and the microscope may be commercially available in some cases. Those of ordinary skill in the art are aware of different types of microscopes that may be obtained, for example, a fluorescence microscope, a phase contrast microscope, a dark field microscope, a polarization microscope, etc. A typical microscope may include components such as a source of light (e.g., a light bulb, a mirror to collect ambient light, or the like), a condenser, a specimen holder (e.g., a stage), and an objective lens. The microscope typically will include other components as well (e.g., focus adjustment mechanisms, multiple objective lenses, projector lenses, eyepieces, etc.).

Certain systems and methods of the invention can be used with a microscope. In one embodiment, a microscope may be modified by introducing one or more optical components, such as an array of microlenses and/or a transmission grating, to the microscope. For instance, the condenser of a microscope may be replaced by an array of microlenses. In some cases, the optical components may be added, modified, and/or removed from the microscope as necessary, in some cases even while one or more samples are present on the microscope. For example, after determining the sample, the array of microlenses may be moved and/or rotated, and/or replaced by a second array of microlenses; the transmission grating may be moved and/or rotated, and/or replaced by a second transmission grating; or the like.

It should be understood that a variety of systems and methods are contemplated within the scope of the instant invention, in addition to the embodiments described above. For example, in one embodiment, light arising from one or more light sources may pass through one or more samples, then through one or more microlenses or other optical components, then through one or more transmission gratings, before being detected. In another embodiment, light arising from one or more light sources may pass through one or more microlenses or other optical components, then through one or more samples, then through one or more transmission gratings, before being detected. In yet another embodiment, light arising from one or more light sources may pass through one or more microlenses or other optical components, then through one or more transmission gratings, then through one or more samples, before being detected. Other variations will be known to one of ordinary skill in the art, for example, involving more than one set of microlenses or other optical components, transmission gratings, etc. If a microfluidic system is used, the microfiuidic system may include one or more channels. As used herein, a "channel" of a microfluidic system is a conduit associated with the microfluidic system that is able to transport one or more fluids from one location to another. Fluids may flow through the channels continuously, randomly, intermittently, etc., and the fluids may contain one or more samples therein. The channel may be a closed channel, or a channel that is open, for example, open to the external environment surrounding the microfluidic system. The channel can include characteristics that facilitate control over fluid transport within the channel, e.g., structural characteristics (e.g., shape), and or physical/chemical characteristics (e.g., hydrophobicity vs. hydrophilicity). The fluid within the channel may partially or completely fill the channel. In some cases the fluid may be held or confined within the channel or a portion of the channel in some fashion, for example, using surface tension (i.e., such that the fluid is held within the channel within a meniscus, such as a concave or convex meniscus). The channel may have any suitable cross-sectional shape that allows for fluid transport, for example, a square channel, a circular channel, a rounded channel, a rectangular channel (e.g., having any aspect ratio), a triangular channel, an irregular channel, etc. The channel may be of any size within the microfluidic system. For example, the channel may have a largest dimension perpendicular to a direction of fluid flow within the channel of less than about 1000 micrometers in some cases, less than about 500 micrometers in other cases, less than about 400 micrometers in other cases, less than about 300 micrometers in other cases, less than about 200 micrometers in still other cases, less than about 100 micrometers in still other cases, or less than about 50 or 25 micrometers in still other cases. The dimensions of the channel may be chosen in certain cases, for example, to allow a certain volumetric or linear flowrate of fluid within the channel. Of course, the number of channels, the shape or geometry of the channels, and the placement of channels within the microfluidic system can be determined by those of ordinary skill in the art. A variety of materials and methods can be used to form the microfiuidic system. For example, the microfiuidic system may be formed from solid materials, in which the channels can be formed via micromachining, film deposition processes such as spin coating and chemical vapor deposition, laser fabrication, photolithographic techniques, etching methods including wet chemical or plasma processes, and the like. See, for example, Scientific American, 248:44-55, 1983 (Angell, et al). In one example, at least a portion of the microfiuidic system is formed of silicon by etching features in a silicon chip. Technologies for precise and efficient fabrication of various microfiuidic systems from silicon are known. In another example, various components of the systems and devices of the invention can be formed of a polymer, for example, an elastomeric polymer such as polydimethylsiloxane ("PDMS"), polytetrafiuoroethylene ("PTFE" or Teflon®), or the like.

Various components of the microfiuidic system may be fabricated from polymeric, flexible and/or elastomeric materials, and can be conveniently formed of a hardenable fluid in some cases, facilitating fabrication via molding (e.g. replica molding, injection molding, cast molding, etc.). The hardenable fluid can be essentially any fluid that can be induced to solidify, or that spontaneously solidifies, into a solid. In one example, the hardenable fluid comprises a polymeric liquid or a liquid polymeric precursor (i.e. a "prepolymer"). Suitable polymeric liquids can include, for example, thermoplastic polymers, thermoset polymers, or mixtures of such polymers heated above their melting point. As another example, a suitable polymeric liquid may include a solution of one or more polymers in a suitable solvent, which solution forms a solid polymeric material upon removal of the solvent, for example, by evaporation. Such polymeric materials, which can be solidified from, for example, a melt state or by solvent evaporation, are well known to those of ordinary skill in the art. A variety of polymeric materials, many of which are elastomeric, are suitable, and are also suitable for forming molds or mold masters, for systems where one or both of the mold masters is composed of an elastomeric material. A non-limiting list of examples of such polymers includes polymers of the general classes of silicone polymers, epoxy polymers, and acrylate polymers. Epoxy polymers are characterized by the presence of a three-membered cyclic ether group commonly referred to as an epoxy group, 1,2-epoxide, or oxirane. For example, diglycidyl ethers of bisphenol A can be used, in addition to compounds based on aromatic amine, triazine, and cycloaliphatic backbones. Another example includes the well-known Novolac polymers. Non-limiting examples of silicone elastomers suitable for use in the microfluidic system include those formed from precursors including the chlorosilanes such as methylchlorosilanes, ethylchlorosilanes, phenylchlorosilanes, etc. Silicone polymers may be used in some cases, for example, the silicone elastomer polydimethylsiloxane (PDMS). Non-limiting examples of PDMS polymers include those sold under the trademark Sylgard by Dow Chemical Co., Midland, MI, for example Sylgard 182, Sylgard 184, Sylgard 186, etc. Also, silicone polymers, such as PDMS, can be elastomeric and thus may be useful for forming very small features with relatively high aspect ratios. Flexible (e.g., elastomeric) molds or masters can be advantageous in this regard.

