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Apparatus and methods relating to spatially light modulated microscopyApparatus and methods relating to spatially light modulated microscopy description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070188856, Apparatus and methods relating to spatially light modulated microscopy. Brief Patent Description - Full Patent Description - Patent Application Claims
RELATED APPLICATIONS [0001] This application claims priority to U.S. Provisional Application No. 60/063,893 filed on Oct. 29, 1997, the contents of which are incorporated herein by reference. FIELD OF THE INVENTION [0002] The field of the present invention is the magnification of images of objects, and more particularly microscopes and the field of microscopy. BACKGROUND OF THE INVENTION [0003] Microscopy is used to produce magnified representations of both dynamic and stationary objects or samples. There are many different modes of microscopy such as brightfield microscopy, darkfield microscopy, phase contrast microscopy, fluorescence microscopy, reflectance or reflected light microscopy and confocal microscopy. All of these forms of microscopy deliver illumination light in a controlled, fashion to the sample and collect as much of the light containing the desired information about the sample as possible. Typically, this is accomplished using Kohler illumination in any of reflectance microscopy, transmission microscopy or epifluorescence microscopy. Both of these methods use appropriately placed diaphragms and lenses to control both the size of the numerical aperture (illumination cone) and the size of the illuminated area of the sample. In Kohler illumination, diaphragms are placed in at least two locations. First, a diaphragm is placed in the conjugate image plane of the sample, a location which permits control of the size of the illuminated area of the sample. Second, a diaphragm is placed in the conjugate image plane of the aperture diaphragm of the objective lens(es) (this location is also a conjugate image plane of the aperture diaphragm of the condensor lens(es)), a location which permits control of the angle(s) of the light illuminating the sample. Typically, any of the diaphragms can be a simple iris (for example, for brightfield microscopy and epillumination fluorescence microscopy), but the diaphragms can also be more complex (for example, in darkfield microscopy, where the diaphragms may comprise cutout rings of different diameters). [0004] An example of a microscope using Kohler illumination is set forth in FIG. 1. In the figure, microscope 2 comprises a light source 4 that emits a plurality of light rays, which have been divided into first light rays 6, second light rays 8 and third light rays 10. The light rays are transmitted along an illumination light path 3 from light source 4 through light source lens 12, adjustable iris field diaphragm 14 and condensor lenses 16. An adjustable iris aperture diaphragm (condensor) 18 can be disposed between upstream and downstream condensor lenses 16. The light then contacts, or impinges upon, sample 20 and then proceeds to pass through objective lenses 22, which objective lenses can comprise an aperture diaphragm (objective) 24 spaced between the objective lenses 22, and then the light rays proceed to a light detector 26. As noted above, the angle of illumination of the sample can be controlled by modulating the light as it passes through conjugate image planes of the aperture diaphragm of the objective lens, which planes can be found, for example, at light source 4 and the upstream aperture diaphragm 18 in FIG. 1, while the location and/or area of illumination of the sample can be controlled by modulating light as it passes through a conjugate image plane of the sample, which plane corresponds to the adjustable iris field diaphragm 14 in FIG. 1. [0005] One preferred form of microscopy is confocal microscopy, in which discreet aperture spots are illuminated in the object plane of the microscope from which transmission, reflected or fluorescent light is then relayed for observation through conjugate apertures in the image plane. In some embodiments, confocal microscopy can result in spatial resolution about 1.3 times better than the optimum resolution obtainable by conventional light microscopy. See, e.g., U.S. Pat. No. 5,587,832. Additionally, confocal microscopy can reduce the interference of stray, out-of-focus light from an observed specimen above or below the focal plane, and can permit optical sectioning of tissue as well as high-resolution 3-D reconstruction of the tissue. The technique can effectively resolve individual cells and living tissue without staining. Confocal microscopy can be performed using mechanical translation of the specimen with fixed optics, or using a fixed specimen and scanning beams manipulated by special rotating aperture disks. See, U.S. Pat. No. 4,802,748, U.S. Pat. No. 5,067,805, U.S. Pat. No. 5,099,363, U.S. Pat. No. 5,162,941. Such disks typically comprise a plurality of apertures, but only one aperture at a time is used for confocal scanning. Still other known confocal scanning systems have used a laser beam rastered with rotating mirrors to scan a specimen or a laser beam that scans a slit rather than a spot; such slit scanning increases imaging speed but slightly degrades resolution. See, U.S. Pat. No. 5,587,832. [0006] Conventional confocal microscopes can be slow to acquire images for certain applications and become even slower as the scan line density increases and the aperture separation decreases. In addition, it is difficult to practically adjust the perimeters of the confocal microscope in commercial systems, and the signal to noise ratio (SNR) is sacrificed to increase the imaging rate. In addition, proper alignment of conventional confocal systems can be critical and difficult to maintain. In addition, laser-based systems are more expensive