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Calibration of fluorescence resonance energy transfer in microscopy

United States Patent

December 9, 2003
View the Complete Patent at the US Patent & Trademark Office
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.
Youvan; Dougalas C. (San Diego, CA), Silva; Christopher M. (Sunnyvale, CA), Bylina; Edward J. (San Diego, CA), Coleman; William J. (San Diego, CA), Dilworth; Michael R. (Santa Cruz, CA), Yang; Mary M. (San Diego, CA)
Kairos Scientific, Inc. (San Diego, CA)
09/ 912,117
July 23, 2001
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH This invention was made at least in part with funds from the Federal Government, Department of Energy. The Government may have certain rights in the invention.