
Proceedings Paper
Localization of protein-protein interactions among three fluorescent proteins in a single living cell: three-color FRET microscopyFormat | Member Price | Non-Member Price |
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Paper Abstract
Förster resonance energy transfer (FRET) methodology has been used for over 30 years to localize protein-protein
interactions in living specimens. The cloning and modification of various visible fluorescent proteins (FPs) has generated
a variety of new probes that can be used as FRET pairs to investigate the protein associations in living cells. However,
the spectral cross-talk between FRET donor and acceptor channels has been a major limitation to FRET microscopy.
Many investigators have developed different ways to eliminate the bleedthrough signals in the FRET channel for one
donor and one acceptor. We developed a novel FRET microscopy method for studying interactions among three
chromophores: three-color FRET microscopy. We generated a genetic construct that directly links the three
FPs - monomeric teal FP (mTFP), Venus and tandem dimer Tomato (tdTomato), and demonstrated the occurrence of mutually
dependent energy transfers among the three FPs. When expressed in cells and excited with the 458 nm laser line, the
mTFP-Venus-tdTomato fusion proteins yielded parallel (mTFP to Venus and mTFP to tdTomato) and sequential (mTFP
to Venus and then to tdTomato) energy transfer signals. To quantify the FRET signals in the three-FP system in a single
living cell, we developed an algorithm to remove all the spectral cross-talk components and also to separate different
FRET signals at a same emission channel using the laser scanning spectral imaging and linear unmixing techniques on
the Zeiss510 META system. Our results were confirmed with fluorescence lifetime measurements and using acceptor
photobleaching FRET microscopy.
Paper Details
Date Published: 13 February 2009
PDF: 10 pages
Proc. SPIE 7183, Multiphoton Microscopy in the Biomedical Sciences IX, 71832E (13 February 2009); doi: 10.1117/12.814877
Published in SPIE Proceedings Vol. 7183:
Multiphoton Microscopy in the Biomedical Sciences IX
Ammasi Periasamy; Peter T. C. So, Editor(s)
PDF: 10 pages
Proc. SPIE 7183, Multiphoton Microscopy in the Biomedical Sciences IX, 71832E (13 February 2009); doi: 10.1117/12.814877
Show Author Affiliations
Yuansheng Sun, Univ. of Virginia (United States)
Cynthia F. Booker, Univ. of Virginia Health System (United States)
Cynthia F. Booker, Univ. of Virginia Health System (United States)
Richard N. Day, Univ. of Virginia Health System (United States)
Ammasi Periasamy, Univ. of Virginia (United States)
Ammasi Periasamy, Univ. of Virginia (United States)
Published in SPIE Proceedings Vol. 7183:
Multiphoton Microscopy in the Biomedical Sciences IX
Ammasi Periasamy; Peter T. C. So, Editor(s)
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