Distance calibration of fluorescence energy-transfer values on cell surfaces

Peter Salga; Andrea Bodnar; Sandor Damjanovich; Laszlo Matyus

doi:10.1117/12.310559

9 June 1998 Distance calibration of fluorescence energy-transfer values on cell surfaces

Peter Salga, Andrea Bodnar, Sandor Damjanovich, Laszlo Matyus

Proceedings Volume 3261, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing V; (1998) https://doi.org/10.1117/12.310559
Event: BiOS '98 International Biomedical Optics Symposium, 1998, San Jose, CA, United States

Abstract

Different kinds of cell surface receptor clusters have been discovered recently using fluorescence resonance energy transfer (FRET) measurements. This method is capable for identifying molecular interactions, however the exact distances remain obscure, because the classical Foerster efficiency-distance relationship is valid only in the case of one donor one acceptor systems. This condition can not be fulfilled when cell surface molecules are labeled with monoclonal antibodies carrying different number of fluorescent donor and acceptor molecules. Our aim was to carry out FRET measurements on such cell surface receptors, where the distances are constant, and the only changing parameter is the donor-acceptor ratio of the used labels. For our experiments we used JY B lymphoblastoid cells, and we labeled the MHC class I heavy chain with KE-2 or W6/32 monoclonal antibodies and the length chain with L-368 monoclonal antibody tagged with different numbers of donor or acceptor molecules. The FRET efficiencies were measured either in a microscope using the photobleaching method or in a fluorescence activated cell sorter. We changed the donor acceptor ratio in a wide range in order to make a suitable calibration curve for other FRET experiments. The obtained calibration curve gives us the possibility to relate FRET efficiencies to real distances of cell surface receptors. Another source of deviation in the FRET efficiencies arise from the selected method. There was a marked difference between the FRET efficiencies measured by flow cytometry and with the photobleaching method even on same cells and between same epitopes.

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Peter Salga, Andrea Bodnar, Sandor Damjanovich, and Laszlo Matyus "Distance calibration of fluorescence energy-transfer values on cell surfaces", Proc. SPIE 3261, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing V, (9 June 1998); https://doi.org/10.1117/12.310559

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KEYWORDS

Fluorescence resonance energy transfer

Energy transfer

Luminescence

Molecules

Monoclonal antibodies

Calibration

Receptors

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