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.

The characteristics of tumour development and metastasis relate not only to genomic heterogeneity but also to spatial heterogeneity, associated with variations in the intratumoural arrangement of cell populations, vascular morphology and oxygen and nutrient supply. While optical (photonic) microscopy is commonly employed to visualize the tumour microenvironment, it assesses only a few hundred cubic microns of tissue. Therefore, it is not suitable for investigating biological processes at the level of the entire tumour, which can be at least four orders of magnitude larger. In this study, we aimed to extend optical visualization and resolve spatial heterogeneity throughout the entire tumour volume. We developed an optoacoustic (photoacoustic) mesoscope adapted to solid tumour imaging and, in a pilot study, offer the first insights into cancer optical contrast heterogeneity in vivo at an unprecedented resolution of <50 μm throughout the entire tumour mass. Using spectral methods, we resolve unknown patterns of oxygenation, vasculature and perfusion in three types of breast cancer and showcase different levels of structural and functional organization. To our knowledge, these results are the most detailed insights of optical signatures reported throughout entire tumours in vivo, and they position optoacoustic mesoscopy as a unique investigational tool linking microscopic and macroscopic observations.

Optoacoustic (photoacoustic) imaging has seen marked technological advances in detection and data analysis, but there is less progress in understanding the photophysics of optoacoustic signal generation of commonly used contrast agents, such as dyes and chromoproteins. This gap blocks the precise development of novel agents and the accurate analysis and interpretation of Multispectral Optoacoustic Tomography (MSOT) images. To close it, we developed a multimodal laser spectrometer (MLS) to enable the simultaneous measurement of optoacoustic, absorbance, and fluorescence spectra. MLS provides reproducible, high-quality optoacoustic (non-radiative) spectra by using correction and referencing workflow. Herein, we employ MLS to analyze several common dyes (Methylene Blue, Rhodamine 800, Alexa Fluor 750, IRDye 800CW and Indocyanine green) and proteins (sfGFP, mCherry, mKate, HcRed, iRFP720 and smURFP) and shed light on their internal conversion properties. Our data shows that the optical absorption spectra do not correlate with the optoacoustic spectra for the majority of the analytes. We determine that for dyes, the transition underlying the high energy shoulder, which mostly correlates with an aggregation state of the dyes, has significantly more optoacoustic signal generation efficiency than the monomer transition. Our analyses for proteins point to a favored vibrational relaxation and optoacoustic signal generation that stems from the neutral or zwitterionic chromophores. We were able to crystalize HcRed in its optoacoustic state, confirming the change isomerization respect to its fluorescence state. Such data is highly relevant for the engineering of tailored contrast agents for optoacoustic imaging. Furthermore, discrepancies between absorption and optoacoustic spectra underline the importance of correct spectral information as a prerequisite for the spectral-unmixing schemes that are often required for in vivo imaging. Finally, optoacoustic spectra of some of the most commonly used proteins and dyes in optical imaging, recorded on our MLS, reveal previously unknown photophysical characteristics, such as unobserved photo-switching behavior.

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.