Fluorescent nanoscopy method
First Claim
1. A method for nanoscopy of dyed objects being placed in a solution or as a slice of a preserved sample being placed in a polymer, wherein a fluorescent microscope having digital video-cameras with suppressing color filters for picking up fluorescence light of the object that are able to detect with a low noise level images of separate fluorescing molecules excited by a laser, the microscope having a computer for recording images received from the video-camera, the method comprising:
- periodically turning one to two thousands of non-fluorescing dye molecules into fluorescing fluorescing dye molecules by a UV flash, wherein the molecules cover object structures, turning the molecules into fluorescing molecules by separating chemical groups which block fluorescence, detecting images of created fluorescing molecules on video-camera frames as separate spots, wherein the molecules photobleach in the process of detection and frames with residual fluorescence are subtracted from frames containing a mixture of fluorescing particles and a residual fluorescence, saving subtracted frames to computer memory to find centers of spots which represent coordinates of dye molecules, repeating the process a number of times to calculate coordinates of all dye molecules in a lighted area of the object and reconstruct a 2-D image or a 3-D image of the object with a resolution better than 20-50 nm.
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Accused Products
Abstract
An analysis of an object dyed with fluorescent coloring agents carried out with the aid of a fluorescent microscope which is modified for improved resolving power and called a nanoscope. The method is carried out with a microscope having an optical system for visualizing and projecting a sample image to a video camera which records and digitizes images of individual fluorescence molecules and nanoparticles at a low noise, a computer for recording and processing images, a sample holder arranged in front of an object lens, a fluorescent radiation exciting source and a set of replaceable suppression filters for separating the sample fluorescent light. Separately fluorescing visible molecules and nanoparticles are periodically formed in different object parts, the laser produces the oscillation thereof which is sufficient for recording the non-overlapping images of the molecules and nanoparticles and for decoloring already recorded fluorescent molecules, wherein tens of thousands of pictures of recorded individual molecule and nanoparticle images, in the form of stains having a diameter on the order of a fluorescent light wavelength multiplied by a microscope amplification, are processed by a computer for searching the coordinates of the stain centers and building the object image according to millions of calculated stain center co-ordinates corresponding to the co-ordinates of the individual fluorescent molecules and nanoparticles. With this invention it is possible to obtain a two-dimensional and a three-dimensional image with a resolving power better than 20 nm and to record a color image by dyeing proteins, nucleic acids and lipids with different coloring agents.
49 Citations
8 Claims
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1. A method for nanoscopy of dyed objects being placed in a solution or as a slice of a preserved sample being placed in a polymer, wherein a fluorescent microscope having digital video-cameras with suppressing color filters for picking up fluorescence light of the object that are able to detect with a low noise level images of separate fluorescing molecules excited by a laser, the microscope having a computer for recording images received from the video-camera, the method comprising:
periodically turning one to two thousands of non-fluorescing dye molecules into fluorescing fluorescing dye molecules by a UV flash, wherein the molecules cover object structures, turning the molecules into fluorescing molecules by separating chemical groups which block fluorescence, detecting images of created fluorescing molecules on video-camera frames as separate spots, wherein the molecules photobleach in the process of detection and frames with residual fluorescence are subtracted from frames containing a mixture of fluorescing particles and a residual fluorescence, saving subtracted frames to computer memory to find centers of spots which represent coordinates of dye molecules, repeating the process a number of times to calculate coordinates of all dye molecules in a lighted area of the object and reconstruct a 2-D image or a 3-D image of the object with a resolution better than 20-50 nm. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8)
Specification
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- Resources
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Current AssigneeSuper Resolution Technologies LLC (Acacia Research Corporation)
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Original AssigneeAndrey Alexeevich Klimov, Dmitry Andreevich Klimov, Evgeniy Andreevich Klimov, Tatiana Vitalyevna Klimova
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InventorsKlimov, Andrey Alexeevich, Klimova, Tatiana Vitalyevna, Klimov, Dmitry Andreevich, Klimov, Evgeniy Andreevich
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Primary Examiner(s)Sines; Brian J
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Application NumberUS11/920,661Publication NumberTime in Patent Office1,607 DaysField of Search436/172, 436/174, 436/164, 977/840, 977/881, 977/868, 977/869, 356/401US Class Current436/172CPC Class CodesG01N 21/6428 Measuring fluorescence of f...G01N 21/6458 Fluorescence microscopy flu...G01N 21/6486 Measuring fluorescence of b...H04N 13/275 from 3D object models, e.g....H04N 7/18 Closed-circuit television [...Y10S 977/84 Manufacture, treatment, or ...Y10S 977/868 with optical meansY10S 977/869 Optical microscopeY10S 977/881 Microscopy or spectroscopy,...Y10T 436/143333 Saccharide [e.g., DNA, etc.]Y10T 436/25 including sample preparation