The methods described herein may be computer implemented in some aspects of the invention. Software and data (for example, image data, spectral data, experimental data, etc.) can be stored on computer readable media and can be executed or accessed at a later time. Software can be used, for example, to obtain data, store data, organize data, correlate data and/or to provide information.

Some of the methods described herein and various embodiments and variations of the methods and acts, individually or in combination, may be defined by computer- readable signals tangibly embodied on or more computer-readable media, for example, non-volatile recording media, integrated circuit memory elements, or a combination thereof. Computer readable media can be any available media that can be accessed by a computer. By way of example, and not limitation, computer readable media may comprise computer storage media and communication media. Computer storage media includes volatile and nonvolatile, removable and non-removable media implemented in any method or technology for storage of information such as computer readable instructions, data structures, program modules or other data. Computer storage media includes, but is not limited to, RAM, ROM, EEPROM, flash memory or other memory technology, CD-ROM, digital versatile disks (DVD), optical storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, other types of volatile and non- volatile memory, any other medium which can be used to store the desired information and which can accessed by a computer, and any suitable combination of the foregoing. Communication media typically embodies computer- readable instructions, data structures, program modules or other data in a modulated data signal such as a carrier wave or other transport mechanism and includes any information delivery media. The term "modulated data signal" means a signal that has one or more of its characteristics set or changed in such a manner as to encode information in the signal. By way of example, and not limitation, communication media includes wired media such as a wired network or direct-wired connection, wireless media such as acoustic, RP, infrared and other wireless media, other types of communication media, and any suitable combination of the foregoing.

Computer-readable signals embodied on one or more computer-readable media may define instructions, for example, as part of one or more programs, that, as a result of being executed by a computer, instruct the computer to perform one or more of the functions described herein, and/or various embodiments, variations and combinations thereof. Such instructions may be written in any of a plurality of programming languages, for example, Java, Visual Basic, C, C#, or C++, Fortran, Pascal, Lisp, Java, Perl, Eiffel, Basic, COBOL, etc., or any of a variety of combinations thereof. The computer-readable media on which such instructions are embodied may reside on one or more of the components of any of systems described herein or known to those skilled in the art, may be distributed across one or more of such components, and may be in transition therebetween. The computer-readable media may be transportable such that the instructions stored thereon can be loaded onto any computer system resource to implement the aspects of the present invention discussed herein. In addition, it should be appreciated that the instructions stored on the computer-readable medium, described above, are not limited to instructions embodied as part of an application program running on a host computer. Rather, the instructions may be embodied as any type of computer code (e.g., software or microcode) that can be employed to program a processor to implement the above-discussed aspects of the present invention.

It should be appreciated that any single component or collection of multiple components of a computer system, for example, the computer system described in relation to Figs. 13 A- 13 B, that perform the functions described herein can be generically considered as one or more controllers that control such functions. The controllers can be implemented in numerous ways, such as with dedicated hardware and/or firmware, using a processor that is programmed using microcode or software to perform the functions recited above, etc. or any suitable combination of the foregoing.

Further, on each of the devices that include one or more components of the systems, each of the components may reside in one or more locations on the system. For example, different portions of the components of these systems may reside in different areas of memory (e.g., RAM, ROM, disk, etc.) on the device. Each of such devices may include, among other components, a plurality of known components such as one or more processors, a memory system, a disk storage system, one or more network interfaces, one or more buses or other internal communication links interconnecting the various components, etc. The systems, and components thereof, may be implemented using a computer system, such as that described below in relation to Figs. 13A-13B.

Various embodiments according to the invention may be implemented on one or more computer systems. These computer systems, may be, for example, general-purpose computers such as those based on Intel PENTIUM-type and XScale-type processors, Motorola PowerPC, Motorola DragonBall, IBM HPC, Sun UltraSPARC, Hewlett- Packard PA-RISC processors, any of a variety of processors available from Advanced Micro Devices (AMD), or any other type of processor. It should be appreciated that one or more of any type of computer system may be used to implement various embodiments of the invention. A general-purpose computer system according to one embodiment of the invention is configured to perform any of the functions described above. It should be appreciated that the system may perform other functions and the invention is not limited to having any particular function or set of functions.

For example, various aspects of the invention may be implemented as specialized software executing in a general -purpose computer system 1000 such as that shown in Figure 3. The computer system 1000 may include a processor 1003 connected to one or more memory devices 1004, such as a disk drive, memory, or other device for storing data. Memory 1004 is typically used for storing programs and/or data during operation of the computer system 1000. Components of computer system 1000 may be coupled by an interconnection mechanism 1005, which may include one or more buses (e.g., between components that are integrated within a same machine) and/or a network (e.g., between components that reside on separate discrete machines). The interconnection mechanism 1005 enables communications (e.g., data, instructions) to be exchanged between system components of system 1000. Computer system 1000 also includes one or more input devices 1002, for example, a keyboard, mouse, trackball, microphone, touch screen, etc. and one or more output devices 1001, for example, a printing device, a display screen, a speaker, etc. In addition, computer system 1000 may contain one or more interfaces (not shown) that connect computer system 1000 to a communication network (in addition or as an alternative to the interconnection mechanism 1005.

The storage system 1006, shown in greater detail in Fig. 13B, typically includes a computer readable and/or writeable nonvolatile recording medium 1101 in which signals are stored that define a program to be executed by the processor or information stored on or in the medium 1101 to be processed by the program. The medium may, for example, be a disk or flash memory. Typically, in operation, the processor causes data to be read from the nonvolatile recording medium 1101, into another memory 1102 that allows for faster access to the information by the processor than does the medium 1101. This memory 1102 is typically a volatile, random access memory such as a dynamic random access memory (DRAM) or static memory (SRAM). It may be located in storage system 1006, as shown, or in memory system 1004, not shown. The processor 1003 generally manipulates the data within the integrated circuit memory 1004, 1102 and then copies the data to the medium 1101 after processing is completed. A variety of mechanisms are known for managing data movement between the medium 1101 and the integrated circuit memory element 1004, 1102, and the invention is not limited thereto. The invention is not limited to a particular memory system 1004 or storage system 1006.

The computer system may include specially-programmed, special-purpose hardware, for example, an application-specific integrated circuit (ASIC). Aspects of the invention may be implemented in software, hardware or firmware, or any combination thereof. Further, such methods, acts, systems, system elements and components thereof may be implemented as part of the computer system described above or as an independent component.

