Spatial heterogeneity of oxygenation and haemodynamics in breast cancer resolved in vivo by conical multispectral optoacoustic mesoscopy.

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.

Longitudinal imaging of T cell-based immunotherapy with multi-spectral, multi-scale optoacoustic tomography.

Most imaging studies of immunotherapy have focused on tracking labeled T cell biodistribution in vivo for understanding trafficking and homing parameters and predicting therapeutic efficacy by the presence of transferred T cells at or in the tumour mass. Conversely, we investigate here a novel concept for longitudinally elucidating anatomical and pathophysiological changes of solid tumours after adoptive T cell transfer in a preclinical set up, using previously unexplored in-tandem macroscopic and mesoscopic optoacoustic (photoacoustic) imaging. We show non-invasive in vivo observations of vessel collapse during tumour rejection across entire tumours and observe for the first time longitudinal tumour rejection in a label-free manner based on optical absorption changes in the tumour mass due to cellular decline. We complement these observations with high resolution episcopic fluorescence imaging of T cell biodistribution using optimized T cell labeling based on two near-infrared dyes targeting the cell membrane and the cytoplasm. We discuss how optoacoustic macroscopy and mesoscopy offer unique contrast and immunotherapy insights, allowing label-free and longitudinal observations of tumour therapy. The results demonstrate optoacoustic imaging as an invaluable tool in understanding and optimizing T cell therapy.

Eigenspectra optoacoustic tomography achieves quantitative blood oxygenation imaging deep in tissues.

Light propagating in tissue attains a spectrum that varies with location due to wavelength-dependent fluence attenuation, an effect that causes spectral corruption. Spectral corruption has limited the quantification accuracy of optical and optoacoustic spectroscopic methods, and impeded the goal of imaging blood oxygen saturation (sO2) deep in tissues; a critical goal for the assessment of oxygenation in physiological processes and disease. Here we describe light fluence in the spectral domain and introduce eigenspectra multispectral optoacoustic tomography (eMSOT) to account for wavelength-dependent light attenuation, and estimate blood sO2 within deep tissue. We validate eMSOT in simulations, phantoms and animal measurements and spatially resolve sO2 in muscle and tumours, validating our measurements with histology data. eMSOT shows substantial sO2 accuracy enhancement over previous optoacoustic methods, potentially serving as a valuable tool for imaging tissue pathophysiology.