Two-photon fluorescence lifetime microscopy (TP-FLIM) is a powerful quantitative imaging technique that that can characterize and analyze the microenvironment changes of biological samples in the fluorescence lifetime dimension. It is not affected by factors such as excitation light intensity, sample bleaching, and fluorophore concentration, and has the advantages of high sensitivity and resolution. Therefore, it has extensive applications in biomedical research, disease diagnosis, and other fields. Second harmonic generation imaging (SHG) possesses the unique characteristics of high spatial resolution and imaging depth inherent in nonlinear optical imaging. SHG occurs in non-centrosymmetric structures, a property exhibited by collagen tissue in biological organisms. Consequently, SHG has been employed for specific tissue structure imaging. This paper combines TP-FLIM and SHG technologies to investigate pathological lung sections. Vector analysis of TP-FLIM is applied to analyze the fluorescence lifetime data of lung collagen fibers. Through TP-FLIM and SHG technologies, the lifetime of collagen fibers in the sample can be calculated and the collagen fiber tissue and non-collagen fiber tissue in the lung tissue sections can be clearly distinguished. This provides a new direction and possibility for indepth understanding of pulmonary fibrosis and other pathological mechanisms related to collagen fiber diseases, offering new insights into pathology and provisional diagnosis.

Applications of two-photon fluorescence lifetime and second harmonic generation imaging technologies in pathological tissue sections