Single molecule detection by in situ hybridization
First Claim
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1. A method for digital imaging fluorescence microscopy detection of an individual nucleic acid probe bound to a nucleic acid target molecule in a sample, said method comprising the steps of:
- (a) obtaining a fluorochrome-labeled nucleic acid probe wherein the number of fluorochromes per probe is predetermined;
(b) contacting said labeled probe with a nucleic acid target molecule under conditions allowing said labeled probe to bind to said target molecule;
(c) removing unbound labeled probes from labeled probes bound to target molecules;
(d) detecting, by digital imaging fluorescence microscopy, said labeled probe bound to said target molecule, wherein said digital imaging fluorescence microscopy includes digitally recording a series of optical sections;
(e) determining the total fluorescence intensity per fluorochrome under imaging conditions;
(f) determining the total fluorescence intensity per probe from said total fluorescence intensity per fluorochrome;
(g) normalizing said total fluorescence intensity per fluorochrome under imaging conditions to a value for a single conjugated fluorochrome whose image is acquired through a series of optical sections and restored, thereby producing a normalized total fluorescence intensity;
(h) producing a prepared image from said series of optical sections by subjecting said series of optical sections, sequentially, to dark current subtraction, background subtraction, and normalization to the first optical section;
(i) producing a restored image;
(j) identifying discrete objects in said restored image, said objects corresponding to target molecules;
(k) comparing said normalized total fluorescence intensity to values of objects in said restored image;
(l) determining the presence of one or more probes in one of said objects, thereby detecting an individual nucleic acid probe bound to a nucleic acid target molecule in a sample.
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Abstract
Disclosed are methods for accurately determining the total emission intensity of a single fluorochrome, under imaging conditions, using a digital imaging fluorescence microscopy system. Also disclosed are methods for detecting and localizing probe-target molecule binding. The detection method has sufficient resolution and sensitivity to locate and detect a single target-bound probe.
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Citations
27 Claims
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1. A method for digital imaging fluorescence microscopy detection of an individual nucleic acid probe bound to a nucleic acid target molecule in a sample, said method comprising the steps of:
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(a) obtaining a fluorochrome-labeled nucleic acid probe wherein the number of fluorochromes per probe is predetermined; (b) contacting said labeled probe with a nucleic acid target molecule under conditions allowing said labeled probe to bind to said target molecule; (c) removing unbound labeled probes from labeled probes bound to target molecules; (d) detecting, by digital imaging fluorescence microscopy, said labeled probe bound to said target molecule, wherein said digital imaging fluorescence microscopy includes digitally recording a series of optical sections; (e) determining the total fluorescence intensity per fluorochrome under imaging conditions; (f) determining the total fluorescence intensity per probe from said total fluorescence intensity per fluorochrome; (g) normalizing said total fluorescence intensity per fluorochrome under imaging conditions to a value for a single conjugated fluorochrome whose image is acquired through a series of optical sections and restored, thereby producing a normalized total fluorescence intensity; (h) producing a prepared image from said series of optical sections by subjecting said series of optical sections, sequentially, to dark current subtraction, background subtraction, and normalization to the first optical section; (i) producing a restored image; (j) identifying discrete objects in said restored image, said objects corresponding to target molecules; (k) comparing said normalized total fluorescence intensity to values of objects in said restored image; (l) determining the presence of one or more probes in one of said objects, thereby detecting an individual nucleic acid probe bound to a nucleic acid target molecule in a sample. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21)
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22. A method for determining the total fluorescence intensity of a single fluorochrome in a digital imaging microscopy system, under imaging conditions, said method comprising the steps of:
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(a) providing a microscope slide and coverslip; (b) providing on the surface of said microscope slide a first fluorescence point source; (c) providing on the surface of said coverslip a second fluorescence point source; (d) preparing a standard solution containing a known concentration of fluorochrome-labeled probes; (e) placing a suitable volume of said standard solution between and in contact with said microscope slide and said coverslip, said first fluorescence point source and said second fluorescence point source being within the field of view of said digital imaging microscopy system; (f) determining a distance between said first and second fluorescence point sources, thereby obtaining a z axis distance between said microscope slide and said coverslip; (g) determining an imaged volume by determining the product of an x axis distance, a y axis distance, and said z axis distance; (h) determining the total amount of probe fluorescence from the imaged volume; (i) calculating the total number of probes in the imaged volume using (1) said known concentration of probes in said standard solution, and (2) said imaged volume; and (j) dividing the total amount of probe fluorescence from said imaged volume by the number of fluorochromes in the imaged volume to determine the total fluorescence intensity of a single fluorochrome under imaging conditions. - View Dependent Claims (23, 24, 25, 26)
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27. A kit for use in determining the total fluorescence intensity of a single fluorochrome in a digital imaging microscopy system under imaging conditions, said kit comprising:
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(a) a spotted microscope slide comprising fluorescent material on at least one surface, said material being deposited on said surface as a discrete slide spot, thereby forming a spotted microscope slide surface, said slide spot having a diameter and a thickness between 1 and 400 nanometers; and (b) a spotted coverslip comprising fluorescent material on at least one surface, said material being deposited on said surface as a discrete coverslip spot, thereby forming a spotted coverslip surface, said coverslip spot having a diameter and a thickness between 1 and 400 nanometers; wherein said spotted microscope slide and said spotted coverslip can be used in combination, with said spotted microscope slide surface and said spotted coverslip surface opposing each other, so that said microscope slide spot and said coverslip spot are simultaneously visible within the field of view of a microscope.
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Specification