public class AlgorithmFRETAcceptorPhotobleach
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 program compares the fluorescence of two 2D images or of two 2D slices inside a 2 slice 3D image - an image before acceptor photobleaching and an image after acceptor photobleaching. In the 3D image the prebleached slice is required to be the first slice and the postbleached slice is required to be the second slice. Before acceptor photobleaching, the donor fluorescence is quenched by FRET with the unbleached acceptor. After destroying the acceptor by photobleaching, the donor fluorescence will increase. Only 1 color will be used from a color image.

An optional registration may be performed before FRET. In this registration the prebleached image is registered to the postbleached image. AlgorithmRegOAR2D is used with the cost function being the only registration parameter the user can vary in the dialog box. Correlation ratio is the default cost function, but the user can also select least squares, normalized cross correlation, or normalized mutual information. The FRET will be performed with the registered prebleached image rather than on the original prebleached image. If the image is a color image, the selected color will determine the transformations for all the colors.

The code here assumes the presence of 1 or 2 or 3 VOI regions - a required donor region and optional background region and optional signal normalization region. The VOIs must all be placed in the postbleached image. Radio buttons in the dialog box are used to select a red photobleached or a blue background VOI or a green signal VOI. Either an ellipse VOI, rectangle VOI, or polyline VOI will be selected from the top MIPAV toolbar. There is no need to hit the NEW_VOI button. The background region will have a smaller average intensity than the donor region.

If a background VOI is present, the average of the prebleached background is subtracted from the average of the prebleached donor region and the average of the postbleached background is subtracted is subtracted from the average of the postbleached donor region. Then, the energy transfer efficiency is calculated as (background subtracted postbleached donor intensity - (background subtracted prebleached donor intensity)/ background subtracted postbleached donor intensity

A signal VOI cannot be used unless a background VOI is also present. Let b1 and s1 be the prebleached values and b2 and s2 be the postbleached values. The following equation is used to convert a donor value from the prebleached image into a donor value on the postbleached image: postbleached = ((s2 - b2)/(s1 - b1)) * prebleached + (b2*s1 - b1*s2)/(s1 - b1)

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.