Probing the photophysical properties of fluorescent proteins using photoacoustic pump-probe spectroscopy and imaging.

Pump-probe excitation of fluorophores has been shown to overcome the limitations of conventional multiwavelength imaging and linear unmixing approaches by providing fluorophore-specific contrast whilst eliminating the dominant background signal of endogenous chromophores. In this study, methods for generating pump-probe signals and images are investigated that rely on changing 1) the pump wavelength whilst keeping the probe wavelength fixed, 2) the probe wavelength whilst keeping the pump wavelength fixed, and 3) the time delay between the pump and probe pulse. Time-resolved PA signals were generated in purified solutions of genetically expressed red fluorescent proteins Katushka, mNeptune, and mCardinal in a cuvette. Spectra of the difference signal amplitude were found to correlate with the absorption and emission spectra. The difference signal plotted as a function of time delay also showed characteristic features for each protein. To demonstrate the capability of multiplexed imaging, the spatial distributions of Katushka and mNeptune were recovered from 2D difference images of a phantom. This study demonstrates that methods based on pump-probe excitation can be used to probe the photophysical properties of fluorophores. By detecting changes in these properties due to a stimulant, such as pH, the methods may find application in biosensing of the cellular microenvironment.