Two‐Photon Fluorescence and Second‐Harmonic Generation Microscopy: Principles and Biomedical Applications

ABSTRACT Two‐photon microscopy (TPM) has revolutionized biological imaging by enabling high‐resolution, deep‐tissue visualization with reduced photobleaching and photodamage. This review discusses complementary nonlinear optical imaging modalities such as two‐photon fluorescence (TPF), second‐harmonic generation (SHG), and fluorescence lifetime imaging (FLIM) microscopy that together provide structural, molecular, and metabolic information. The review begins by introducing the basic principles, instrumentation, and capabilities of two‐photon fluorescence microscopy and FLIM. Followed by the development of SHG microscopy, highlighting advances from conventional SHG imaging to polarization‐resolved, circular dichroism, and Stokes–Mueller polarimetric approaches. The biomedical applications of these complementary techniques are subsequently discussed, followed by an overview of multimodal imaging strategies that combine structural, molecular, and functional information. Collectively, these modalities represent a versatile and rapidly evolving imaging toolkit with broad potential to advance biomedical research, disease diagnosis, and precision medicine.