Computer program for analyzing donor photobleaching FRET image series
Gergely Szentesi
Department of Biophysics and Cell Biology, Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary
Cell Biophysics Research Group of the Hungarian Academy of Sciences, Hungary
Search for more papers by this authorGyörgy Vereb
Department of Biophysics and Cell Biology, Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary
Search for more papers by this authorGábor Horváth
Department of Biophysics and Cell Biology, Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary
Search for more papers by this authorAndrea Bodnár
Cell Biophysics Research Group of the Hungarian Academy of Sciences, Hungary
Search for more papers by this authorÁkos Fábián
Department of Biophysics and Cell Biology, Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary
Search for more papers by this authorJános Matkó
Department of Immunology, Eötvös Loránd University, Budapest, Hungary
Search for more papers by this authorRezső Gáspár
Department of Biophysics and Cell Biology, Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary
Search for more papers by this authorSándor Damjanovich
Department of Biophysics and Cell Biology, Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary
Cell Biophysics Research Group of the Hungarian Academy of Sciences, Hungary
Search for more papers by this authorCorresponding Author
László Mátyus
Department of Biophysics and Cell Biology, Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary
Department of Biophysics and Cell Biology, Medical and Health Science Center, University of Debrecen, P.O. Box 39, H-4012 Debrecen, HungarySearch for more papers by this authorAttila Jenei
Department of Biophysics and Cell Biology, Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary
Search for more papers by this authorGergely Szentesi
Department of Biophysics and Cell Biology, Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary
Cell Biophysics Research Group of the Hungarian Academy of Sciences, Hungary
Search for more papers by this authorGyörgy Vereb
Department of Biophysics and Cell Biology, Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary
Search for more papers by this authorGábor Horváth
Department of Biophysics and Cell Biology, Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary
Search for more papers by this authorAndrea Bodnár
Cell Biophysics Research Group of the Hungarian Academy of Sciences, Hungary
Search for more papers by this authorÁkos Fábián
Department of Biophysics and Cell Biology, Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary
Search for more papers by this authorJános Matkó
Department of Immunology, Eötvös Loránd University, Budapest, Hungary
Search for more papers by this authorRezső Gáspár
Department of Biophysics and Cell Biology, Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary
Search for more papers by this authorSándor Damjanovich
Department of Biophysics and Cell Biology, Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary
Cell Biophysics Research Group of the Hungarian Academy of Sciences, Hungary
Search for more papers by this authorCorresponding Author
László Mátyus
Department of Biophysics and Cell Biology, Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary
Department of Biophysics and Cell Biology, Medical and Health Science Center, University of Debrecen, P.O. Box 39, H-4012 Debrecen, HungarySearch for more papers by this authorAttila Jenei
Department of Biophysics and Cell Biology, Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary
Search for more papers by this authorAbstract
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 (β2-microglobulin and heavy chain) of major histocompatibility complex (MHC) class I glycoproteins and FRET between MHC I and MHC II 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. © 2005 International Society for Analytical Cytology
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