Two-photon microscopy (TPM) has become an indispensable tool in the life sciences, offering exceptional spatial resolution, deep tissue penetration, and low phototoxicity. It provides a revolutionary solution for long-term dynamic observation of living cells and tissues. This article systematically explains the working principles of TPM and reviews its applications in cutting-edge fields such as neuroscience, immunology, oncology, regenerative science and plant biology, highlighting its remarkable multidisciplinary adaptability. Building on this foundation, we further explore the breakthrough directions and future prospects for the development of this technology.
Bu et al. (2026) studied this question.