Localization of protein-protein interactions among three fluorescent proteins in a single living cell: three-color fret microscopy

Yuansheng Sunl, Cynthia F. SunlBooker, Richard Day, Ammasi Periasamy

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


F0̈rster 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, themTFP-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.

Original languageEnglish
Title of host publicationProgress in Biomedical Optics and Imaging - Proceedings of SPIE
StatePublished - 2009
EventMultiphoton Microscopy in the Biomedical Sciences IX - San Jose, CA, United States
Duration: Jan 25 2009Jan 27 2009


OtherMultiphoton Microscopy in the Biomedical Sciences IX
CountryUnited States
CitySan Jose, CA


  • Acceptor photobleaching FRET
  • F0̈rster resonance energy transfer (FRET)
  • Fluorescence lifetime imaging (FLIM)-FRET microscopy
  • Microscopy
  • Monomeric teal fluorescent protein (mTFP)
  • Protein-protein interactions
  • Spectral FRET microscopy
  • Tandem dimer Tomato (tdTomato)
  • Three-color FRET microscopy
  • Venus

ASJC Scopus subject areas

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

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