Fig. 6: Live-cell time-lapse imaging of selective organelle dynamics with Carbow-switch.
a Photo-selective mitochondrial imaging in normal condition. Live U2OS cells were labeled with Mito-switch and selectively switched on by 405 nm irradiation (dashed lines in Mito-closed channels). Time-lapse SRS imaging of selected mitochondria showed the decrease of intensity after 10 min in PBS. Overlay image of mitochondria at 0 min (red) and 10 min (green) showed the dispersed pattern with partial overlap at two time points. b Photo-selective mitochondrial imaging under oxidative stress. Live U2OS cells labeled with Mito-switch were selectively switched on by 405 nm irradiation (dashed lines in Mito-closed channels). Time-lapse SRS imaging of mitochondria in selected cells showed compact distribution with strong signal after 30 min treatment in H2O2. Overlay image at 0 min (red) and 30 min (green) indicated mitochondrial aggregation with time (white arrowhead). c Photo-selective lysosomal imaging during stress granule formation. Live U2OS cells expressing EGFP-ATXN2 were labeled with Lyso-switch and selectively switched on by 405 nm irradiation (dashed lines in Lyso-closed channels). Time-lapse fluorescence imaging of EGFP showed bright puncta formation of stress granules in the cytoplasm during 30 min sodium arsenite treatment. Time-lapse SRS imaging of lysosomes showed heterogeneous distributions in selected cells. In cells with many stress granules, lysosomal distribution remained scattered (thick arrow). In cells with few stress granules, lysosomes formed clusters in the perinuclear region (thin arrow). Overlay image of selected lysosomes (red) and EGFP-ATXN2 (green) at both 0 min and 30 min showed little overlap between lysosomes and stress granules. All protein CH3 were imaged by SRS to show the cell morphology. Scale bar: 20 μm. All experiments were repeated three times independently with similar results.
