Labeling subcellular structures in living specimens using live-cell incompatible dyes with excellent optical properties

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Despite the urgent needs of imaging living specimens for cutting-edge biological research, most of the existing fluorescent labeling methods suffer from either poor optical properties or complicated operations to realize cell-permeability and specificity. Here, we introduce a method to overcome this tradeoff by incubating living cells and tissues with fluorescent dyes, no matter if they are cell-permeable or not, at particular conditions (concentration and temperature) without physical cell-penetration or chemical modifications. Based on this method, the mitochondrial labeling capability of Atto dyes, especially Atto 647N with extraordinary optical properties, together with interesting interactions between organelles is revealed. These results indicate the great potential of using dyes, which are normally considered “live-cell incompatible”, to capture the morphology and dynamics of subcellular structures in living specimens.