BACKGROUND: Chronic kidney disease (CKD) is a global burden affecting both children and adults. Novel imaging modalities hold great promise to visualize and quantify structural, functional, and molecular organ damage. The aim of the study was to visualize and quantify murine renal vasculature using label-free raster scanning optoacoustic mesoscopy (RSOM) in explanted organs from mice with renal injury. MATERIAL AND METHODS: For the experiments, freshly bisected kidneys of alpha 8 integrin knock-out (KO) and wildtype mice (WT) were used. A total of n=7 female (n=4 KO, n=3 WT) and n=6 male animals (n=2 KO, n=4 WT) aged 6 weeks were examined with RSOM optoacoustic imaging systems (RSOM Explorer P50 at SWL 532nm and/or ms-P50 imaging system at 532 nm, 555 nm, 579 nm, and 606 nm). Images were reconstructed using a dedicated software, analyzed for size and vascular area and compared to standard histologic sections. RESULTS: RSOM enabled mapping of murine kidney size and vascular area, revealing differences between kidney sizes of male (m) and female (f) mice (merged frequencies (MF) f vs. m: 52.42±6.24 mm2 vs. 69.18±15.96 mm2, p=0.0156) and absolute vascular area (MF f vs. m: 35.67±4.22 mm2 vs. 49.07±13.48 mm2, p=0.0036). Without respect to sex, the absolute kidney area was found to be smaller in knock-out (KO) than in wildtype (WT) mice (WT vs. KO: MF: p=0.0255) and showed a similar trend for the relative vessel area (WT vs. KO: MF p=0.0031). Also the absolute vessel areas of KO compared to WT were found significantly different (MF p=0.0089). A significant decrease in absolute vessel area was found in KO compared to WT male mice (MF WT vs. KO: 54.37±9.35 mm2 vs. 34.93±13.82 mm2, p=0.0232). In addition, multispectral RSOM allowed visualization of oxygenated and deoxygenated parenchymal regions by spectral unmixing. CONCLUSION: This study demonstrates the capability of RSOM for label-free visualization of differences in vascular morphology in ex vivo murine renal tissue at high resolution. Due to its scalability optoacoustic imaging provides an emerging modality with potential for further preclinical and clinical imaging applications.