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):
- 6661909
- Application Number:
- 09/912,117
- 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:
- 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 = {Tue Dec 09 00:00:00 EST 2003},
month = {Tue Dec 09 00:00:00 EST 2003}
}