Delving into the microscopic world has been revolutionized: This review explores two-photon fluorescence microscopy, a powerful tool enabling noninvasive, three-dimensional study of biological specimens with submicrometer resolution. This technology leverages the simultaneous absorption of two photons to excite fluorophores, resulting in unique advantages like reduced photodamage and enhanced penetration depth. The process generates high-contrast images and provides a novel method to trigger localized photochemical reactions. This offers significant advancements over traditional microscopy techniques, providing more detailed and less disruptive imaging of biological structures and processes. As two-photon microscopy continues to find an increasing number of applications in both biology and medicine, this review provides a valuable resource for researchers seeking to understand its principles and applications. This research has significant implications for disease diagnosis, drug development, and the fundamental understanding of biological systems, appealing to readers in **biomedical engineering**, cell biology, and genetics.
As a review appearing in the Annual Review of Biomedical Engineering, this article effectively bridges the gap between engineering principles and biological applications. It also connects to work published in other journals, particularly those specializing in optics, biophysics, and cell biology.