Photoswitchable proteins have so far been barely investigated in the
field of optoacoustic (OptA) mesoscopy. In particular, the unmixing
performance and achievable depth with photoswitchable
bacteriophytochromes in an OptA mesoscopy setup with a single-element
detector remain uncharted. Therefore, this study investigates the
switching kinetics and unmixing performance at two wavelengths of
photoswitchable bacteriophytochromes expressed by E. coli cells
in an OptA mesoscopy set up with a single-element spherically focused 25
MHz piezoelectric detector at different depths using a light scattering
phantom. We found that the signal-to-noise ratio dropped by
approximately 5 dB/mm as a consequence of declining detector
sensitivity, attenuation of the acoustic wave, and decreasing fluence.
This limited unmixing with the current system parameters to a depth of
2-4 millimeters. The analysis of the switching kinetics indicates that
an increased number of laser pulses was needed to switch the protein
between the ON- and OFF states as depth increases. Based on these
analyzed switching kinetics, recommendations can be made regarding the
pulsing schemes needed at different depths. The knowledge gained on the
required pulsing schemes for successful protein switching, unmixing
performance, and achievable depth can be leveraged in future
ex-vivo/in-vivo studies using photoswitchable proteins combined with
OptA mesoscopy.