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

János Roszik, Duarte Lisboa, János Szöllosi, György Vereb

Research output: Contribution to journalArticle

46 Citations (Scopus)


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 FRETefficiency 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.

Original languageEnglish
Pages (from-to)761-767
Number of pages7
JournalCytometry Part A
Issue number9
Publication statusPublished - Sep 1 2009



  • 3D image processing
  • Confocal laser scanning microscopy
  • FRET calibration
  • Fluorescence resonance energy transfer
  • ImageJ
  • Intensity-based FRET
  • Ratiometric FRET

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Histology
  • Cell Biology

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