Absolute two-photon absorption spectra and two-photon brightness of orange and red fluorescent proteins

J Phys Chem B. 2009 Jan 29;113(4):855-9. doi: 10.1021/jp8087379.


Fluorescent proteins with long emission wavelengths are particularly attractive for deep tissue two-photon microscopy. Surprisingly, little is known about their two-photon absorption (2PA) properties. We present absolute 2PA spectra of a number of orange and red fluorescent proteins, including DsRed2, mRFP, TagRFP, and several mFruit proteins, in a wide range of excitation wavelengths (640-1400 nm). To evaluate 2PA cross section (sigma(2)), we use a new method relying only on the optical properties of the intact mature chromophore. In the tuning range of a mode-locked Ti:sapphire laser, 700-1000 nm, TagRFP possesses the highest two-photon cross section, sigma(2) = 315 GM, and brightness, sigma(2)phi = 130 GM, where phi is the fluorescence quantum yield. At longer wavelengths, 1000-1100 nm, tdTomato has the largest values, sigma(2) = 216 GM and sigma(2)phi = 120 GM, per protein chain. Compared to the benchmark EGFP, these proteins present 3-4 times improvement in two-photon brightness.

Publication types

  • Letter
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Color
  • Luminescent Proteins / chemistry*
  • Models, Molecular
  • Molecular Conformation
  • Photons
  • Spectrometry, Fluorescence
  • Spectrophotometry


  • Luminescent Proteins