Optical histopathology by label-free multiphoton-excitation microscopic imaging as a promising analytic method for cancer and inflammatory diseases

Remarkable progress has been made in the analysis of dynamic biological information using fluorescent intravital imaging techniques. This minimally invasive technology, which allows for real-time visualization of tissue images within the living body, should bring significant advantages for life science research and human tissue studies. Multiphoton excitation microscopy (MPM), which has been mainly employed for intravital imaging in biological research, is a promising technique for the fluorescence imaging of human tissues. Although fluorescent labeling by genetic modification and the administration of fluorescent reagents is difficult in human tissue imaging, label-free MPM can generate image files composed of nonlinear optical phenomena and autofluorescent signals from living bodies using only near-infrared excitation. Label-free MPM can image human tissues in three dimensions in real time without the need for tissue removal, fixation, or staining. This technique has demonstrated utility in the histopathological examination of several organs and tissues. Additionally, it correlates well with quantitative image analysis, including artificial intelligence- and deep learning-driven methods. These features indicate that label-free MPM has sufficient potential for the advancement of histopathology through high-dimensional analysis. The clinical implementation of label-free MPM will alter histopathological examinations in clinical settings, advancing the discipline of histopathology.