Ultrahigh resolution multicolor colocalization of single fluorescent probes
First Claim
1. A method for the colocalization of two or more species of interest in a sample comprising:
- a) exciting the species of interest with a single wavelength of light thereby causing the two or more species of interest to emit light of a distinctive emission characteristic, b) separating the distinctive emission characteristics of the two or more species of interest, c) detecting the emitted light from the two or more species of interest, d) moving the sample a predetermined distance, e) repeating steps a) through d) a predetermined number of times thereby creating a multitude of representations of the excitation point spread function (PSF), f) determine the geometric center of the representations of the excitation PSF for at least two species of the two or more species of interest, g) computing the distance between the geometric centers of the representations of the excitation PSF for the species of interest.
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Abstract
A novel optical ruler based on ultrahigh-resolution colocalization of single fluorescent probes is described. Two unique families of fluorophores are used, namely energy-transfer fluorescent beads and semiconductor nanocrystal (NC) quantum dots, that can be excited by a single laser wavelength but emit at different wavelengths. A novel multicolor sample-scanning confocal microscope was constructed which allows one to image each fluorescent light emitter, free of chromatic aberrations, by scanning the sample with nanometer scale steps using a piezo-scanner. The resulting spots are accurately localized by fitting them to the known shape of the excitation point-spread-function of the microscope.
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Citations
26 Claims
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1. A method for the colocalization of two or more species of interest in a sample comprising:
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a) exciting the species of interest with a single wavelength of light thereby causing the two or more species of interest to emit light of a distinctive emission characteristic, b) separating the distinctive emission characteristics of the two or more species of interest, c) detecting the emitted light from the two or more species of interest, d) moving the sample a predetermined distance, e) repeating steps a) through d) a predetermined number of times thereby creating a multitude of representations of the excitation point spread function (PSF), f) determine the geometric center of the representations of the excitation PSF for at least two species of the two or more species of interest, g) computing the distance between the geometric centers of the representations of the excitation PSF for the species of interest. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26)
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Specification