Two-Photon Excitation Spectra of Various Fluorescent Proteins within a Broad Excitation Range

Two-photon excitation fluorescence laser-scanning microscopy is the preferred method for studying dynamic processes in living organ models or even in living organisms. Thanks to near-infrared and infrared excitation, it is possible to penetrate deep into the tissue, reaching areas of interest relevant to life sciences and biomedicine. In those imaging experiments, two-photon excitation spectra are needed to select the optimal laser wavelength to excite as many fluorophores as possible simultaneously in the sample under consideration. The more fluorophores that can be excited, and the more cell populations that can be studied, the better access to their arrangement and interaction can be reached in complex systems such as immunological organs. However, for many fluorophores, the two-photon excitation properties are poorly predicted from the single-photon spectra and are not yet available, in the literature or databases. Here, we present the broad excitation range (760 nm to 1300 nm) of photon-flux-normalized two-photon spectra of several fluorescent proteins in their cellular environment. This includes the following fluorescent proteins spanning from the cyan to the infrared part of the spectrum: mCerulean3, mTurquoise2, mT-Sapphire, Clover, mKusabiraOrange2, mOrange2, LSS-mOrange, mRuby2, mBeRFP, mCardinal, iRFP670, NirFP, and iRFP720