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Evaluation of intensity-based ratiometric FRET in image cytometry—Approaches and a software solution

  1. János Roszik1,
  2. Duarte Lisboa1,
  3. János Szöllősi1,2,
  4. György Vereb1,*

Article first published online: 9 JUL 2009

DOI: 10.1002/cyto.a.20747

Issue

Cytometry Part A

Cytometry Part A

Volume 75A, Issue 9, pages 761–767, September 2009

Additional Information

How to Cite

Roszik, J., Lisboa, D., Szöllősi, J. and Vereb, G. (2009), Evaluation of intensity-based ratiometric FRET in image cytometry—Approaches and a software solution. Cytometry Part A, 75A: 761–767. doi: 10.1002/cyto.a.20747

Author Information

  1. 1

    Department of Biophysics and Cell Biology, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary

  2. 2

    Cell Biology and Signaling Research Group of the Hungarian Academy of Sciences, University of Debrecen, Debrecen, Hungary

*Department of Biophysics and Cell Biology, Medical and Health Science Center, University of Debrecen, Debrecen, P.O. Box 39, Nagyerdei krt. 98, Debrecen H-4012, Hungary

Publication History

  1. Issue published online: 13 AUG 2009
  2. Article first published online: 9 JUL 2009
  3. Manuscript Accepted: 3 MAY 2009
  4. Manuscript Revised: 23 APR 2009
  5. Manuscript Received: 16 DEC 2008

Funded by

  • European Community. Grant Numbers: EU FP6 LSHBCT-2004-503467, EU FP6 LSHC-CT-2005-018914, EU FP6 MRTN-CT-2005-019481
  • Hungarian National Research Fund. Grant Numbers: K62648, K75752, K68763

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Keywords:

  • fluorescence resonance energy transfer;
  • ratiometric FRET;
  • intensity-based FRET;
  • FRET calibration;
  • confocal laser scanning microscopy;
  • ImageJ;
  • 3D image processing

Abstract

The intensity-based ratiometric FRET (fluorescence resonance energy transfer) method is a powerful technique for following molecular interactions in living cells. Since it is not based on irreversibly destroying the donor or the acceptor fluorophores, the time course of changes in FRET efficiency values can be monitored by this method. ImageJ, a sophisticated software tool for many types of image processing allows users to extend it with programs for various purposes. Implementing intensity-based ratiometric FRET with ImageJ vastly enhances the applicability of the FRET method. We developed an efficient ImageJ plugin, RiFRET, which calculates FRET efficiency on a pixel-by-pixel basis from ratiometric FRET images. It allows the user to correct for channel cross-talk (bleed-through) and to calculate FRET from image stacks, i.e., from 3D data sets. Semiautomatic processing for larger datasets is also included in the program. Furthermore, several options for calibrating FRET efficiency calculations were tested and their applicability to various expression systems is discussed. Although the ratiometric FRET method is widely applied, our plugin is the first freely available software for evaluating such FRET data. The program is user friendly and provides reliable, standardized results. © 2009 International Society for Advancement of Cytometry

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