Fluorescence resonance energy transfer (FRET) imaging of a single living cell using green fluorescent protein

Ammasi Periasamy, Steve A. Kay, Richard Day

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

7 Citations (Scopus)

Abstract

Fluorescence resonance energy transfer (FRET) imaging microscopy is a unique tool to visualize the spatiotemporal dynamics of protein interactions in living cells. Genetic vectors that encode protein fusions with green fluorescent protein (GFP) provide a method for imaging protein localization in living cells. We used FRET to study dimerization of the pituitary-specific transcription factor Pit-1 fused to GFP and BFP. A fusion protein containing GFP separated from BFP by 29 amino acids served as a positive control for FRET. Transcriptional activity of the GFP- and BFP-Pit-1 fusion proteins was demonstrated by their ability to activate the prolactin gene promoter. Using optimized excitation and emission filters, cells expressing the fluorescently-tagged Pit-1 proteins were imaged with a back- thinned, back-illuminated CCD chip that has about 50% quantum efficiency in the blue range. 2D FRET images acquired at the focal plane demonstrated Pit-1 protein associations in the nucleus of living cells. The significance of 2- and 3-D energy transfer imaging from these living cells is discussed.

Original languageEnglish (US)
Title of host publicationProceedings of SPIE - The International Society for Optical Engineering
Pages58-66
Number of pages9
Volume2983
DOIs
StatePublished - 1997
Externally publishedYes
EventFunctional Imaging and Optical Manipulation of Living Cells - San Jose, CA, United States
Duration: Feb 10 1997Feb 10 1997

Other

OtherFunctional Imaging and Optical Manipulation of Living Cells
CountryUnited States
CitySan Jose, CA
Period2/10/972/10/97

Fingerprint

Energy Transfer
resonance fluorescence
Green Fluorescent Proteins
Fluorescence
energy transfer
Cells
Imaging
proteins
Proteins
Protein
Imaging techniques
Cell
Fusion reactions
Transcription Factor Pit-1
Fusion
fusion
Genetic Vectors
Dimerization
Fluorescence Resonance Energy Transfer
Quantum efficiency

Keywords

  • Blue fluorescent protein
  • CCD camera
  • Green fluorescent protein
  • Huorescence resonance energy transfer (FRET)
  • Microscopy imaging
  • Pit-1 protein and dimerization

ASJC Scopus subject areas

  • Applied Mathematics
  • Computer Science Applications
  • Electrical and Electronic Engineering
  • Electronic, Optical and Magnetic Materials
  • Condensed Matter Physics

Cite this

Periasamy, A., Kay, S. A., & Day, R. (1997). Fluorescence resonance energy transfer (FRET) imaging of a single living cell using green fluorescent protein. In Proceedings of SPIE - The International Society for Optical Engineering (Vol. 2983, pp. 58-66) https://doi.org/10.1117/12.274324

Fluorescence resonance energy transfer (FRET) imaging of a single living cell using green fluorescent protein. / Periasamy, Ammasi; Kay, Steve A.; Day, Richard.

Proceedings of SPIE - The International Society for Optical Engineering. Vol. 2983 1997. p. 58-66.

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

Periasamy, A, Kay, SA & Day, R 1997, Fluorescence resonance energy transfer (FRET) imaging of a single living cell using green fluorescent protein. in Proceedings of SPIE - The International Society for Optical Engineering. vol. 2983, pp. 58-66, Functional Imaging and Optical Manipulation of Living Cells, San Jose, CA, United States, 2/10/97. https://doi.org/10.1117/12.274324
Periasamy A, Kay SA, Day R. Fluorescence resonance energy transfer (FRET) imaging of a single living cell using green fluorescent protein. In Proceedings of SPIE - The International Society for Optical Engineering. Vol. 2983. 1997. p. 58-66 https://doi.org/10.1117/12.274324
Periasamy, Ammasi ; Kay, Steve A. ; Day, Richard. / Fluorescence resonance energy transfer (FRET) imaging of a single living cell using green fluorescent protein. Proceedings of SPIE - The International Society for Optical Engineering. Vol. 2983 1997. pp. 58-66
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