Optical train and method for TIRF single molecule detection and analysis
First Claim
1. An apparatus for single molecule analysis, the apparatus comprising:
- a support having a sample located thereon;
at least two lasers that produce light at distinct wavelengths;
a collimator for directing said light onto said sample through a total internal reflection objective;
and at least one detector for detecting each of said wavelengths in said fluorescent emissions.
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Accused Products
Abstract
In one aspect the invention relates to an apparatus for analyzing the presence of a single molecule using total internal reflection. In one embodiment an apparatus for single molecule analysis includes a support having a sample located thereon; two sources of light at distinct wavelengths, a collimator for directing the light onto the sample through a total internal reflection objective; a receiver for receiving a fluorescent emission produced by a single molecule in the sample in response to the light; and a detector for detecting each of the wavelengths in the fluorescent emission. In another embodiment the apparatus further comprises a focusing laser for maintaining focus of the objective on the sample.
60 Citations
59 Claims
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1. An apparatus for single molecule analysis, the apparatus comprising:
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a support having a sample located thereon;
at least two lasers that produce light at distinct wavelengths;
a collimator for directing said light onto said sample through a total internal reflection objective;
and at least one detector for detecting each of said wavelengths in said fluorescent emissions. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 55)
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17. An apparatus for analysis of single molecules, the apparatus comprising:
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a first laser;
a band-pass filter in optical communication with said first laser;
at least one first lens in optical communication with said band-pass filter;
a second laser;
a second band-pass filter in optical communication with said second laser;
at least one second lens in optical communication with said second band-pass filter;
a dichroic beam combiner in optical communication with said at least one first lens and said at least one second lens;
a collimator in optical communication with said dichroic beam combiner;
a field stop in optical communication with said collimator;
an illumination dichroic lens for passing light from said first and second lasers to an objective for focusing on a sample and for passing fluorescent emissions from said sample to a camera dichroic filter, said camera dichroic filter for passing light of a first wavelength to a first camera and light of a second wavelength to a second camera; and
a computer in communication with said first and second cameras for analyzing said fluorescent emissions. - View Dependent Claims (18, 19, 20, 21)
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22. An apparatus for analyzing the presence of a single molecule using total internal reflection comprising:
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a sample plate having a sample located thereon;
a light source providing two wavelengths of light;
a sizing collimator producing a spot of collimated light of a defined size on said sample;
a receiver for reducing non-fluorescent light in the fluorescent image produced by said sample by each of said wavelengths of light; and
a detector in optical communication with said receiver, said detector positioned to detect said fluorescent image produced by said sample by each of said wavelengths of light. - View Dependent Claims (23, 24, 25, 26, 27)
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28. An apparatus for analyzing the presence of a single molecule using total internal reflection comprising:
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a sample plate having a sample located thereon;
means for providing two wavelengths of light;
means for producing a spot of collimated light of a defined size on said sample;
means for receiving a fluorescent image produced by said sample by each of said wavelengths of light and reducing non-fluorescent light; and
means for detecting said fluorescent image produced by said sample by each of said wavelengths of light. - View Dependent Claims (29, 30)
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31. An apparatus for analyzing the presence of a single molecule comprising:
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a sample plate having a sample located thereon;
a first laser providing a fluorescence stimulating wavelength of light;
a second laser providing a second wavelength of light;
a collimator producing, from said first laser, a spot of collimated light of a defined size on said sample;
a detector for detecting a fluorescent image produced by said sample in response to said spot of collimated light; and
an autofocus module adjusting the focus of the fluorescent image in response to the light from said second laser. - View Dependent Claims (32, 34)
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33. An apparatus for analyzing the presence of a single molecule comprising:
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means for holding a sample;
means for providing a fluorescence stimulating wavelength of light;
means for providing a second wavelength of light;
means for producing, from said means for providing a fluorescence stimulating wavelength of light, a spot of collimated light of a defined size on said sample;
means for detecting a fluorescent image produced by said sample; and
means for adjusting the focus of the fluorescent image in response to the light from said means for providing a second wavelength of light.
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35. A method for analyzing a single molecule comprising the steps of:
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providing a sample;
producing light at two distinct wavelengths;
directing said light at two distinct wavelengths onto said sample through a total internal reflection objective;
receiving fluorescent emissions produced by a single molecule in said sample in response to said light at two distinct wavelengths; and
detecting said fluorescent emissions. - View Dependent Claims (36, 37)
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38. A method for analyzing a single molecule comprising the steps of:
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providing a sample;
producing light at a first wavelength;
directing said light at said first wavelength onto said sample through a total internal reflection objective;
receiving a fluorescent emission produced by a single molecule in said sample in response to said light at said first wavelength;
autofocusing the fluorescent emission; and
detecting said fluorescent emission.
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39. A method for sequencing a nucleic acid, comprising the steps of:
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(a) attaching a nucleic acid comprising a first optically-detectable label to a surface;
(b) exposing said nucleic acid to a first wavelength of light;
(c) determining the location of said nucleic acid based upon the response of said first optically-detectable label to said first wavelength of light;
(d) exposing said nucleic acid to a polymerase and a nucleotide comprising a second optically-detectable label;
(e) removing unincorporated nucleotides;
(f) exposing said nucleic acid to a second wavelength of light using total internal reflection;
(g) determining the location of said second optically-detectable label based upon the response of said second optically-detectable label to said second wavelength of light;
(h) removing or inactivating said second optically-detectable label; and
(i) repeating steps (d) through (h) for second and subsequent nucleotides. - View Dependent Claims (40, 41, 42, 43, 44, 45, 46, 47, 48, 49)
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50. A method for sequencing a nucleic acid, comprising the steps of:
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(a) attaching a nucleic acid to a surface;
(b) exposing said surface to a first wavelength of light;
(c) autofocusing an image of said surface in response to said first wavelength of light;
(d) exposing said nucleic acid to a polymerase and a nucleotide comprising a first optically-detectable label;
(e) removing any unincorporated nucleotide;
(f) exposing said nucleic acid to a second wavelength of light;
(g) determining the location of said first optically-detectable label based upon the response of said first optically-detectable label to said second wavelength of light;
(h) removing or inactivating said first optically-detectable label; and
(i) repeating steps (d) through (h) for second and subsequent nucleotides. - View Dependent Claims (51, 52, 53, 54)
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56. An apparatus for single molecule analysis, the apparatus comprising:
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a support having a sample located thereon;
at least two lasers that produce light at distinct wavelengths;
a collimator for directing said light onto said sample through a total internal reflection objective; and
at least one detector for detecting each of said wavelengths. - View Dependent Claims (57, 58, 59)
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Specification