Two-photon uncaging of bioactive compounds: Starter guide to an efficient IR light switch

Triggering physiological responses with a light switch has become a reality with the development of smart molecular probes such as photolabile protecting groups (PPGs), able to "uncage" biological ligands on demand. To make the light switch virtually harmless and confine the excitation to the single-cell level, the caged ligands can be released using two-photon (2P) absorption and 2P microscopy using red/infrared light. This exceptional level of precision however comes at the cost of a reduced photosensitivity and a poor compatibility of early PPGs with 2P excitation. This review aims to provide a tutorial guidebook to the design of 2P-sensitive PPGs suitable for optobiology by discussing challenges, strategies and progress in uncaging of bioactive compounds. To do so, we first recall the photo-physical principles governing 2P absorption, and the resulting ground rules in the design of efficient 2P absorbing organic dyes. We then detail how following these guidelines has led to tremendous progress in the development of a new generation of caged compounds, and the implications in the fields of biophotonics, from neurology to targeted therapy.