European Biophysics Journal

, Volume 27, Issue 4, pp 377–389

Intensity-based energy transfer measurements in digital imaging microscopy

  • Péter Nagy
  • György Vámosi
  • Andrea Bodnár
  • Stephen J. Lockett
  • János Szöllősi
ARTICLE

Abstract

Investigation of protein-protein associations is important in understanding structure and function relationships in living cells. Using Förster-type resonance energy transfer between donor and acceptor labeled monoclonal antibodies we can assess the cell surface topology of membrane proteins against which the antibodies were raised. In our current work we elaborated a quantitative image microscopic technique based on the measurement of fluorescence intensities to calculate the energy transfer efficiency on a pixel-by-pixel basis. We made use of the broad excitation and emission spectrum of cellular autofluorescence for background correction of images. In addition to the reference autofluorescence images (UV background) we recorded three fluorescent images (donor, acceptor and energy transfer signal) of donor-acceptor double labeled samples, and corrected for spectral spillage of the directly excited donor and acceptor fluorescence into the energy transfer image. After careful image registration we were able to calculate the energy transfer efficiency on a pixel-by-pixel basis. In this paper, we also present a critical comparison between results obtained with this method and other approaches (photobleaching and flow cytometric energy transfer measurements).

Key words Energy transfer Protein association Digital imaging microscopy Background correction 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Copyright information

© Springer-Verlag Berlin Heidelberg 1998

Authors and Affiliations

  • Péter Nagy
    • 1
  • György Vámosi
    • 1
  • Andrea Bodnár
    • 1
  • Stephen J. Lockett
    • 3
  • János Szöllősi
    • 1
  1. 1.Department of Biophysics and Cell Biology, University Medical School of Debrecen, H-4012 Debrecen, Hungary e-mail: nagyp@jaguar.dote.huHU
  2. 2.Biophysical Research Group of the Hungarian Academy of Sciences, University Medical School of Debrecen, H-4012 Debrecen, HungaryHU
  3. 3.Lawrence Berkeley Laboratory, Berkeley, California

Personalised recommendations