Sequential azide/diyne cycloadditions proved highly effective for the macrocyclization of a bis-azido aza-dipyrrin. Macrocyclic aza-dipyrrin could be produced in 30 min at rt in water with changes in fluorescence intensity and lifetimes measurable upon reaction. Live cell microscopy showed that aza-dipyrrins were suitable for confocal and STED super-resolution imaging and a bioorthogonal response to macrocyclization could be detected in cellular compartments. These results will encourage a broader examination of the sensing and imaging uses of aza-dipyrrins.