Simplified Instrument Calibration for Wide-Field Fluorescence Resonance Energy Transfer (FRET) Measured by the Sensitized Emission Method

Audrey Menaesse, Daniel Sumetsky, Nicolas Emanuely, Jeremy L. Stein, Evan M. Gates, Brenton D. Hoffman, Nada N. Boustany

Research output: Contribution to journalArticlepeer-review

10 Scopus citations


Fӧrster (or fluorescence) resonance energy transfer (FRET) is a quantifiable energy transfer in which a donor fluorophore nonradiatively transfers its excitation energy to an acceptor fluorophore. A change in FRET efficiency indicates a change of proximity and environment of these fluorophores, which enables the study of intermolecular interactions. Measurement of FRET efficiency using the sensitized emission method requires a donor–acceptor calibrated system. One of these calibration factors named the G factor, which depends on instrument parameters related to the donor and acceptor measurement channels and on the fluorophores quantum efficiencies, can be determined in several different ways and allows for conversion of the raw donor and acceptor emission signals to FRET efficiency. However, the calculated value of the G factor from experimental data can fluctuate significantly depending on the chosen experimental method and the size of the sample. In this technical note, we extend the results of Gates et al. (Cytometry Part A 95A (2018) 201–213) by refining the calibration method used for calibration of FRET from image pixel data. Instead of using the pixel histograms of two constructs with high and low FRET efficiency to determine the G factor, we use pixel histogram data from one construct of known efficiency. We validate this method by determining the G factor with the same constructs developed and used by Gates et al. and comparing the results from the two approaches. While the two approaches are equivalent theoretically, we demonstrate that the use of a single construct with known efficiency provides a more precise experimental measurement of the G factor that can be attained by collecting a smaller number of images.

Original languageEnglish (US)
Pages (from-to)407-416
Number of pages10
JournalCytometry Part A
Issue number4
StatePublished - Apr 2021

All Science Journal Classification (ASJC) codes

  • Pathology and Forensic Medicine
  • Histology
  • Cell Biology


  • FRET efficiency
  • calibration
  • fluorescence microscopy
  • intensity-based method
  • molecular imaging


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