Small-molecule fluorophores are powerful tools for biological research. They have enabled researchers to study cellular architecture and decipher biological processes. Responsive fluorescent sensors have enabled the study of a wide range of analytes and their effects on biological phenomena in situ. The application of fluorescent sensors to studies in living organisms is complicated by challenges such as biocompatibility, chemostability, photostability and sufficient penetration of light through living tissues. Translation to in vivo imaging is therefore not straightforward and requires innovative approaches. Recent advances in the design of fluorophores with improved photophysical properties and the development of long-wavelength-emitting fluorophore scaffolds that can be modularly functionalised with targeting and sensing groups have allowed the application of fluorogenic, ratiometric and reversible sensors in vivo.