Previously we have found that the two-photon absorption (2PA) spectra of red fluorescent proteins have enhanced vibronic transitions and are about twofold broader, compared to the corresponding one-photon absorption (1PA) spectra. We explained these observations by the dependence of the permanent dipole moment change (Deltamu) on a particular vibrational coordinate (in the Herzberg-Teller spirit). Here we demonstrate that this effect is responsible for the 2PA spectral shapes of many fluorescent dyes including rhodamines, ATTO dyes, fluorescein, oxazines, acridines, pyronines, resorufin, and thionine. By measuring the two-photon polarization ratio as a function of excitation wavelength in these dyes, we were able to separate the transitions with Deltamu perpendicular to the long molecular axis (“FC”-type) and parallel to it (“HT”-type). We have found the previously undetected “HT-ghost” transitions under the pure electronic envelope. These are the 0-0 transitions of the "HT" progression build on the BLA frequency that are non-vanishing if the corresponding Huang-Rhys factor is not zero.
Red fluorescent proteins (RFPs) and biosensors built upon them provide an attractive advantage for two-photon laser microscopy because they can report from deeper layers of tissue compared to green fluorescent proteins. Using mCherry RFP we show that although the shorter wavelength excitation (740–800 nm) is several times more efficient compared to longer wavelength excitation (1000–1200 nm), the photobleaching of chromophore occurs much faster for the former. This can be explained by a different photobleaching mechanism: with higher energy-photons, 3-4 photons are sufficient to reach an energy threshold (ionization potential) and photodetach an electron from the chromophore.
Red fluorescent proteins (RFPs) and genetically encoded biosensors built upon them present additional imaging wavelengths and can report from deeper layers of tissues compared to green fluorescent proteins (GFPs). However, the fluorescence quantum yield (QY) of the popular red variants such as mCherry and mPlum are low (0.1 – 0.2). Same is true for a number of red genetically encoded calcium indicators (GECIs) even in their bright state. The theoretically proposed physical mechanism of nonradiative relaxation involves intramolecular charge transfer (CT) from the phenolate to imidazolinone ring in the excited state that is coupled to the chromophore twisting about the methine bridge bonds. Such twisting can result in transition (hopping) between potential energy surfaces of the excited state and the ground state at their conical intersection. An alternative pathway (supported by our MD simulations) can involve accidental drops of the CT dark excited state (S2) energy below the bright S1 state in the course of temporal fluctuations of the protein surrounding. If the CT state governs nonradiative relaxation, strong local electric field directed from phenolate to imidazolinone can block this process resulting in the QY increase. We use two-photon spectroscopy to evaluate the components of the protein electric field in the plane of the RFP chromophore and demonstrate that in a series of RFPs nonradiative relaxation rate is correlated with the field component along the long chromophore axis. This provides useful guidelines for engineering brighter RFP probes.
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