public class AlgorithmFRETBleedThrough
extends AlgorithmBase

Fluorescence Resonance Energy Transfer FRET refers to the nonradiative transfer of energy from an excited state donor fluorescent molecule to a nearby acceptor fluorescent molecule. The energy transfer efficiency E, 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: E = 1/[1 + (r/R0)**6] (eq. 1) where R0, the Forster critical distance, is the distance at which E = 0.5. We also have E = 1 - FDA/FD, (eq. 2) where FDA is the donor fluorescence in the presence of an acceptor and FD is the donor fluorescence in the absence of the acceptor. The equivalent of an acceptor's absence can be created by photobleaching the acceptor. Thus, a method using equation 2, can be performed on a single sample by measuring the donor fluorescence before and after photobleaching the acceptor molecules.

Because FRET falls off as the sixth power of the distance between the donor and the acceptor, no FRET occurs for distances greater than 2R0. Since R0 is on the order of 10 to 70 Angstroms, by performing FRET measurements it is possible to distinguish proteins that are merely nearby in the same compartment from those proteins that are interacting with each other.

This code is a port of the MATLAB routine TwoD_FRET_and_2FP_bleed_thru_from_1FP provided by Dr. Stephen Lockett.

The code here requires 3 images - image with a FP1 dye taken with a FP1 filter, image with a FP1 dye taken with a FRET filter, and image with a FP1 dye taken with an FP2 filter. The code is run with a set of 3 images with donor dye only to obtain the DFP to FRET bleed thru and the DFP to AFP bleed thru. The code is run with a set of 3 images with acceptor dye only to obtain the AFP to FRET bleed thru and the AFP to DFP bleed thru. When these 4 bleed thru parameters have been obtained, then a set of 3 images containing both donor and acceptor dye is used with AlgorithmFRETEfficiency to obtain a FRET efficiency on each active VOI. So a set of 9 images is used to obtain the FRET efficiency. Only 1 user selected color will be used from a color image.

The code here assumes the presence of 2 or more VOI regions - a backgorund region and 1 or more active regions whose areas are used for the bleedthrough calculations. If a pixel is saturated in any of the 3 images, then that pixel will be excluded from all calculations. The VOIs must all be placed in the source image and the source image must be an image of the 1FP taken with a 1FP filter. That is, the source image must either be an image with donor dye only taken with a donor fluorescent peak filter or an image with acceptor dye only taken with an acceptor fluorescent peak filter. Radio buttons in the dialog box are used to select an active VOI or a blue background VOI. Either an ellipse VOI, rectangle VOI, levelset VOI, or polyline VOI will be selected from the top MIPAV toolbar. Hit the NEW_VOI button to create other active VOIs. Just do not use blue as an active VOI color. The background region will have a smaller average intensity than the active region.

denom = mean(FP1 dye with FP1 filter active VOI) - mean(FP1 dye with FP1 filter background VOI)

FRET bleed through = [mean(FP1 dye with FRET filter active VOI) - mean(FP1 dye with FRET filter background VOI)]/denom

FP2 bleed through = [mean(FP1 dye with FP2 filter active VOI) - mean(FP1 dye with FP2 filter background VOI)]/ denom

The code outputs the FP1 to FRET bleed thru and the FP1 to FP2 bleed thru for each active VOI region.

Reference: 1.) "Imaging Protein-Protein Interactions Using Fluorescence Energy Transfer Microscopy" by Anne K. Kenworthy, Methods, 24, 2001, pp. 289 - 296.

2.) "Quantitative Fluorescence Resonance Energy Transfer Measurements Using Fluorescence Microscopy" by Gerald W. Gordon, Gail Berry, Xiao Huan Liang, Beth Levine, and Brian Herman, Biophysical Journal, Volume 74, May, 1998, pp. 2702-2713.