Simultaneous Ultraviolet Photoacoustic and Auto‐Fluorescence Microscopy for High‐Contrast Histological Imaging

Abstract Ultraviolet (UV) photoacoustic microscopy has attracted lots of attention since it can provide histological images for disease diagnosis without any tissue processing or staining, holding great potential for rapid histopathology in hospitals. However, sometimes, the nuclear contrast in the images is relatively low due to the high UV absorption of various surrounding biomolecules (e.g., heme, myoglobin, lipids, etc.), resulting in low diagnostic accuracy. Here, a label‐free dual‐modality imaging system with ultraviolet photoacoustic and auto‐fluorescence (PAAF) microscopy is proposed, which can obtain photon absorption‐induced PA and AF images simultaneously using only one UV pulsed laser. With the opposite contrast acquired in the PA and AF images and the image fusion technique, this proposed PAAF microscopy enables high‐contrast and high‐sensitivity histological imaging for various tissues, even under a low excitation energy of 0.7 nJ and a high pulse‐to‐pulse energy fluctuation of ≈30%. Mouse brain, kidney, liver, lung, and human lung tissues processed by different clinical protocols have been imaged to demonstrate the versatility of PAAF microscopy, showing its promising applications in surgical pathology.