Multi-Spectral Optoacoustic Tomography (MSOT) merges the power of high-resolution imaging at tissue depths of several millimeters to centimeters with the advantages of optical imaging, in large part by exploiting spectral detection of endogenous molecules in tissue or exogenous photoabsorbing probes. Current advances in fast-tuning laser technology, image reconstruction and spectral detection schemes have yielded real-time optoacoustic (photoacoustic) imaging spanning applications from microscopy to human imaging. This progress has generated an unprecedented need for imaging probes and reporter gene approaches optimized for optoacoustic signal generation. New classes of probes are emerging and create new opportunities for visualizing morphological and pathophysiological features in vivo, in a non-invasive manner. Here we review recent progress in optoacoustic probes and discuss applications and challenges for biological imaging as well as prospects for clinical translation.

We introduce a selective and cell-permeable calcium sensor for photoacoustics (CaSPA), a versatile imaging technique that allows for fast volumetric mapping of photoabsorbing molecules with deep tissue penetration. To optimize for Ca-dependent photoacoustic signal changes, we synthesized a selective metallochromic sensor with high extinction coefficient, low quantum yield, and high photobleaching resistance. Micromolar concentrations of Ca lead to a robust blueshift of the absorbance of CaSPA, which translated into an accompanying decrease of the peak photoacoustic signal. The acetoxymethyl esterified sensor variant was readily taken up by cells without toxic effects and thus allowed us for the first time to perform live imaging of Ca fluxes in genetically unmodified cells and heart organoids as well as in zebrafish larval brain via combined fluorescence and photoacoustic imaging.

Figure 1. Relaxometric Ca 2+ (full squares) and Mg 2+ (empty triangles) titration curves of Gd2L 1 (A), Gd2L 2 (B), and Gd2L 3 (C) performed at 25°C and 11.75 T. The lines correspond to the fit as explained in the text.