Assessment of performance characteristics of photoswitchable bacteriophytochromes at different depths using...

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.