Although computer system 1000 is shown by way of example as one type of computer system upon which various aspects of the invention may be practiced, it should be appreciated that aspects of the invention are not limited to being implemented on the computer system as shown in Fig. 13. Various aspects of the invention may be practiced on one or more computers having a different architecture or components that that shown in Fig. 13.

Computer system 1000 may be a general-purpose computer system that is programmable using a high-level computer programming language. Computer system 1000 may be also implemented using specially programmed, special purpose hardware. In computer system 1000, processor 1003 is typically a commercially available processor such as the well-known Pentium class processor available from the Intel Corporation. Many other processors are available. Such a processor usually executes an operating system which may be, for example, the Windows® 95, Windows® 98, Windows NT®, Windows® 2000 (Windows® ME), Windows® XP, Windows CE® or Pocket PC® operating systems available from the Microsoft Corporation, MAC OS System X available from Apple Computer, the Solaris Operating System available from Sun Microsystems, Linux available from various sources, UNIX available from various sources, or Palm OS® available from Palmsource, Inc. Many other operating systems may be used.

The processor and operating system together define a computer platform for which application programs in high-level programming languages are written. It should be understood that the invention is not limited to a particular computer system platform, processor, operating system, or network. Also, it should be apparent to those skilled in the art that the present invention is not limited to a specific programming language or computer system. Further, it should be appreciated that other appropriate programming languages and other appropriate computer systems could also be used.

One or more portions of the computer system may be distributed across one or more computer systems (not shown) coupled to a communications network. These computer systems also may be general-purpose computer systems. For example, various aspects of the invention may be distributed among one or more computer systems configured to provide a service (e.g., servers) to one or more client computers, or to perform an overall task as part of a distributed system. For example, various aspects of the invention may be performed on a client-server system that includes components distributed among one or more server systems that perform various functions according to various embodiments of the invention. These components may be executable, intermediate (e.g., IL) or interpreted (e.g., Java) code which communicate over a communication network (e.g., the Internet) using a communication protocol (e.g.,

TCP/IP).

It should be appreciated that the invention is not limited to executing on any particular system or group of systems. Also, it should be appreciated that the invention is not limited to any particular distributed architecture, network, or communication protocol.

Various embodiments of the present invention may be programmed using an object-oriented programming language, such as SmallTalk, Java, C++, Ada, or C# (C-

Sharp). Other object-oriented programming languages may also be used. Alternatively, functional, scripting, and/or logical programming languages may be used. Various aspects of the invention may be implemented in a non-programmed environment (e.g., documents created in HTML, XML or other format that, when viewed in a window of a browser program, render aspects of a graphical-user interface (GUI) or perform other functions). Various aspects of the invention may be implemented as programmed or non-programmed elements, or any combination thereof. Further, various embodiments of the invention may be implemented using Microsoft.NET technology available from

Microsoft Corporation.

The following are incorporated herein by reference: U.S. Patent Application

Serial No. 10/384,080, filed March 7, 2003, entitled "Microlens for Projection Lithography and Method of Preparation Thereof," by Wu, et ah, published as U.S. Patent

Application Publication No. 2004/0027675 on February 12, 2004; U.S. Patent

Application Serial No. 10/120,847, filed April 10, 2002, entitled "Microlens for

Projection Lithography and Method of Preparation Thereof," by Wu, et a ; U.S.

Provisional Patent Application Serial No. 60/283,102, filed April 10, 2001, entitled "Microlens for Projection Lithography and Method of Preparation Thereof," by Wu, et ah ; and U.S. Provisional Patent Application Serial No. 60/682,459, filed May 19, 2005, entitled "Microsystem Spectroscopy," by Damean, et ah

The following examples are intended to illustrate certain embodiments of the present invention, but do not exemplify the full scope of the invention. EXAMPLE 1

This example describes an optical method that resolves wavelength, time, and/or location simultaneously for samples in a microsystem. In this technique, continuous spectra of wavelengths are analyzed at multiple positions in the field of view of a microscope. This example thus demonstrates a new and flexible method for analyzing the composition of samples at a number of locations, and/or in a number of samples, simultaneously and/or continuously. This particular example combines an array of convex microlenses, which concentrate light on specific locations in a sample, with a transmission grating, which disperses the light that leaves the sample into a continuous spectrum. Fig. IA shows a schematic diagram and photograph of the setup. An array of microlenses, with diameters of about 50 micrometers, was fabricated in an opaque background by reflowing photoresist (with an index of refraction of 1.59), followed by electroplating of nickel around the microlenses. The sample chamber was constructed from poly(dimethylsiloxane) (PDMS), which is optically transparent, using known soft lithography techniques. A commercially available transmission grating, made of an index-matched epoxy on glass, in which the pitch (spacing) was sufficiently small (about 11 micrometers) to disperse different wavelengths of visible light into resolvable spatial positions, was also used. Further details are shown below.

An optical microscope served both as the light source and the detector in these experiments. A tungsten-halogen lamp acted as the light source, and a black-and-white charge-coupled device (CCD) camera, which typically has higher sensitivity than color cameras, captured the image containing the spectra. The time resolution of this experiment was determined by the acquisition time of the camera, which was 1 to 10 milliseconds, depending on the level of illumination of the light source. In this optical setup, the condenser of the microscope was removed from the light path, and microlenses were used as mini-condensers. By focusing the objective lens near the microlenses, the diffraction spectrum could be captured in the primary image plane. The diffraction spectrum is normally observed only at the rear focal plane (and its conjugate planes) (Fig. IB).

In Fig. IB, an illumination pathway of the optical setup is illustrated that leads to facile imaging of the diffraction spectrum using a brightfield microscope. In a regular arrangement for observing diffraction in a microscope, the objective lens is focused on a transmission grating; as a result, an image of the grating forms at the primary image plane and is observed via the eyepiece, and an image of the diffracted light forms at the rear focal plane of the objective lens, observable by using a Bertrand lens. In this example, however, by focusing the objective lens near the microlenses rather than the transmission grating, the optical plane of the condenser iris, which is conjugate to the rear focal plane, is nearly co-planar with the specimen plane, which is conjugate to the primary image plane. As a result, the image of the diffracted light, which is normally observed only in the rear focal plane, also forms on the primary image plane, and can be captured by a camera.

