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Fluorescence is an essential imaging modality for labelling and visualising cells and sub-cellular structures. Multicolour labelling is especially challenging due to differences in physicochemical and photophysical behaviour of structurally unrelated fluorophores in the heterogeneous environments found in sub-cellular compartments. Herein, we report the conjugation of three azide-bearing BODIPYs with similar core structures but widely different emission wavelengths (green, red and NIR) to tyrosine residues of a model globular protein (BSA)
via
a common linking methodology. The resulting BODIPY–BSA conjugates have demonstrated multi-wavelength fluorescence emission for biological applications. Fluorescence imaging was performed in HeLa cells through live cell confocal microscopy imaging, with good intracellular location visualisation observed.