Stimulated Emission Depletion Imaging Reveals Mitochondrial Phenotypic Heterogeneity under Apoptosis Stimuli across Living Glioma Models

The mitochondrial phenotypes contribute to the understanding of disease mechanisms and treatments, which are typically characterized through the omics methods. However, the high dynamics and phenotypic heterogeneity of mitochondria require high-resolution characterization within individual living cells. Therefore, we introduce a fluorescence analysis method, based on two-color and fluorescence lifetime stimulated emission depletion (STED) super-resolution imaging, to explore mitochondrial phenotypic heterogeneity in human (U87) and mouse (GL261) glioma models. Furthermore, we used rotenone and etoposide to simulate the effects of antitumor drugs, inducing apoptosis through mitochondrial dysfunction, respectively. The two-color labeling introduces intracellular parameters to qualitatively visualize changes in mitochondrial morphology, while fluorescence lifetime reflects the status of mitochondria and their microenvironment from the perspective of probe characteristics. This method reveals mitochondria phenotypic heterogeneity induced by the apoptotic stimuli in human and mouse glioma models from a morphological perspective.