This system is able to produce magnified and high-quality images of the diffracted light that are suitable for data processing, and it can function using a brightfield microscope with no requirements for special optics. By analyzing the intensity at each pixel of the image of the first-order diffracted light, and converting the pixel number to wavelength, the absorbance of the sample vs. wavelength in the visible spectrum could be obtained. In this system, the focal distance of the microlenses was determined to be about 179 micrometers. The lenses helped to concentrate light into the sample when placed directly above the lenses. In addition, because the transmission grating was about 3 mm above the sample, well past the focal point of the microlenses, the microlenses helped to spread the light at the grating to illuminate a large number of grooves and hence increase the resolution of the diffracted light.

The spectral resolving power of the system was characterized using two lasers for the light source, rather than broad-spectrum light from the microscope. Using the formula of spectral resolving power R = λ / Δλ, where Δλ denotes the full- width half- maximum at a particular wavelength λ, R was determined to be about 21 (Figs. 1C and ID), which is comparable to the spectral resolving power of other integrated optical microspectrometers. These figures show the measurement of spectral resolution using two lasers of 532 nm (-1) and 635 nm (+1). The system also included a straight cylindrical microlens, with the transmission grating positioned such that the grooves are aligned parallel to the long axis of the microlens. In the optical image of the diffraction spectrum (Fig. 1C), numbers correspond to the diffracted orders. In the processed spectrum of intensity vs. pixel number (Fig. ID), the arrows point to the full-width half- maxima that were measured as Δλ, and used to calculate R.

Fabrication of the array of microlenses proceeded as follows. Glass substrates were cleaned by sonication in acetone and methanol for 5 min. A thin layer of titanium (2 nm) followed by gold (10 nm) was deposited by e-beam evaporation on the glass slides. A thin layer (~4 micrometers) of Microposit 1818 photoresist (Shipley Co. Inc., Marlborough, MA, U.S.A.) was spin-coated at 2000 rpm for 40 s on the gold-coated slides, and baked on a hotplate at 105 0C for 3.5 min. The photoresist was exposed through a transparency mask (patterned with 50 micrometer circles for the fabrication of spherical microlenses, or 50 x 500 micrometer rectangles for the fabrication of cylindrical microlenses, as further discussed below) for 12 s in a Karl Suss mask aligner with a UV-light source (365 nm-405 nm). Posts of photoresist were obtained after development in Microposit 351 developer (Shipley Co. Inc., Marlborough, MA, U.S.A.) diluted 5:1 with MQ water. In a reflow procedure, the slides were heated at 150 0C for 30 min to melt the photoresist to form lenses (the refractive index of the photoresist is 1.59). Approximately 100 nm of nickel was deposited onto the exposed gold area for ~2 min at a current density of 10 niA/cm2 in a warm (~45 0C) nickel sulfamate electroplating bath. The apparatus was prepared as follows. The transmission grating employed (92 grooves/mm; Edmund Industrial Optics, Barrington, NJ, U.S.A.) is commonly used for laser beam division and multiple laser line separation in visible wavelengths; it diffracts the beam into multiple orders: -1, 0, and +1. According to the manufacturer's specifications, the distribution of light at 632 nm through this grating was 45% for 0 order, and 20% for -1 and +1 orders.

The array of microlenses was placed on the stage of a Leica DMRX optical microscope so that the microlenses are facing up. The microscope was operated in the transmission mode using a L 4Ox / 0.60 D Plan ∞/0 objective lens (Leitz Wetzlar). For measuring the absorbance of various dilutions of bromophenol blue, the transmission grating was placed above and in contact with the array of microlenses, and the dye samples were placed between the light source of the microscope and the microlenses (this arrangement was appropriate because the spectra of specific locations in the sample were not analyzed in this example). For analyzing samples in micro wells or microchannels, the PDMS sample chamber was placed on the array of microlenses, and the transmission grating above the sample chamber.

The microscope was connected to a black-and-white CCD camera (Hamamatsu ORCAER, Hamamatsu Photonics K.K., Japan). An acquisition time of 1 to 10 milliseconds was used, depending on the level of illumination. The images collected by this camera were analyzed with the MetaMorph software package (Universal Imaging Corp., Downingtown, PA, U.S.A.). For clarity of visualization, images were also captured using a color camera (Nikon Digital Camera DXM1200, Japan). As a reference for spectra measurements, a benchtop spectrophotometer was used

(HP 8453, Hewlett-Packard, Wilmington, DE, U.S.A.). A green laser diode (532 nm) and a red laser diode (635 nm) (Edmund Industrial Optics, Barrington, NJ, U.S.A.) were used as concurrent light sources. A glass slide containing the cylindrical microlens was placed onto the stage of the microscope, on top of which was placed a transmission grating with the grooves oriented along the long axis of the microlenses. The focus of the objective lens was changed until a clear image of the diffraction spectra was observed, and an image of the spectra was captured with the black-and-white CCD camera.

EXAMPLE 2 In this example, various diffraction spectra were analyzed using the apparatus of

Example 1. These diffraction spectra resulted from a number of different patterns and geometries of microlenses. An array of spherical microlenses, combined with a transmission grating, generated an array of discrete spots (corresponding to the zeroth order of diffraction, or light that passed straight through the grating) with two accompanying diffraction spectra (corresponding to the +1 and -1 orders of diffracted light) (Figs. 2A and 2B). The grating used in this study did not efficiently produce higher orders of diffracted light, and therefore avoided overcrowding of diffraction spectra. It was found that blue light diffracted to a lesser extent than red light, with an observed linear dispersion of 0.4 micrometers/nm in the first-order diffracted light. Overall, the pattern of diffracted light demonstrated that an array of spherical microlenses can be used to obtain visible spectra at multiple discrete locations of a sample in a microsystem, such as microwells and microarrays, and for analyzing the same sample at multiple locations for signal averaging.

The pattern of diffracted light that was generated by a cylindrical microlens and a transmission grating showed that the +1 and -1 orders of diffracted light were manifested as continuous lines (Figs. 2C and 2D). Moreover, for a cylindrical microlens that turned at a right angle, the grating could be oriented at 45° to each segment of the microlens to produce a continuous set of spectra for both segments (Figs. 2E and 2F). Thus, the pattern of diffracted light suggested that cylindrical microlenses can be useful for measuring a continuous set of spectra for samples in microfiuidic systems with a variety of geometries of microchannels. Now referring to Fig. 2 in more detail, Fig. 2A is an optical micrograph of an array of spherical microlenses (50 micrometers in diameter, spaced at 100 micrometer pitch). The inset shows a schematic of a transmission grating (92 grooves/mm; the grooves are not drawn to scale for clarity. Fig. 2B is a recorded image of the diffraction spectra. These diagrams show the orientation of the transmission grating with respect to the microlens. For clarity of visualization, a color CCD camera was used to image the spectra, and no sample chamber was used (such that the transmission grating lay directly on top of the microlenses). Three orders of diffraction (0, +1, and -1) were visible for each microlens. The image was taken with an objective lens of 2Ox magnification. The scale bar represents 50 micrometers. Figs. 2C and 2D are similar to Figs. 2A and 2B, but with a straight cylindrical microlens. Figs. 2E and 2F are also similar to Figs. 2A and 2B, but with a cylindrical microlens with a right-angle turn. The images in Fig. 2C and Fig. 2E were taken with an objective lens of 4Ox magnification. The scale bar represents 25 micrometers.

