Diazirine groups-strained three-membered rings containing two doubly bound nitrogen atoms-release nitrogen gas upon thermal or photochemical excitation, revealing reactive carbenes that can undergo C-H, O-H, or N-H insertion to nearby substrates (Fig. ). First popularized by Brunner for use in biological target identification, trifluoromethyl aryl diazirines have been used extensively to search for protein-binding partners for bioactive small molecules (Fig. ). Thanks to their small size (relative to alternative photoactivatable groups like benzophenones), excellent stability (in the absence of bright light), lack of toxicity, and desirable activation wavelength (ca. 365 nm; considerably longer than that required for activation of benzophenones or aryl azides), trifluoromethyl aryl diazirines have emerged as the 'gold standard' for target identification experiments. Reagents developed on the trifluoromethyl aryl diazirine scaffold are also now frequently employed for proximity mapping of T-cell protein environments, and for the functionalization and crosslinking of low-functionality aliphatic polymers. Overview of diazirine reactivity and utility in chemical biology. A: Photochemical or thermal activation of diazirine reagents reveals reactive carbenes that can engage in X-H insertions to nearby substrate molecules. B: Photoaffinity labelling of target proteins with diazirine-enabled small molecules allows for identification of macromolecular binding partners.