Computer program for analyzing donor photobleaching FRET image series

Background: The photobleaching fluorescence resonance energy transfer (pbFRET) technique is a spectroscopic method to measure proximity relations between fluorescently labeled macromolecules using digital imaging microscopy. To calculate the energy transfer values one has to determine the bleaching time constants in pixel-by-pixel fashion from the image series recorded on the donor-only and donor and acceptor double-labeled samples. Because of the large number of pixels and the time-consuming calculations, this procedure should be assisted by powerful image data processing software. There is no commercially available software that is able to fulfill these requirements. Methods: New evaluation software was developed to analyze pbFRET data for Windows platform in National Instrument LabVIEW 6.1. This development environment contains a mathematical virtual instrument package, in which the Levenberg-Marquardt routine is also included. As a reference experiment, FRET efficiency between the two chains (beta 2-microglobulin and heavy chain) of major histocompatibility complex (MHC) class I glycoproteins and FRET between MHC I and MHC 11 molecules were determined in the plasma membrane of JY, human B lymphoma cells. Results: The bleaching time constants calculated on pixel-by-pixel basis can be displayed as a color-coded map or as a histogram from raw image format, Conclusion: In this report we introduce a new version of pbFRET analysis and data processing software that is able to generate a full analysis pattern of donor photobleaching image series under various conditions. (c) 2005 International Society for Analytical Cytology.