EXAMPLE 3 In this example, quantitative absorbance values of the sample were extracted over a range of visible wavelengths by analyzing the first-order diffracted light. To measure the absorbance spectrum of a given location of the sample in the field of view, the pixel intensities were measured along a line placed orthogonal to the corresponding diffraction spectrum, and converted to absorbance values as a function of wavelength. For these experiments, a dye in a quartz cuvette served as the sample. Using bromophenol blue dye as a standard for calibration (see below for details), the pixels of the camera image were converted to wavelength in nm. A resolution of 1.2 nm per pixel, using a 4Ox objective lens, was obtained over the range of wavelengths from 450 nm to 700 nm. The wavelength spectrum of bromophenol blue obtained from this analysis, with an absorption maximum of 595 nm, agreed closely with that measured by a benchtop spectrophotometer, with an absorption 6 maximum of 599 nm), and an average deviation of 0.02 absorbance units over the full range of wavelengths (Fig. 3 A, which shows a comparison of wavelength spectra of 6.0 mM bromophenol blue as measured using an embodiment of the invention and a benchtop spectrophotometer).

In these experiments, a quartz cuvette with a 100 micrometer path length was used to hold a sample of bromophenol blue (6.0 mM), and was placed above the transmission grating. The sample is normally placed between the microlenses and the grating; this arrangement allowed for precise alignment of the microlenses to specific locations of the sample, and thus was useful for the analysis of samples in micro wells or microchannels where such spatial control is needed. For purposes of calibration, where samples (such as optical filters) are homogeneous across the entire field of view, the sample can be placed between the light source and the microlenses, or between the grating and the objective lens. A black-and-white CCD camera, for maximum sensitivity, was used to capture the images of the diffraction spectra. Water in the quartz cuvette was used as a blank, and the absorbance values were calculated by taking the logarithm of the ratio of transmissions of the blank and sample. The average difference between these two sets of data was 0.02 absorbance units, and the absorbance maximum was found to be at 595 nm, compared to 599 nm using the benchtop spectrophotometer.

At the absorption maximum, these experiments for different concentrations of bromophenol blue (from 0.4 mM to 6.0 mM) yielded absorbance values that closely matched those obtained by the benchtop spectrophotometer over a range of dilutions of bromophenol blue, with an average deviation between the two methods of 0.03 absorbance units (Fig. 3B). The absorbance values were measured at 595 nm, the absorption maximum of bromophenol blue. The dashed line corresponds to an ideal correlation, in the case of both instruments yielding the same absorbance value for every dilution. The spectral measurements of dilutions of bromophenol blue were as follows.

Dilutions of bromophenol blue (Sigma-Aldrich, Inc., St. Louis, MO, U.S.A.) were made in MQ water. The following concentrations of bromophenol blue were used: 0.4 mM, 0.8 mM, 1.5 mM, 3.0 mM and 6.0 mM. A 100 micrometer quartz cell (Starna Cells, Inc., Atascadero, CA, U.S.A.) was used as the sample holder. For each dilution, two measurements were made: one with a benchtop spectrophotometer, and one with the apparatus as used in this example. For each dilution, the CCD camera was used to capture an image of the spectra. An image was also taken for a sample consisting of MQ water, which corresponded to a blank measurement. To reduce the noise in these images caused by scratches and dust on the CCD camera and on the lenses of the microscope, an image of concentrated black ink was collected to serve as a background for all other measurements. The images collected for each dilution were similar to the image shown in Fig.

6 A, which shows images of the first-order diffracted light as recorded by a black-and- white CCD camera for two concentrations of bromophenol blue. The images were processed with MetaMorph software package, and analyzed the intensity of light along a line that scanned along one spectrum (see Fig. 7). This line was placed in the same location for all images to ensure proper comparison. The intensity of the transmitted light of each pixel along the scanning line was measured for each image. After subtraction of the intensity values of the transmitted light corresponding to the black ink (denoted as Ib) from the intensity values of the transmitted light of the sample (denoted as I) and MQ water (denoted as Io), the results displayed in Fig. 6B were obtained. The plots in Fig. 6B show the intensity of the transmitted light vs. pixel number along the line scan for MQ water, for 3.0 mM and 6.0 mM of bromophenol blue. The error bars for each pixel (from successive readings of the light) were less than 20 a.u. (a.u. for arbitrary units).

The absorbance of the various bromophenol blue dilutions was calculated for each pixel as -log((/-/έ)/(/ø-/i)). The results are displayed in Fig. 6C as absorbance vs. pixel number (with an absorbance maximum at pixel #150 in this image). The error in absorbance for each pixel number were less than 0.01. To convert the pixel number to wavelength in run, the line spectrum of a calibration standard was measured, rhodamine suspended in cured PDMS (poly(dimethylsiloxane), Sylgard 184, Dow Corning, Midland, MI, U.S.A.). Fig. 7 presents the spectrum in the absence and presence of the calibration standard, and the line scan. The absorbance spectrum of the standard measured was matched to that measured by the benchtop spectrophotometer using two well-defined wavelengths: at 549 nm where the transmission curve reaches a minimum, and at 608 nm where this curve changes its curvature and approaches a straight line (Fig. 8). In this manner, the pixel number was paired to a specific wavelength, as: 549 nm — > pixel #112 and 608 nm — > pixel #161. These results gave the correspondence of 450 nm-700 nm to the pixel range 30-237 on the line scan, with one pixel corresponding to 1.2 nm. From Fig. 6C, the absorbance maximum of bromophenol blue was measured to be 595 nm, whereas the benchtop spectrophotometer showed that the absorbance maximum was 599 nm. Thus, the absorbance maximum was resolved to an error of 4 nm, or about 3 pixels. Other methods of calibration, including the use of lasers and optical filters as described elsewhere gave similar results (data not shown). The absorbances at 450 nm to 700 nm for different dilutions of bromophenol blue were calculated from the spectral images (Fig. 6A). Over this range of wavelengths, the absorbance values showed good agreement with those obtained by a benchtop spectrophotometer, with the average difference between these two sets of data being 0.02 a.u. for a sample of 6 mM of bromophenol blue. The absorbance maximum of bromophenol blue was measured to be 595 nm, in close agreement with 599 nm determined using the benchtop spectrophotometer.

