Reversibly switchable fluorescent proteins (rsFPs) have had a revolutionizing effect on life science imaging due to their contribution to subdiffraction-resolution optical microscopy (nanoscopy). Initial studies showed that their use as labels could also be highly beneficial for emerging Photo- or Optoacoustic imaging. It could be shown that their use in Optoacoustics i) strongly improves the imaging CNR due to modulation and locked-in detection, ii) facilitates fluence calibration affording precise measurements of physiological parameters and finally iii) could boost spatial resolution following similar concepts as used for nanoscopy. However, rsFPs show different photophysical behavior in optoacoustics than in optical microscopy because optoacoustics requires pulsed illumination and depends on signal generation via non-radiative energy decay channels. This implies that rsFPs optimized for fluorescence imaging may not be ideal for optoacoustics. Here, we analyze the photophysical behavior of a broad range of rsFPs with optoacoustics and analyze how the experimental factors central to optoacoustic imaging influence the different types of rsFPs. Finally, we discuss how knowledge of the switching behavior can be exploited for various OA imaging approaches using sophisticate temporal unmixing schemes.
