Activatable Two-Photon-Excited Molecular Fluorescent Probes for Near-Infrared Biosensing and Bioimaging

Two-photon-excited fluorescence imaging (TPEFI) is a rapidly advancing technique for detecting analytes and visualizing biological conditions in real time. Compared to conventional one-photon excitation, TPEFI offers advantages, such as deeper tissue penetration, reduced photodamage, high sensitivity, and superior temporal resolution, making it highly suitable for in vivo imaging applications. The integration of biostimuli-responsive elements into two-photon fluorophores has enabled the development of bioactivatable two-photon-excited small molecules that are effective in near-infrared bioimaging for monitoring diverse biological processes and diseases. This review highlights recent progress (2019–2024) in the design and application of two-photon-excited near-infrared fluorescent probes specifically developed for activity-based bioimaging. We provide a succinct overview of both chemically and enzymatically activated probes by discussing their structural design principles, bioresponsive characteristics, two-photon absorption and emission properties, and their use in vitro and in vivo for monitoring specific biomedical conditions and diseases.