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Title: Calibration of fluorescence resonance energy transfer in microscopy

Abstract

Imaging hardware, software, calibrants, and methods are provided to visualize and quantitate the amount of Fluorescence Resonance Energy Transfer (FRET) occurring between donor and acceptor molecules in epifluorescence microscopy. The MicroFRET system compensates for overlap among donor, acceptor, and FRET spectra using well characterized fluorescent beads as standards in conjunction with radiometrically calibrated image processing techniques. The MicroFRET system also provides precisely machined epifluorescence cubes to maintain proper image registration as the sample is illuminated at the donor and acceptor excitation wavelengths. Algorithms are described that pseudocolor the image to display pixels exhibiting radiometrically-corrected fluorescence emission from the donor (blue), the acceptor (green) and FRET (red). The method is demonstrated on samples exhibiting FRET between genetically engineered derivatives of the Green Fluorescent Protein (GFP) bound to the surface of Ni chelating beads by histidine-tags.

Inventors:
; ; ; ; ;
Issue Date:
Research Org.:
Kairos Scientific, Inc., San Diego, CA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1174620
Patent Number(s):
6,661,909
Application Number:
09/912,117
Assignee:
Kairos Scientific, Inc. (San Diego, CA) OSTI
Resource Type:
Patent
Country of Publication:
United States
Language:
English
Subject:
46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND TECHNOLOGY

Citation Formats

Youvan, Dougalas C., Silva, Christopher M., Bylina, Edward J., Coleman, William J., Dilworth, Michael R., and Yang, Mary M. Calibration of fluorescence resonance energy transfer in microscopy. United States: N. p., 2003. Web.
Youvan, Dougalas C., Silva, Christopher M., Bylina, Edward J., Coleman, William J., Dilworth, Michael R., & Yang, Mary M. Calibration of fluorescence resonance energy transfer in microscopy. United States.
Youvan, Dougalas C., Silva, Christopher M., Bylina, Edward J., Coleman, William J., Dilworth, Michael R., and Yang, Mary M. Tue . "Calibration of fluorescence resonance energy transfer in microscopy". United States. https://www.osti.gov/servlets/purl/1174620.
@article{osti_1174620,
title = {Calibration of fluorescence resonance energy transfer in microscopy},
author = {Youvan, Dougalas C. and Silva, Christopher M. and Bylina, Edward J. and Coleman, William J. and Dilworth, Michael R. and Yang, Mary M.},
abstractNote = {Imaging hardware, software, calibrants, and methods are provided to visualize and quantitate the amount of Fluorescence Resonance Energy Transfer (FRET) occurring between donor and acceptor molecules in epifluorescence microscopy. The MicroFRET system compensates for overlap among donor, acceptor, and FRET spectra using well characterized fluorescent beads as standards in conjunction with radiometrically calibrated image processing techniques. The MicroFRET system also provides precisely machined epifluorescence cubes to maintain proper image registration as the sample is illuminated at the donor and acceptor excitation wavelengths. Algorithms are described that pseudocolor the image to display pixels exhibiting radiometrically-corrected fluorescence emission from the donor (blue), the acceptor (green) and FRET (red). The method is demonstrated on samples exhibiting FRET between genetically engineered derivatives of the Green Fluorescent Protein (GFP) bound to the surface of Ni chelating beads by histidine-tags.},
doi = {},
journal = {},
number = ,
volume = ,
place = {United States},
year = {2003},
month = {12}
}

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