Integrated ultraviolet photoacoustic and spectroscopic autofluorescence microscopy for label-free histological and molecular imaging

Microstructural and molecular information of tissue specimens is essential for both clinical diagnosis and basic research. However, conventional histological examination suffers from time-consuming sample preparation involving micrometer-thick sectioning and complex chemical staining. Here, we develop a multimodal microscopy system by integrating ultraviolet photoacoustic microscopy (UV-PAM) and spectroscopic autofluorescence microscopy (SS-AFM) for label-free inspections of slide-free specimens. The UV-PAM subsystem sensitively measures the optical absorption of DNA/RNA, and the SS-AFM subsystem spectroscopically identifies fluorescence emissions of endogenous fluorophores, enabling comprehensive visualization of co-registered histological and molecular characteristics. The multimodal microscopy provides rapid microstructural imaging of cell nuclei, cytoplasm, and the extracellular matrix, matching standard histology views using hematoxylin and eosin staining. Moreover, molecular differentiation of nucleotides, tryptophan, and collagen subtypes is achieved using multimodal microscopy, which is verified by the picrosirius red staining method. Potentially, the proposed technology holds promise for accurate intraoperative histopathological assessments of neoplasms.