Light fluence normalization in turbid tissues via temporally unmixed multispectral optoacoustic tomography

Discerning the accurate distribution of chromophores and biomarkers by means of optoacoustic imaging is commonly challenged by the highly heterogeneous excitation light patterns resulting from strong spatial variations of tissue scattering and absorption. Here we used the light-fluence dependent switching kinetics of reversibly switchable fluorescent proteins (RSFPs), in combination with real-time acquisition of volumetric multi-spectral optoacoustic data to correct for the light fluence distribution deep in scattering media. The new approach allows for dynamic fluence correction in time-resolved imaging, e.g., of moving organs, and can be extended to work with a large palette of available synthetic and genetically encoded photochromic substances for multiplexed wavelength-specific fluence normalization.