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The reactive partnership between azides and strained alkynes is at the forefront of bioorthogonal reactions, with their in situ cellular studies often achieved through the use of off to on fluorophores with fluorescence microscopy. In this work, the first demonstration of a bioorthogonal, macrocycle-forming reaction occurring within the nuclear envelope of live cells has been accomplished, utilising on/on fluorescence lifetime imaging microscopy for real-time continuous observation of the transformation. The fluorescent, macrocyclic BF 2 azadipyrromethene was accessible through a double 1,3-dipolar cycloaddition within minutes, between a precursor bis-azido substituted fluorophore and Sondheimer diyne in water or organic solvents. Photophysical properties of both the starting bis-azide BF 2 azadipyrromethene and the fluorescent macrocyclic products were obtained, with near identical emission wavelengths and intensities, but different lifetimes. In a novel approach, the progress of the live-cell bioorthogonal macrocyclization was successfully tracked through a fluorescence lifetime change of 0.6 ns from starting material to products, with reaction completion achieved within 45 min. The continuous monitoring and imaging of this bioorthogonal transformation in the nuclear membrane and invaginations, of two different cancer cell lines, has been demonstrated using a combination of fluorescence intensity and lifetime imaging with phasor plot analysis. As there is a discernible difference in fluorescence lifetimes between starting material and products, this approach removes the necessity for off-to-on fluorogenic probes when preparing for bioorthogonal cell-imaging and microscopy., Competing Interests: Authors declare no conflict of interest exist., (This journal is © The Royal Society of Chemistry.)