Triplet-triplet annihilation upconversion based nanocapsules for bioimaging under excitation by red and deep-red light

Non-toxic and biocompatible triplet-triplet annihilation upconversion based nanocapsules (size less than 225nm) were successfully fabricated by the combination of miniemulsion and solvent evaporation techniques. A first type of nanocapsules displays an upconversion spectrum characterized by the maximum of emission at λ_max=550nm under illumination by red light, λ_exc=633nm. The second type of nanocapsules fluoresces at λ_max=555nm when excited with deep-red light, λ_exc=708nm. Conventional confocal laser scanning microscopy (CLSM) and flow cytometry were applied to determine uptake and toxicity of the nanocapsules for various (mesenchymal stem and HeLa) cells. Red light (λ_exc=633nm) with extremely low optical power (less than 0.3μW) or deep-red light (λ_exc=708nm) was used in CLSM experiments to generate green upconversion fluorescence. The cell images obtained with upconversion excitation demonstrate order of magnitude better signal to background ratio than the cell images obtained with direct excitation of the same fluorescence marker. Avoiding phototoxicity: Polymeric nanocapsules demonstrating efficient triplet-triplet annihilation photon upconversion are used for confocal imaging of living cells at ultra low excitation intensity. This drastically reduces the cell autofluorescence and the collateral phototoxicity.