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.

Pupillometry, a noninvasive measure of arousal, complements human functional MRI (fMRI) to detect periods of variable cognitive processing and identify networks that relate to particular attentional states. Even under anesthesia, pupil dynamics correlate with brain-state fluctuations, and extended dilations mark the transition to more arousable states. However, cross-scale neuronal activation patterns are seldom linked to brain state-dependent pupil dynamics. Here, we complemented resting-state fMRI in rats with cortical calcium recording (GCaMP-mediated) and pupillometry to tackle the linkage between brain-state changes and neural dynamics across different scales. This multimodal platform allowed us to identify a global brain network that covaried with pupil size, which served to generate an index indicative of the brain-state fluctuation during anesthesia. Besides, a specific correlation pattern was detected in the brainstem, at a location consistent with noradrenergic cell group 5 (A5), which appeared to be dependent on the coupling between different frequencies of cortical activity, possibly further indicating particular brain-state dynamics. The multimodal fMRI combining concurrent calcium recordings and pupillometry enables tracking brain state-dependent pupil dynamics and identifying unique cross-scale neuronal dynamic patterns under anesthesia.

Photoacoustic imaging (PAI) is an attractive imaging modality that can volumetrically map the distribution of photoabsorbing molecules with deeper tissue penetration than multiphoton microscopy. To enable dynamic sensing of divalent cations via PAI, we have engineered a new reversible near-infrared probe that is more sensitive to calcium as compared to other biologically relevant cations. The metallochromic compound showed a strong reduction of its peak absorbance at 765 nm upon addition of calcium ions that was translated into robust signal changes in photoacoustic images. Therefore, the heptamethine cyanine dye will be an attractive scaffold to create a series of metallochromic sensors for molecular PAI.

Photocontrol of reversibly switchable fluorescent proteins (RSFPs) was used to program optoacoustic signal time courses that were temporally unmixed to increase the proteins' contrast-to-noise-ratios (CNRs) in optoacoustic imaging. In this way, two variants of the RSFP Dronpa with very similar optoacoustic spectra could be readily discriminated in the presence of highly absorbing blood. Addition of temporal unmixing to multispectral optoacoustic tomography (tuMSOT) in conjunction with synthetic or genetically encoded photochromic contrast agents and customized photoswitching schedules can increase the performance of multiplexed and high-contrast molecular optoacoustic imaging.

Extensive in vivo imaging studies investigate the hippocampal neural network function, mainly focusing on the dorsal CA1 region given its optical accessibility. Multi-modality fMRI with simultaneous hippocampal electrophysiological recording reveal broad cortical correlation patterns, but the detailed spatial hippocampal functional map remains lacking given the limited fMRI resolution. In particular, hemodynamic responses linked to specific neural activity are unclear at the single-vessel level across hippocampal vasculature, which hinders the deciphering of the hippocampal malfunction in animal models and the translation to critical neurovascular coupling (NVC) patterns for human fMRI. We simultaneously acquired optogenetically-driven neuronal Ca2+ signals with single-vessel blood-oxygen-level-dependent (BOLD) and cerebral-blood-volume (CBV)-fMRI from individual venules and arterioles. Distinct spatiotemporal patterns of hippocampal hemodynamic responses were correlated to optogenetically evoked and spreading depression-like calcium events. The calcium event-related single-vessel hemodynamic modeling revealed significantly reduced NVC efficiency upon spreading depression-like (SDL) events, providing a direct measure of the NVC function at various hippocampal states.

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.

There is growing interest in genetically expressed reporters for in vivo studies of bacterial colonization in the context of infectious disease research, studies of the bacterial microbiome or cancer imaging and treatment. To empower non-invasive high-resolution bacterial tracking with deep tissue penetration, we herein use the genetically controlled biosynthesis of the deep-purple pigment Violacein as a photobleaching-resistant chromophore label for in vivo optoacoustic (photoacoustic) imaging in the near-infrared range. We demonstrate that Violacein-producing bacteria can be imaged with high contrast-to-noise in strongly vascularized xenografted murine tumors and further observe that Violacein shows anti-tumoral activity. Our experiments thus identify Violacein as a robust bacterial label for non-invasive optoacoustic imaging with high potential for basic research and future theranostic applications in bacterial tumor targeting.

Background and objectives:Intervertebral disc herniation is a common disease in chondrodystrophic dogs, and a similar neurologic condition also occurs in humans. Percutaneous laser disc ablation (PLDA) is a minimally invasive procedure used increasingly for prevention of disc herniation. PLDA is performed on thoracolumbar discs to which the same laser energy is applied regardless of their mineral content. Knowledge of individual disc mineral composition would allow laser energy dosage adjustments and more accurate treatment of degenerative discs. Usually, PLDA is guided by radiography/fl uoroscopy, which has a limited sensitivity of approximately 60 % for identifi cation of mineralized discs. An imaging or sensing technology that provides a more accurate pre-operative in-situ assessment of the disc mineralization, and potentially rapid post-operative feedback, could optimize the outcome of the PLDA procedure. A sensing technology of needle-probing single-fi ber refl ectance (SFR) spectroscopy is therefore proposed that is considered to be compatible with PLDA work fl ow. The objective of this study was to demon strate the feasibility of this technology in assessing the increased light scattering associated with mineralization in intervertebral discs in chondrodystrophoid canine species. Materials and methods: A pilot study was performed on a total of 21 intervertebral discs from two cadaveric dogs ( " Dog A " and " Dog B " ). The discs were imaged by computed tomography (CT), radiography, and SFR spectroscopy, before histopathologic examination. SFR spectroscopy in the visible/near-infrared band was performed on the nucleus pulposus of the intervertebral disc through a 20-gauge spinal needle placed percutaneously for PLDA. A normalization method was applied to the raw remission spectra to extract a dimension-less and wavelength-dependent intensity profi le in the 500 -950 nm spectral range. Results: In total, six discs were determined to be degenerative on histopathology, fi ve discs of " Dog A " and one disc of " Dog B " . CT diagnosed all six degenerated discs, whereas radiography missed two of the fi ve degenerated discs of " Dog A " . The wavelength-dependent mean scattering intensity profi les of the six degenerated discs were noticeably higher than the mean scattering intensity profi les of the 15 " normal " or insignifi cantly mineralized discs over the entire spectral range. The mean scattering intensities, averaged over each of the entire profi les, were 2.79 ± 0.58 (mean ± SD) for the six degenerated discs and 1.48 ± 0.37 for the 15 " normal " or insignifi cantly mineralized discs. A two-sample t -test showed p < 0.001 for the difference of the averaged scattering intensity between these both groups of discs. Conclusions: SFR spectroscopy measurements indicate that the increase of light scattering intensity across the entire 500 -950 nm spectral range is associated with the mineralization in canine intervertebral discs. However, the scattering characteristics of the nucleus pulposus measured in this stu...