Calibration of fluorescence resonance energy transfer in microscopy
- San Jose, CA
- Sunnyvale, CA
- Moutain View, CA
- Santa Cruz, CA
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.
- Assignee:
- Kairos Scientific, Inc. (San Diego, CA)
- Patent Number(s):
- US 6456734
- Application Number:
- 09/092,316
- OSTI ID:
- 874767
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
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
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