Intensity-based energy transfer measurements in digital imaging microscopy

Péter Nagy, G. Vámosi, Andrea Bodnár, Stephen J. Lockett, J. Szöllősi

Research output: Contribution to journalArticle

65 Citations (Scopus)

Abstract

Investigation of protein-protein associations is important in understanding structure and function relationships in living cells. Using Forster-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 pixelby-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).

Original languageEnglish
Pages (from-to)377-389
Number of pages13
JournalEuropean Biophysics Journal
Volume27
Issue number4
DOIs
Publication statusPublished - 1998

Fingerprint

Energy Transfer
Microscopy
Fluorescence
Photobleaching
Fluorescence Resonance Energy Transfer
Membrane Proteins
Proteins
Monoclonal Antibodies
Antibodies

Keywords

  • Background correction
  • Digital imaging microscopy
  • Energy transfer
  • Protein association

ASJC Scopus subject areas

  • Biophysics

Cite this

Intensity-based energy transfer measurements in digital imaging microscopy. / Nagy, Péter; Vámosi, G.; Bodnár, Andrea; Lockett, Stephen J.; Szöllősi, J.

In: European Biophysics Journal, Vol. 27, No. 4, 1998, p. 377-389.

Research output: Contribution to journalArticle

Nagy, Péter ; Vámosi, G. ; Bodnár, Andrea ; Lockett, Stephen J. ; Szöllősi, J. / Intensity-based energy transfer measurements in digital imaging microscopy. In: European Biophysics Journal. 1998 ; Vol. 27, No. 4. pp. 377-389.
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