Fig. 7A is an image of the zeroth-order and first-order diffracted light, taken with a color camera, with no sample, and the line that is used to scan the intensity of the wavelength spectrum. Fig. 7B is an image of the zeroth order and first-order diffracted light with the calibration standard as sample. Figs. 7C and 7D correspond to the conditions of Figs. 7A and 7B, respectively, but the images are taken with a black-and- white CCD camera and are used for data processing. Note that the first-order diffracted light and the zeroth-order light shown in each image do not correspond to the same microlens, but the same sample is placed over all the microlenses.

Fig. 8 uses the calibration standard shown in Fig. 7. Fig. 8A is a transmission spectrum of the calibration standard obtained with a benchtop spectrophotometer. The error bars were less than 2% for all measured values. Fig. 8B is the illumination intensity vs. pixel number obtained from images collected by the black-and- white CCD camera) for the calibration standard. The average error from all measurements was 3%. The vertical lines show the assignments of pixel numbers to wavelengths in nm that were subsequently used for the processing of images of samples.

EXAMPLE 4 To demonstrate the use of an embodiment of the invention in a microsystem, in this example, the wavelength spectra of different dyes were measured in a set of microwells by analyzing their diffracted light. In this experiment, a set of cylindrical microlenses were used (Fig. 4A, which shows an optical micrograph of an array of cylindrical microlenses, and a schematic of transmission grating with its orientation with respect to the microlenses (inset)). Four 90 micrometer deep microwells were fabricated in a poly(dimethylsiloxane) microchip connected by a system of microchannels.

Each microchannel has a 100 micrometer wide section that acted as a microwell, and two 50 micrometer wide sections which connect to the inlets and outlets. Four 50- micrometers wide cylindrical microlenses were aligned to the microwells by slowly moving the glass plate containing the microlenses. As described previously, the images were recorded with a black-and-white CCD camera and analyzed to produce spectra of illumination vs. pixel number for each well. This data were processed as described in the previous section to produce absorbance spectra. For clarity of visualization, color pictures were also taken.

Three different dyes and a water sample as a blank were added (Fig. 4B) to the microwells. For the four microwells, from left to right, the dyes were bromophenol blue (BPB), orange green (OG), Janus green (JG) (Sigma-Aldrich, Inc., St. Louis, MO, U.S.A.), and water (H2O) as a blank. These were saturated solutions dissolved in phosphate-buffered saline (Sigma-Aldrich, Inc., St. Louis, MO, U.S.A.).

After aligning the microlenses to the microwells, and placing the grating on the sample chamber so that the grooves are parallel to the long axis of the microlenses (Fig. 4A), four diffraction spectra were recorded on one image using a black-and-white CCD camera. For clarity of visualization, images of the diffraction spectra were also captured using a color CCD camera. Fig. 4C shows recorded images of the diffracted spectra (only 0 and +1 orders are shown) using the color CCD camera. Analysis of the pixel intensities of the first-order diffracted light, followed by conversion of pixel number to wavelengths using a calibration standard, revealed absorbance maxima of 626 nm, 436 nm, and 601 nm (with an error of +9 nm) for BPB, OG, and JG, respectively. The expected results from measurements using a benchtop spectrophotometer were 610 nm, 448 nm, and 575 nm.

Analysis of the diffraction spectra showed the absorbance maxima of the three dyes to be within an average of 18 nm of those recorded by a benchtop spectrophotometer. One factor that contributed to the deviation between the experimental and expected results was the referencing of data to a blank in a different microwell (for example, water in one channel served as the blank for a sample in a different channel). In addition, non-uniform contacts between the sample chamber and optical components (i.e., microlenses and transmission grating), as well as roughness (at the micron scale) of the surface of the microchannels, can scatter the light.

EXAMPLE 5 By talcing a series of images over time, in this example, an embodiment of the invention was used to analyze the absorbance spectra at specific locations of a sample in a microchannel during dynamic events. In this system, the dynamic switching of dye- containing fluid streams in laminar flow were monitored.

A 150 micrometer wide and 74 micrometer deep microchannel was fabricated in PDMS by soft lithography; the PDMS piece was plasma oxidized and sealed to a -glass plate. Next, a permanently aligned microlens/microchannel system was constructed as follows. An ABM mask aligner was used to align a 50 micrometer wide cylindrical microlens to the center of the microchannel. Double-sided tape was used to attach the microlens to a quartz plate, and placed the PDMS/glass microchannel on the chuck. A few drops of OG125 (Epoxy Technology, Billerica, MA), which acts as a UV-curable adhesive, were placed onto the glass plate. After aligning the microlens to the microchannel on the microscope, the PDMS/glass microchannel was brought into full contact with the microlens. The system was exposed to UV-light (365 nm, at 50 mW/cm2) for a total of 5 min. The exposure was staged in 30-second intervals, in between which minor adjustments in alignment were made. After the adhesive was cured, the aligned microlens and microchannel were removed from the quartz plate and chuck, respectively, by cutting the double-sided tape.

The microlens/microchannel system was placed on the stage of a brightfield microscope, and the solutions were pumped through the microchannels using two digitally controlled syringe pumps (Harvard Apparatus PhD2000) at flow rates of 0.1 to 1 mL/hour. The images of the diffraction spectra were recorded using a black-and-white camera, at 2.5 frames/second for 8 seconds. For clarity of visualization, color images of the laminar flow and the diffraction spectra were also captured. Also, images of diffraction spectra using phosphate-buffered saline in the microchannels were taken as a reference, as well as diffraction spectra using two different optical filters as samples which acted as calibration standards. Line scans were drawn across the diffraction spectra, and the pixel intensities were processed as described in the previous sections to produce absorbance spectra.

The percentage compositions of fluorescein and sulforhodamine were calculated as follows. The absorbance values of pure sulforhodamine at 485 nm {AsuifOι48s) and 560 nm (Asuifojβo) were measured as the absorbance values of the fluid in the microchannel at t=0 seconds, and the absorbance values of pure fluorescein at 485 nm {AflUOΛ485) and 560 nm (Afluor,56o) were measured as the absorbance values of the fluid in the microchannel at /=8 seconds. At each time measurement, the percentage compositions of sulforhodamine (%suϊfό) and fluorescein (%fluor) were calculated by solving the following system of equations:

A '■448855 = — {Afluor,485) X (%fluor) + {AsuιfoAS5) X (%Sulfθ) A '■5s660ύ == (Afluorjβo) x (%fluor) + (Asuιfθι560) x (%sulfό).

