Ultraviolet photoacoustic microscopy (UV-PAM) provides label-free imaging of cell nuclei with the superior optical absorption of DNAs and RNAs. UV-PAM does not require any additional staining process to visualize the structures of tissues and cells. However, conventional UV-PAM is limited in imaging speed to be used in real pre-clinical and clinical situations. To overcome this limitation, we developed an UV-PAM adopting a high-speed scanning module. We demonstrated the applicability of the system as an intraoperative surgical margin assessment tool by imaging formalinfixed paraffine-embedded (FFPE) sections of human colon cancer tissues and comparing these results to conventional optical microscopic images. Additionally, three more types of cancerous tissue such as liver, uterus, and kidney were photoacoustically examined. The imaging results successfully showed the characteristic features of cancerous and normal tissues, allowing rapid diagnosis of cancer. UV-PAM has achieved sufficient spatial resolution to distinguish the individual nuclei in human tissue, but it is challenging to resolve the closely adjacent nuclei. Therefore, we applied tissue expansion technology, which enables nanoscale imaging of subcellular components beyond the resolution of optical microscopy, to our developed system. A mouse brain section was physically expanded by hydrogel-tissue hybridization, and consequently distances between cell nuclei in the tissue increased. We could separate densely distributed nuclei in the hippocampus of mouse brain better compared to conventional UV-PAM. In short, we developed a novel PA imaging system with the enhanced temporal and spatial resolution by combining fast scanning modality and tissue expansion technology with UV-PAM. ? COPYRIGHT SPIE. Downloading of the abstract is permitted for personal use only.1