Difference between revisions of "Microscopy: Fluorescent Resonance Energy Transfer (FRET) Bleed Through and Efficiency"
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Revision as of 13:43, 31 July 2012
This page is a stub.
Refer to thh HTML version of this MIPAV algorithm http://mipav.cit.nih.gov/documentation/HTML%20Algorithms/MicroscopyFRETBleedThroughEfficiency.html
Summary
This section provides information on and discusses how to use the following two FRET algorithms:
- FRET Bleed Through algorithm
- FRET Efficiency algorithm
Used consecutively, the FRET Bleed Through algorithm uses two sets of three 2D images and the FRET Efficiency algorithm uses one set of three 2D images to measure effects dependent on the proximity of fluorescent-labeled molecules.
You must first run the FRET Bleed Through algorithm twice: once on acceptor-dyed images and once on donor-dyed images. Using the results achieved from running this algorithm, you then use the FRET Efficiency algorithm to process images that were dyed with both the donor and acceptor dyes to obtain the FRET efficiency.
Background
Fluorescent resonance energy transfer (FRET) refers to the non-radiative transfer of energy from an excited fluorochrome, called a donor, to a nearby fluorescent molecule, called an acceptor. The FRET technique measures the fluorescence signals of the donor, the acceptor, and the FRET signal. If FRET occurs, the donor channel signal is quenched and the acceptor channel signal is sensitized or increased.
The energy transfer efficiency E is conventionally defined as the number of energy transfer events divided by the number of photons absorbed by the donor, is related to the distance R between the acceptor and donor by:
<math>
E= \frac{1}{\left [ 1 + \frac{r}{R_0}^6 \right ]}
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