Label-free histopathological diagnosis of frozen sections based on multi-excitation and broad-emission autofluorescence imaging

Tumor surgery requires rapid intraoperative histopathological diagnosis to guide treatment and improve patient prognosis. Autofluorescence (AF) imaging offers a label-free approach with strong potential for rapid intraoperative use; however, most studies to date have been performed on deparaffinized sections based on bandpass emission settings. Its compatibility with frozen sections-which is essential for time-constrained intraoperative settings-remains unclear. Here, we systematically compared AF imaging of frozen sections with conventional deparaffinized sections, finding that the bandpass-filtered AF signal of frozen sections was weak and susceptible to photobleaching. To address this issue, we demonstrated multi-excitation and broad-emission (MEBE) AF imaging of frozen sections and showed that MEBE AF imaging of frozen sections enabled reliable tissue morphology assessment. This approach potentially provides diagnostic quality comparable to standard H&E staining and paves the way for rapid intraoperative histopathological diagnosis. We further evaluated the approach in liver disease models and found that MEBE AF imaging accurately delineated tumor boundaries in liver cancer and assessed injury severity in acute liver injury, consistent with histopathological confirmation. Our study bridges MEBE AF imaging of frozen and deparaffinized sections, along with preliminary exploration in liver disease diagnosis. The findings will advance AF-based histopathology for clinical translation, enhancing surgical efficiency and patient prognosis.