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.

High resolution, multiplexed experiments are a staple in cellular imaging. Analogous experiments in animals are challenging, however, due to significant scattering and autofluorescence in tissue at visible (VIS, 350–700 nm) and near-infrared (NIR, 700–1000 nm) wavelengths. Here, we enable real-time, non-invasive multicolor imaging experiments in animals through the design of optical contrast agents for the shortwave infrared (SWIR, 1000–2000 nm) region and complementary advances in imaging technologies. We developed tunable, SWIR-emissive flavylium polymethine dyes and established structure-photophysical property relationships for this class of bright SWIR contrast agents. In parallel, we designed an imaging system with variable NIR/SWIR excitation and single-channel detection, facilitating video-rate multicolor SWIR imaging for optically guided surgery and imaging of awake and moving mice with multiplexed detection. Optimized dyes matched to 980 nm and 1064 nm lasers, combined with the clinically approved indocyanine green, enabled real-time, three-color imaging with high temporal and spatial resolutions.

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.

Fluorescence imaging is currently being actively developed for surgical guidance; however, it remains underutilized for diagnostic and endoscopic surveillance of incipient colorectal cancer in high-risk patients. Here we demonstrate the utility and potential for clinical translation of a fluorescently labeled cathepsin-activated chemical probe to highlight gastrointestinal lesions. This probe stays optically dark until it is activated by proteases produced by tumor-associated macrophages and accumulates within the lesions, enabling their detection using an endoscope outfitted with a fluorescence detector. We evaluated the probe in multiple murine models and a human-scale porcine model of gastrointestinal carcinogenesis. The probe provides fluorescence-guided surveillance of gastrointestinal lesions and augments histopathological analysis by highlighting areas of dysplasia as small as 400 µm, which were visibly discernible with significant tumor-to-background ratios, even in tissues with a background of severe inflammation and ulceration. Given these results, we anticipate that this probe will enable sensitive fluorescence-guided biopsies, even in the presence of highly inflamed colorectal tissue, which will improve early diagnosis to prevent gastrointestinal cancers.