FLIM-FRET microscopy to visualize transcription factor interactions in the nucleus of the living cell

Richard Day, Ignacio A. Demarco, Ty C. Voss, Ye Chen, Ammasi Periasamy

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

2 Citations (Scopus)

Abstract

Wide-field fluorescence microscopy was used to monitor the co-localization of the homeodomain (HD) transcription factor Pit-1 and the basic-leucine zipper protein CCAAT/enhancer binding protein alpha (C/EBPα), each labeled with fluorescent proteins (FP) in the living cell nucleus. Fluorescence resonance energy transfer (FRET) microscopy was used to resolve the angstrom-scale spatial relationships of these expressed proteins, and the effect of a Pit-1 point mutation on the interaction with C/EBPα was characterized. Two-photon excitation fluorescence lifetime imaging microscopy (2p-FLIM) was then used as an independent method to detect these protein interactions. The excited-state lifetime for the cyan FP (CFP) labeling C/EBPα was determined, and the measurements were repeated in cells co-expressing yellow FP (YFP) labeled-proteins. The CFP lifetime was decreased in the presence of the YFP acceptor, which is consistent with donor quenching by FRET. This was verified by acceptor photobleaching, which caused a shift in the donor lifetime to that similar to the donor alone. However, a significant limitation of this technique was demonstrated by the observation that high-energy 2p-excitation resulted in CFP photobleaching and a parallel decrease in its excited-state lifetime. The key question is whether the sensitivity of this imaging approach will be sufficient to acquire significant data from living cells expressing physiological levels of the labeled proteins.

Original languageEnglish (US)
Title of host publicationProceedings of SPIE - The International Society for Optical Engineering
EditorsA. Periasmy, P.T.C. So
Pages36-43
Number of pages8
Volume5323
DOIs
StatePublished - 2004
Externally publishedYes
EventProgress in Biomedical Optics and Imaging - Multiphoton Microscopy in the Biomedical Sciences IV - San Jose, CA, United States
Duration: Jan 25 2004Jan 27 2004

Other

OtherProgress in Biomedical Optics and Imaging - Multiphoton Microscopy in the Biomedical Sciences IV
CountryUnited States
CitySan Jose, CA
Period1/25/041/27/04

Fingerprint

Transcription factors
resonance fluorescence
Microscopic examination
energy transfer
Cells
microscopy
proteins
Proteins
nuclei
interactions
Photobleaching
life (durability)
Excited states
excitation
Imaging techniques
Fluorescence Resonance Energy Transfer
Excitation energy
Fasteners
Fluorescence microscopy
zippers

Keywords

  • Colocalization
  • Fluorescence lifetime imaging microscopy (FLIM)
  • Fluorescence resonance energy transfer (FRET) microscopy
  • Green fluorescent protein (GFP)
  • Transcription factors
  • Two-photon (2p) excitation

ASJC Scopus subject areas

  • Electrical and Electronic Engineering
  • Condensed Matter Physics

Cite this

Day, R., Demarco, I. A., Voss, T. C., Chen, Y., & Periasamy, A. (2004). FLIM-FRET microscopy to visualize transcription factor interactions in the nucleus of the living cell. In A. Periasmy, & P. T. C. So (Eds.), Proceedings of SPIE - The International Society for Optical Engineering (Vol. 5323, pp. 36-43) https://doi.org/10.1117/12.540016

FLIM-FRET microscopy to visualize transcription factor interactions in the nucleus of the living cell. / Day, Richard; Demarco, Ignacio A.; Voss, Ty C.; Chen, Ye; Periasamy, Ammasi.

Proceedings of SPIE - The International Society for Optical Engineering. ed. / A. Periasmy; P.T.C. So. Vol. 5323 2004. p. 36-43.

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

Day, R, Demarco, IA, Voss, TC, Chen, Y & Periasamy, A 2004, FLIM-FRET microscopy to visualize transcription factor interactions in the nucleus of the living cell. in A Periasmy & PTC So (eds), Proceedings of SPIE - The International Society for Optical Engineering. vol. 5323, pp. 36-43, Progress in Biomedical Optics and Imaging - Multiphoton Microscopy in the Biomedical Sciences IV, San Jose, CA, United States, 1/25/04. https://doi.org/10.1117/12.540016
Day R, Demarco IA, Voss TC, Chen Y, Periasamy A. FLIM-FRET microscopy to visualize transcription factor interactions in the nucleus of the living cell. In Periasmy A, So PTC, editors, Proceedings of SPIE - The International Society for Optical Engineering. Vol. 5323. 2004. p. 36-43 https://doi.org/10.1117/12.540016
Day, Richard ; Demarco, Ignacio A. ; Voss, Ty C. ; Chen, Ye ; Periasamy, Ammasi. / FLIM-FRET microscopy to visualize transcription factor interactions in the nucleus of the living cell. Proceedings of SPIE - The International Society for Optical Engineering. editor / A. Periasmy ; P.T.C. So. Vol. 5323 2004. pp. 36-43
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