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

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

doi:10.1117/12.540016

21 June 2004 FLIM-FRET microscopy to visualize transcription factor interactions in the nucleus of the living cell

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

Proceedings Volume 5323, Multiphoton Microscopy in the Biomedical Sciences IV; (2004) https://doi.org/10.1117/12.540016
Event: Biomedical Optics 2004, 2004, San Jose, CA, United States

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/EBPa), 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/EBPa 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/EBPa 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.

Citation Download Citation

Richard N. Day, Ignacio A. Demarco, Ty C. Voss, Ye Chen, and Ammasi Periasamy "FLIM-FRET microscopy to visualize transcription factor interactions in the nucleus of the living cell", Proc. SPIE 5323, Multiphoton Microscopy in the Biomedical Sciences IV, (21 June 2004); https://doi.org/10.1117/12.540016

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