Sulforhodamine Nanothermometer for Multiparametric Fluorescence Lifetime Imaging Microscopy

Live cells function within narrow limits of physiological temperature (T) and O₂ and metabolite concentrations. We have designed a cell-permeable T-sensitive fluorescence lifetime-based nanoprobe based on lipophilic sulforhodamine, which stains 2D and 3D cell models, shows cytoplasmic localization, and has a robust response to T (∼0.037 ns/K). Subsequently, we evaluated the probe and fluorescence lifetime imaging microscopy (FLIM) technique for combined imaging of T and O₂ gradients in metabolically active cells. We found that in adherent 2D culture of HCT116 cells intracellular T and O₂ are close to ambient values. However, in 3D spheroid structures having size >200 μm, T and O₂ gradients become pronounced. These microgradients can be enhanced by treatment with mitochondrial uncouplers or dissipated by drug-induced disaggregation of the spheroids. Thus, we demonstrate the existence of local microgradients of T in 3D cell models and utility of combined imaging of O₂ and T.