21 June 2004 FLIM-FRET microscopy to visualize transcription factor interactions in the nucleus of the living cell
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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.
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Richard N. Day, Richard N. Day, Ignacio A. Demarco, Ignacio A. Demarco, Ty C. Voss, Ty C. Voss, Ye Chen, Ye Chen, Ammasi Periasamy, 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); doi: 10.1117/12.540016; https://doi.org/10.1117/12.540016

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