Comparison of one- and two-photon fluorescence resonance energy transfer microscopy

A. Periasamy, M. Elangovan, Richard Day

Research output: Chapter in Book/Report/Conference proceedingConference contribution

Abstract

The physics and chemistry of fluorescent resonance energy transfer (FRET) have been well studied theoretically and experimentally for many years, but only with recent technical advances has it become feasible to apply FRET in biomedical research. FRET microscopy is a better method for studying the structure and localization of proteins under physiological conditions than are X-ray diffraction, nuclear magnetic resonance, or electron microscopy. In this study, we used four different light microscopy techniques to visualize the interactions of the transcription factor CAATT/enhancer binding protein alpha (C/EBPα) in living pituitary cells. In wide-field, confocal, and two-photon microscopy the FRET image provides 2-D spatial distribution of steady-state protein-protein interactions. The two-photon imaging technique provides a better FRET signal (less bleed through and photo bleaching) compared to the other two techniques. This information, although valuable, falls short of revealing transient interactions of proteins in real time. We will discuss the advantage of fluorescence lifetime methods to measure FRET signals at the moment of the protein-protein interactions at a resolution on the order of subnanoseconds, providing high temporal, as well as spatial resolution.

Original languageEnglish (US)
Title of host publicationProceedings of SPIE - The International Society for Optical Engineering
EditorsA. Periasamy, P.T.C. So
Pages366-372
Number of pages7
Volume4262
DOIs
StatePublished - 2001
Externally publishedYes
EventMultiphoton Microscopy in the Biomedical Sciences - San Jose, CA, United States
Duration: Jan 21 2001Jan 23 2001

Other

OtherMultiphoton Microscopy in the Biomedical Sciences
CountryUnited States
CitySan Jose, CA
Period1/21/011/23/01

Fingerprint

resonance fluorescence
Energy transfer
Microscopic examination
Photons
energy transfer
microscopy
proteins
Proteins
photons
Photobleaching
interactions
Transcription factors
Spatial distribution
Electron microscopy
bleaching
Optical microscopy
Fluorescence Resonance Energy Transfer
temporal resolution
imaging techniques
Physics

Keywords

  • CAATT/enhancer binding protein alpha (C/EBPα)
  • Confocal
  • Fluorescence lifetime imaging (FLIM)
  • Fluorescence resonance energy transfer (FRET)
  • Microscopy
  • Protein-protein interactions
  • Two-photon excitation
  • Wide-field

ASJC Scopus subject areas

  • Electrical and Electronic Engineering
  • Condensed Matter Physics

Cite this

Periasamy, A., Elangovan, M., & Day, R. (2001). Comparison of one- and two-photon fluorescence resonance energy transfer microscopy. In A. Periasamy, & P. T. C. So (Eds.), Proceedings of SPIE - The International Society for Optical Engineering (Vol. 4262, pp. 366-372) https://doi.org/10.1117/12.424577

Comparison of one- and two-photon fluorescence resonance energy transfer microscopy. / Periasamy, A.; Elangovan, M.; Day, Richard.

Proceedings of SPIE - The International Society for Optical Engineering. ed. / A. Periasamy; P.T.C. So. Vol. 4262 2001. p. 366-372.

Research output: Chapter in Book/Report/Conference proceedingConference contribution

Periasamy, A, Elangovan, M & Day, R 2001, Comparison of one- and two-photon fluorescence resonance energy transfer microscopy. in A Periasamy & PTC So (eds), Proceedings of SPIE - The International Society for Optical Engineering. vol. 4262, pp. 366-372, Multiphoton Microscopy in the Biomedical Sciences, San Jose, CA, United States, 1/21/01. https://doi.org/10.1117/12.424577
Periasamy A, Elangovan M, Day R. Comparison of one- and two-photon fluorescence resonance energy transfer microscopy. In Periasamy A, So PTC, editors, Proceedings of SPIE - The International Society for Optical Engineering. Vol. 4262. 2001. p. 366-372 https://doi.org/10.1117/12.424577
Periasamy, A. ; Elangovan, M. ; Day, Richard. / Comparison of one- and two-photon fluorescence resonance energy transfer microscopy. Proceedings of SPIE - The International Society for Optical Engineering. editor / A. Periasamy ; P.T.C. So. Vol. 4262 2001. pp. 366-372
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