than white light systems, but such laser systems do not offer a selection of illumination wavelengths and can also lead to photo toxicity and rapid photo bleaching. [0007] Thus, there has gone unmet a need for improved microscopy systems, including confocal microscopy systems, wherein the angle of illumination of a sample can be easily and rapidly controlled. There has also gone unmet a need for a microscope that can easily and rapidly control the quantity of light reaching the sample, including both varying the absolute quantity of light as well as the location on the sample upon which the quantity of light impinges. The present invention provides these and other advantages. SUMMARY OF THE INVENTION [0008] The present invention provides microscopes that have significant advantages in controlling the light that contacts a sample and/or that is detected emanating from a sample. The improved control includes enhanced, selective control of the angle of illumination, the quantity of light and the location of light reaching the sample and/or detector. The present invention provides these advantages by placing one or more spatial light modulators in the illumination and/or detection light path of the microscope at one or both of the conjugate image plane of the aperture diaphragm of the objective lens and the conjugate image plane of the sample. [0009] Thus, in one aspect the present invention provides microscopes comprising a spatial light modulator comprising an illumination array of individual light transmission pixels, the spatial light modulator disposed in an illumination light path of the microscope at a conjugate image plane of an aperture diaphragm of an objective lens to provide an upstream spatial light modulator. [0010] In some embodiments, the microscopes selectively control the angle of illumination of a sample and the angle of detection of light emanating from the sample, wherein the upstream spatial light modulator is operably connected to a modulator controller containing computer-implemented programming that controls transmissive characteristics of the spatial light modulator to select a desired angle of illumination and detection of the sample and wherein a selected portion of the individual light transmission pixels corresponding to the desired angle of illumination and detection is on. In other embodiments, the microscopes selectively control a quantity of light reaching a sample, the quantity being less than all the light emitted by a light source located at a beginning of the illumination light path, wherein the upstream spatial light modulator is operably connected to a modulator controller containing computer-implemented programming that controls transmissive characteristics of the spatial light modulator to select a desired quantity of illumination and a selected portion of the individual light transmission pixels corresponding to the desired quantity of illumination is on. [0011] In further embodiments, the microscopes are transmission, or reflectance microscopes. The microscopes can comprise a lens disposed in the illumination light path between a light source and the spatial light modulator, the light source disposed at a conjugate image plane of an aperture diaphragm of the objective lens that is located upstream from the upstream spatial light modulator. The microscopes can further comprise a second spatial light modulator that is disposed in a detection light path, located at a downstream conjugate image plane of an aperture diaphragm of the objective lens and operably connected to a second modulator controller containing computer-implemented programming that controls transmissive characteristics of the second spatial light modulator. Preferably, the first modulator controller is operably connected to the second modulator controller such that the second light modulator selectively controls the light in the detection light path to correspond to the desired angle selected by the first modulator controller; the various controllers discussed herein can be separate controllers or can be a single controller. [0012] In still other embodiments, the microscopes provide darkfield microscopy, wherein the upstream spatial light modulator is operably connected to a first modulator controller containing computer-implemented programming that controls transmissive characteristics of the spatial light modulator to select a desired pattern for darkfield microscopy and a selected portion of the individual light transmission pixels corresponding to the desired pattern for darkfield microscopy is on, and a second spatial light modulator is disposed in a detection light path, located at a downstream conjugate image plane of an aperture diaphragm of the objective lens and operably connected to a second modulator controller containing computer-implemented programming that controls transmissive characteristics of the second spatial light modulator to select a complementary pattern of the individual light transmission pixels of the second spatial light modulator to complement the pattern of individual light transmission pixels of the first spatial light modulator that are on, thereby providing a complementary pattern of the individual light transmission pixels of the second spatial light modulator that are on. [0013] In more embodiments, the microscopes alternate between darkfield microscopy and brightfield microscopy. This can be achieved, for example, wherein the first modulator controller and the second modulator controller control the transmissive characteristics of the first and second spatial light modulators to switch between brightfield microscopy and at least one desired pattern for darkfield microscopy. For this and other embodiments that comprise cycling or a method, the microscopes preferably perform the method or cycle, for example, to alternate back and forth between darkfield microscopy and brightfield microscopy, with a cycle or method