An orange fluorescent protein with a large stokes shift for single-excitation multicolor FCCS and FRET imaging

Daria M. Shcherbakova, Mark A. Hink, Linda Joosen, Theodorus W.J. Gadella, Vladislav V. Verkhusha

Research output: Contribution to journalArticlepeer-review

170 Scopus citations


Multicolor imaging based on genetically encoded fluorescent proteins (FPs) is a powerful approach to study several dynamic processes in a live cell. We report a monomeric orange FP with a large Stokes shift (LSS), called LSSmOrange (excitation/emission at 437/572 nm), which fills up an existing spectral gap between the green-yellow and red LSSFPs. Brightness of LSSmOrange is five-fold larger than that of the brightest red LSSFP and similar to the green-yellow LSSFPs. LSSmOrange allows numerous multicolor applications using a single-excitation wavelength that was not possible before. Using LSSmOrange we developed four-color single-laser fluorescence cross-correlation spectroscopy, solely based on FPs. The quadruple cross-correlation combined with photon counting histogram techniques allowed quantitative single-molecule analysis of particles labeled with four FPs. LSSmOrange was further applied to simultaneously image two Förster resonance energy transfer pairs, one of which is the commonly used CFP-YFP pair, with a single-excitation laser line. The combination of LSSmOrange-mKate2 and CFP-YFP biosensors enabled imaging of apoptotic activity and calcium fluctuations in real time. The LSSmOrange mutagenesis, low-temperature, and isotope effect studies revealed a proton relay for the excited-state proton transfer responsible for the LSS phenotype.

Original languageEnglish (US)
Pages (from-to)7913-7923
Number of pages11
JournalJournal of the American Chemical Society
Issue number18
StatePublished - May 9 2012

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry


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