| Summary: | Abstract Wound healing is a well‐coordinated process, necessitating efficient formation of new blood vessels. Vascularization defects are therefore a major risk factor for chronic, non‐healing wounds. The dynamics of mammalian tissue revascularization, vessel maturation, and remodeling remain poorly understood due to lack of suitable in vivo imaging tools. A label‐free large‐scale optoacoustic microscopy (LSOM) approach is developed for rapid, non‐invasive, volumetric imaging of tissue regeneration over large areas spanning up to 50 mm with a depth penetration of 1.5 mm. Vascular networks in dorsal mouse skin and full‐thickness excisional wounds are imaged with capillary resolution during the course of healing, revealing previously undocumented views of the angiogenesis process in an unperturbed wound environment. Development of an automatic analysis framework enables the identification of key features of wound angiogenesis, including vessel length, diameter, tortuosity, and angular alignment. The approach offers a versatile tool for preclinical research in tissue engineering and regenerative medicine, empowering label‐free, longitudinal, high‐throughput, and quantitative studies of the microcirculation in processes associated with normal and impaired vascular remodeling, and analysis of vascular responses to pharmacological interventions in vivo.
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