posted on 2018-08-06, 00:00authored byPaul Vetschera, Kanuj Mishra, Juan Pablo Fuenzalida-Werner, Andriy Chmyrov, Vasilis Ntziachristos, Andre C. Stiel
Reversibly
switchable fluorescent proteins (rsFPs) have had a revolutionizing
effect on life science imaging due to their contribution to sub-diffraction-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 contrast-to-noise ratio 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
nonradiative 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 optoacoustic imaging approaches using sophisticated temporal
unmixing schemes.