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AssigneeKairos Scientific, Inc.
InventorsDougalas C. Youvan, Christopher M. Silva, Edward J. Bylina, William J. Coleman, Michael R. Dilworth, Mary M. Yang
Estimating the term…
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.
A FRET calibration standard from which a FRET efficiency, E, can be determined from optical measurements of the FRET calibration standard, comprising:
The FRET standard of claim 2, where the FRET standard relates a measured intensity of acceptor fluorescence sensitization to a corresponding amount of donor quenching according to:
Dependencies are inferred from the claim text.