A straight cylindrical lens was aligned to the center of a microchannel (Figs. 5A-

5C), in which fluorescein and sulforhodamine B flow side by side. These figures show optical micrographs of a microchannel (Fig. 5A) and of a microlens aligned to the microchannel (Fig. 5B), and a schematic diagram of a transmission grating (Fig. 5C) with the grooves parallel to the microchannel. The composition of the fluid in the microchannel was changed by varying the flow rate of each dye (Figs. 5D-5F). These figures are optical micrographs of laminar flow of fluorescein and sulforhodamine B in the microchannel at different times. 3 mM solutions of fluorescein and sulforhodamine B (Sigma- Aldrich, Inc., St. Louis, MO, U.S.A.) dissolved in phosphate-buffered saline were used. The composition of dyes in the microchannel could be varied by changing the flow rate of each dye. By capturing images of the diffraction spectra (Figs. 5G-5I) while varying the flow rates, the full absorbance spectra of an arbitrary location in the microchannel were measured over time (Fig. 5J). Figs. 5G-5I are images of the diffraction spectra with different compositions of dyes in the microchannel (which can be roughly observed in the zeroth-order spectra). The zeroth and first order-diffracted spectra are shown. In Fig. 5 J, the wavelength spectra of a specific location in the microchannel is shown, as a change in flow rates fills a microchannel primarily with sulforhodamine B to one filled primarily with fluorescein, over the course of 8 seconds.

The spectra were obtained by processing, from a black-and-white image, the pixel intensities along a line scan of the diffraction spectrum. Processing of the data showed the dynamic changes in the composition of fluids in the channel over 8 seconds (Figs. 5K-5L). The absorbance values recorded at 485 nm and 560 nm over the course of the switching of flow rates is shown in Fig. 5K). The percentage composition of fluorescein and sulforhodamine B in the microchannel during the experiment, as calculated from the measurements in Fig. 5 J, are shown in Fig. 5L. Although images were captured every 0.4 seconds in this experiment, the time resolution of the system could be much less, for example, based on the acquisition time of the CCD camera, which can be as low as 1 to 10 milliseconds.

EXAMPLE 6 This example illustrates the orientation of a transmission grating relative to the array of microlenses. The orientation of the transmission grating relative to the microlenses can influence the pattern of diffracted light (Fig. 9). The spectral direction was orthogonal to the grooves of the transmission grating. Phi (φ) is defined as the angle between the spectral and the horizontal direction. Examples of various angles of Phi (φ) are shown as the transmission grating is rotated relative to the array of spherical microlenses.

Certain distinctive patterns appeared in various images for particular Phi (φ) angles. These particular angles could be determined by the arrangement of the spherical microlenses in a quadratic array and also by the direction of the transmission grating. The angles were be determined using elementary calculus. For example, in the situation presented in Fig. 9B, the spectral direction contained two microlenses placed in the opposite nodes of a rectangle having the width equal with the distance between two neighboring microlenses and the length equal with three times this distance. In this particular situation, Phi (φ) was roughly 72°.

Thus, these arrangements can be predicted. In the case of cylindrical microlenses, similar results can be obtained. For some arrangements, certain angles may be avoided, for example, if the angles cause complications in image processing, e.g., due to overlap. In certain cases, however, the angles may be chosen such that the diffraction spectra are kept reasonably close.

EXAMPLE 7 Transmission gratings having different pitches were examined in this example. The transmission gratings studied had groove spacings of 80, 92, and 110 grooves/mm (Edmund Industrial Optics, Barrington, NJ) (Fig. 10).

For comparison, in this example, the transmission gratings were arranged relative to the array of microlenses such that similar situations as that presented in Fig. 9C were studied, where Phi (φ) was about 63°. (This angle was arbitrarily chosen). Fig. 1OB (92 grooves/mm) is comparable with Fig. 9C, showing that pairs of two spectra overlap. If other gratings are used, the spectra may show a higher degree of overlap (Fig. 1OA, 80 grooves/mm), or no overlap (Fig. 1OC, 110 grooves/mm), at the same angle.

EXAMPLE 8 In this example, a thicker sample was analyzed without loss in spectral dispersion. In an apparatus similar to those previously described, a grating was placed at the focus of the microlenses. The focal length of the microlenses was about 135 micrometers. This requirement was met using a microfiuidic channel having the depth of about 270 micrometers, so that the microlenses focus was in the center of the channel. As a consequence, samples having thicknesses as large as 270 micrometers could be placed in the light path between the transmission grating and the array of microlenses. For comparison, in commercial bench-top spectrophotometers, the path lengths of the samples are typically 1 mm to 10 mm.

Preliminary results showed that the sample could be placed anywhere in the light path (e.g., the sample can be placed between the grading and the microlenses, between the grating and the microscope objective, and between the microscope light and the microlenses, etc.) without significantly changing in the resulting spectra. An advantage of placing the sample between the transmission grating and the array of microlenses is that localized information could be collected from the sample. For instance, circular areas having diameters of 5-10 micrometers that are the dimensions of the focus points created by the microlenses could be studied. Some of these results are shown in Fig. 11, which shows an array of spectra when the distance between the transmission grating and the array of microlenses was varied. In these figures, the distance was 0 mm (Fig. 1 IA), 1.5 mm (Fig. HB), or 2.1 mm (Fig. C).

While several embodiments of the present invention 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 functions 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 present invention. 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 teachings of the present invention 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 embodiments of the invention 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, the invention may be practiced otherwise than as specifically described and claimed. The present invention is 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 scope of the present invention.

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.