time of less than the time that is needed for the detector, such as a human, eye or video camera, to acquire an image, for example less than about 0.03 seconds, thereby providing real-time viewing if desired. In other preferred embodiments, the microscopes alternate back and forth between darkfield microscopy and brightfield microscopy without refocusing. [0014] In still more embodiments for this other aspects of the present invention (unless expressly stated otherwise or clear from the context, all embodiments of the present invention can be mixed and matched), the microscopes comprise a light detector located at a downstream end of a detection light path, the light detector comprising a detection array of individual detection pixels. In come embodiments, the detection array of individual detection pixels corresponds to and is aligned with the illumination array of individual light transmission pixels in the upstream spatial light modulator and the detection array of individual detection pixels is operably connected to a detector controller and the illumination array of individual light transmission pixels in the spatial light modulator is connected to the modulator controller, such that the modulator controller contains computer-implemented programming that selects a plurality of desired angles of illumination of the sample and the detector controller contains computer-implemented programming that detects the changes in intensity in the detection array of individual detection pixels corresponding to the plurality of desired angles of illumination and detection and therefrom determines a three-dimensional image of the sample. In other embodiments, which can be combined with the previous embodiments, the modulator controller selects a plurality of desired angles of illumination of the sample to provide a plurality of images of the sample at a corresponding plurality of different depths within the sample and a reconstruction controller comprises computer-implemented programming that tomographically reconstructs the different images to provide a three dimensional image of the sample. [0015] In more embodiments, the modulator controller selects the plurality of desired angles of illumination of the sample such that the plurality of images of the sample at a corresponding plurality of different depths are obtained without moving the sample, a condensor lens or an objective lens. [0016] In other embodiments, the light detector is a charge-coupled device, charge-injection device, video camera and the microscopes can comprise one or more photomultiplier tubes and or ocular eyepieces located at a downstream end of the detection light path. The spatial light modulator can be, for example, a digital micromirror device, microshutter or a liquid crystal device. [0017] In another aspect, the present invention provides microscopes comprising a variable field iris, the microscopes comprising a spatial light modulator comprising an illumination array of individual light transmission pixels and disposed in an illumination light path at a conjugate image plane of a sample, to provide an upstream spatial light modulator, and that is operably connected to a modulator controller containing computer-implemented programming that controls transmissive characteristics of the upstream spatial light modulator to select a plurality of desired field iris settings. In some embodiments for this and other aspects of the invention, the individual light transmission pixels of the illumination array are in the on/off status corresponding to the desired field iris setting. [0018] In a further aspect, the present invention provides microscopes that project a selected image into an image plane of a sample of the microscope, the microscope comprising a spatial light modulator comprising an illumination array of individual light transmission pixels and disposed in a first illumination light path at a conjugate image plane of the sample, to provide a first upstream spatial light modulator, wherein the upstream spatial light modulator is operably connected to at least one controller containing computer-implemented programming that varies an on/off status of the individual light transmission pixels of the illumination array to correspond to the selected image, the microscope further comprising a second illumination light path that provides illumination light to the sample. The selected image can be projected onto the sample or adjacent to the sample or elsewhere if desired. [0019] In certain embodiments, the upstream spatial light modulator is disposed in both the first illumination light path and the second illumination light path and a first portion of the individual light transmission pixels of the illumination array provides illumination light to the sample and second portion of the individual light transmission pixels of the illumination array are in an on/off status corresponding to the selected image. [0020] In further embodiments, wherein the microscopes further comprise a light detector disposed downstream from the sample in a detection light path at a conjugate image plane of the sample, wherein the spatial light modulator and the light detector are operably connected to at least one controller containing computer-implemented programming that controls transmissive characteristics of the upstream spatial light modulator and that compiles the light intensity detection data provided by the light detector, the controller selectively varies the on/off status of individual light transmission pixels of the spatial light modulator to vary the light intensity impinging on selected spots of the sample and thereby vary the intensity of light emanating from the selected spots of the sample and impinging on at least one pixel of the light detector. Continue reading about Apparatus and methods relating to spatially light modulated microscopy... 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