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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:
 [1];  [2];  [1];  [3];  [4];  [1]
  1. San Jose, CA
  2. Sunnyvale, CA
  3. Moutain View, CA
  4. Santa Cruz, CA
Issue Date:
OSTI Identifier:
874767
Patent Number(s):
6456734
Application Number:
09/092,316
Assignee:
Kairos Scientific, Inc. (San Diego, CA)
Patent Classifications (CPCs):
G - PHYSICS G01 - MEASURING G01N - INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
Resource Type:
Patent
Country of Publication:
United States
Language:
English
Subject:
calibration; fluorescence; resonance; energy; transfer; microscopy; imaging; hardware; software; calibrants; methods; provided; visualize; quantitate; amount; fret; occurring; donor; acceptor; molecules; epifluorescence; microfret; compensates; overlap; spectra; characterized; fluorescent; beads; standards; conjunction; radiometrically; calibrated; image; processing; techniques; provides; precisely; machined; cubes; maintain; proper; registration; sample; illuminated; excitation; wavelengths; algorithms; described; pseudocolor; display; pixels; exhibiting; radiometrically-corrected; emission; blue; green; method; demonstrated; samples; genetically; engineered; derivatives; protein; gfp; bound; surface; chelating; histidine-tags; energy transfer; resonance energy; /382/

Citation Formats

Youvan, Douglas 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., 2002. Web.
Youvan, Douglas 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, Douglas 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/874767.
@article{osti_874767,
title = {Calibration of fluorescence resonance energy transfer in microscopy},
author = {Youvan, Douglas 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 = {2002},
month = {9}
}

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