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

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. What is claimed is:

Claims

1. A method, comprising acts of: acquiring an image of one or more samples; acquiring diffraction spectra of a plurality of discrete locations of the one or more samples; and correlating the diffraction spectra and the image.
2. The method of claim 1, comprising passing a plurality of isolated light beams through the plurality of discrete locations.
3. The method of claim 2, wherein the plurality of isolated light beams are produced by passing light through a plurality of microlenses.
4. The method of claim 3 , wherein the distance between the plurality of microlenses and the one or more samples is adjustable.
5. The method of claim 3, wherein each of the microlenses produces an isolated light beam.
6. The method of claim 2, wherein the plurality of isolated light beams are passed through a common lens.
7. The method of claim 1, comprising passing a plurality of isolated light beams through a transmission grating to produce the diffraction spectra.
8. The method of claim 7, wherein the distance between the transmission grating and the one or more samples is adjustable.
9. The method of claim 7, wherein at least a portion of the transmission grating is non-linear.
10. The method of claim I3 wherein the image is a microscopic image.
11. The method of claim 1 , wherein the act of acquiring an image comprises acquiring an image using a camera.
12. The method of claim 1, comprising acquiring a plurality of images of the one or more samples at different times.
13. The method of claim 1, comprising acquiring a plurality of diffraction spectra of the one or more samples at different times.
14. The method of claim 1, wherein the one or more samples is positioned on a microscope.
15. The method of claim 1, wherein the one or more samples is contained within a microfiuidic device.
16. A method, comprising: passing a first plurality of isolated light beams through at least first and second distinct regions of one or more samples; diffracting the isolated light beams using a first transmission grating; replacing the first transmission grating with a second transmission grating; passing a second plurality of isolated light beams through the one or more samples; and diffracting the isolated light beams using the second transmission grating.
17. The method of claim 16, wherein the first plurality of isolated light beams are produced by passing light through a first plurality of microlenses.
18. The method of claim 17, wherein the distance between the first plurality of microlenses and the one or more samples is adjustable.
19. The method of claim 17, wherein each of the microlenses of the first plurality of microlenses produces an isolated light beam of the first plurality of isolated light beams.
20. The method of claim 16, wherein the distance between the first transmission grating and the one or more samples is adjustable.
21. The method of claim 16, wherein at least a portion of the first transmission grating is non-linear.
22. The method of claim 16, further comprising acquiring an image of the one or more samples.
23. The method of claim 16, wherein the one or more samples is positioned on a microscope.
24. The method of claim 16, wherein the one or more samples is contained within a microfluidic device.
25. A method, comprising: passing a first plurality of isolated light beams through at least first and second distinct regions of one or more samples; diffracting the isolated light beams using a transmission grating; moving the transmission grating; passing a second plurality of isolated light beams through the one or more samples; and diffracting the isolated light beams using the transmission grating.
26. The method of claim 25, wherein the first plurality of light beams are produced by passing light through a first plurality of microlenses.
27. The method of claim 26, wherein the distance between the first plurality of microlenses and the one or more samples is adjustable.
28. The method of claim 26, wherein each of the microlenses of the first plurality of microlenses produces an isolated light beam of the first plurality of isolated light beams.
29. The method of claim 25, wherein the distance between the first transmission grating and the one or more samples is adjustable.
30. The method of claim 25, wherein at least a portion of the transmission grating is non-linear.
31. The method of claim 25, further comprising acquiring an image of the one or more samples.
32. The method of claim 25, wherein the one or more samples is positioned on a microscope.
33. The method of claim 25, wherein the one or more samples is contained within a microfiuidic device.
34. A method, comprising: passing light through a first plurality of microlenses to produce a first plurality of isolated light beams; passing the first plurality of isolated light beams through at least first and second distinct regions of one or more samples; diffracting the first plurality of isolated light beams using a transmission grating; replacing the first plurality of microlenses with a second plurality of microlenses; passing light through the second plurality of microlenses to produce a second plurality of isolated light beams; passing the second plurality of isolated light beams through the one or more samples; and diffracting the second plurality of isolated light beams using the transmission grating.
35. The method of claim 34, wherein the distance between the first plurality of microlenses and the one or more samples is adjustable.
36. The method of claim 34, wherein each of the microlenses of the first plurality of microlenses produces an isolated light beam of the first plurality of isolated light beams.
37. The method of claim 34, wherein the distance between the transmission grating and the one or more samples is adjustable.
38. The method of claim 34, wherein at least a portion of the transmission grating is non-linear.
39. The method of claim 34, further comprising acquiring an image of the one or more samples.
40. The method of claim 34, wherein the one or more samples is positioned on a microscope.
41. The method of claim 34, wherein the one or more samples is contained within a microfluidic device.
42. A method, comprising: directing a plurality of isolated light beams through at least first and second distinct regions of one or more samples; and diffracting at least a portion of the isolated light beams passed through the one or more samples using a single transmission grating.
43. The method of claim 42, comprising passing a plurality of isolated light beams through a plurality of discrete locations of the one or more samples.
44. The method of claim 42, wherein the plurality of isolated light beams are produced by passing light through a plurality of microlenses.
45. The method of claim 44, wherein the distance between the plurality of microlenses and the one or more samples is adjustable.
46. The method of claim 44, wherein each of the microlenses produces an isolated light beam.
47. The method of claim 42, wherein the distance between the transmission grating and the one or more samples is adjustable.
48. The method of claim 42, wherein at least a portion of the transmission grating is non-linear.
49. The method of claim 42, further comprising acquiring an image of the one or more samples.
50. The method of claim 42, wherein the one or more samples is positioned on a microscope.
51. The method of claim 42, wherein the one or more samples is contained within a microfluidic device.
52. The method of claim 42, comprising acquiring a plurality of diffraction spectra of the one or more samples at different times.
53. A method, comprising: passing a plurality of isolated light beams through at least first and second distinct regions of one or more samples; and diffracting at least a portion of the isolated light beams passed through one or more samples using a ήon-linear transmission grating.
54. The method of claim 53, comprising passing a plurality of light beams through a plurality of discrete locations of the one or more samples.
55. The method of claim 53, wherein the plurality of light beams are produced by passing light through a plurality of microlenses.
56. The method of claim 55, wherein the distance between the plurality of microlenses and the one or more samples is adjustable.
57. The method of claim 55, wherein each of the microlenses produces an isolated light beam.
58. The method of claim 53, further comprising acquiring an image of the one or more samples.
59. The method of claim 53, wherein the distance between the non-linear transmission grating and the one or more samples is adjustable.
60. The method of claim 53, wherein the one or more samples is positioned on a microscope.
61. The method of claim 53, wherein the one or more samples is contained within a microfluidic device.
62. The method of claim 53, comprising acquiring a plurality of diffraction spectra of the one or more samples at different times.
63. An apparatus, comprising: a microscope comprising a plurality of microlenses and a diffraction grating, wherein the position of the diffraction grating, relative to the microlenses, is adjustable.
64. The article of claim 63, wherein the position is adjustable by adjusting the microscope.
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