Multiphoton FLIM: A reliable FRET detection tool in cell biological applications

R. V. Krishnan, Eva Biener, V. E. Centonze, A. Gertler, B. Herman

Research output: Contribution to journalConference articlepeer-review

2 Scopus citations


Fluorescence lifetime imaging microscopy (FLIM) using multiphoton excitation is emerging as a reliable quantitative tool for measuring fluorescence resonance energy transfer (FRET) in living cells. By virtue of being free from spectroscopic artifacts encountered in conventional FRET detection methods, multiphoton FLIM methods offer the advantages of high spatial and temporal resolution, faster data acquisition and data analysis. We compare the FRET results obtained by two different methods namely (i) multiphoton excitation lifetime-based FRET and (ii) single photon excitation intensity-based acceptor photobleaching FRET. Using the same biological samples, we apply these two different methods in understanding the growth hormone receptor dimerization kinetics at the cell surface of human embryonic kidney cells. We conclude that the multiphoton FLIM using the streak-camera approach provides the best ability to monitor FRET in dynamic situations where high temporal and spatial resolution are required with minimal photodamage/phototoxicity.

Original languageEnglish (US)
Pages (from-to)63-70
Number of pages8
JournalProceedings of SPIE - The International Society for Optical Engineering
StatePublished - 2004
EventProgress in Biomedical Optics and Imaging - Multiphoton Microscopy in the Biomedical Sciences IV - San Jose, CA, United States
Duration: Jan 25 2004Jan 27 2004


  • Acceptor photobleaching FRET
  • FRET
  • Multiphoton FLIM
  • Streak camera


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