Photochromic variants of fluorescent proteins are opening the way to a number of opportunities for high-sensitivity regioselective studies in the cellular environment and may even lead to applications in information and communication technology. Yet, the detailed photophysical processes at the basis of photoswitching have not been fully clarified. In this paper, we used synthetic FP chromophores to clarify the photophysical processes associated with the photochromic behavior. In particular, we investigated the spectral modification of synthetic chromophore analogues of wild-type green fluorescent protein (GFP), Y66F GFP (BFPF), and Y66W GFP (CFP) upon irradiation in solutions of different polarities. We found that the cis-trans photoisomerization mechanism can be induced in all the chromophores. The structural assignments were carried out both by NMR measurements and DFT calculations. Remarkably, we determined for the first time the spectra of neutral trans isomers in different solvents. Finally, we calculated the photoconversion quantum yields by absorption measurements under continuous illumination at different times and by a nanosecond laser-flash photolysis method. Our results indicate that cis-trans photoisomerization is a general mechanism of FP chromophores whose efficiency is modulated by the detailed mutant-specific protein environment.

Green fluorescent protein (GFP) and its variants have been used as fluorescent reporters in a variety of applications for monitoring dynamic processes in cells and organisms, including gene expression, protein localization, and intracellular dynamics. GFP fluorescence is stable, species-independent, and can be monitored noninvasively in living cells by fluorescence microscopy, flow cytometry, or macroscopic imaging techniques. Owing to the presence of a phenol group on the chromophore, most GFP variants display pH-sensitive absorption and fluorescence bands. Such behavior has been exploited to genetically engineer encodable pH indicators for studies of pH regulation within specific intracellular compartments that cannot be probed using conventional pH-sensitive dyes. These pH indicators contributed to shedding light on a number of cell functions for which intracellular pH is an important modulator. In this review we discuss the photophysical properties that make GFPs so special as pH indicators for in vivo use and we describe the probes that are utilized most by the scientific community.

Polarity-dependent fluorescent probes are recently attracting interest for high-resolution cell imaging. Following a stepwise rational approach, we prepared and tested a toolbox of new coumarin derivatives tailored to in vivo imaging applications. Our compounds are characterized by a donor-(coumarin core)-acceptor molecular structure, where the electron donor is represented by alkylether or naphthyl groups, and the electron acceptor is represented by benzothiazene and cyano groups. Prior to synthesis, the substitution patterns were screened by computational methods to provide functional fluorescent derivatives easy to synthesize, and with excitation in the visible region of spectrum. We set up a robust synthetic procedure tunable on the substitution patterns to achieve. These coumarins possess excellent fluorescence quantum yields (up to 0.95), high molar extinction coefficients (up to 46,000 M(-1) cm(-1)), and large Stokes shifts. Furthermore, they display strong solvatochromism, being almost non-emissive in water and very fluorescent in less polar media (up to 780-fold enhancement in brightness). The solvatochromism of these compounds can be accounted for by a photophysical method encompassing two communicating excited states. When tested on cultured cells, the results showed that the developed coumarins were not harmful and their photophysical properties were unchanged compared to free solution. According to the determined solvatochromic properties, the coumarin fluorescence was detected only in the most lipophilic environments of the cell. The prepared compounds represent remarkable tools to investigate subtle biochemical processes in the cell environment after appropriate conjugation to biomolecules, and at the same time constitute the basis for further engineering of a new generation of biosensors.