(a) Cytoplasmic Ca2+-level response of SNAP-Sec61β expressing SiR-labeled cells under negative control conditions (no excitation or STED illumination). (b) Positive control using Ionomycin. (c) STED-irradiated cells using an 8-kHz resonant scanner. (d) STED-irradiated cells with ROS scavenging buffer added. (e-l) Representative fluorescence (e,g-i,k,l) and brightfield (f,j) images of a HeLa cell before and after STED irradiation in ROS scavenging buffer visualizing cell viability via cell morphology and ER movement. Scale bars: 10 μm (j), 5 μm (l). Confocal images (e,i), STED images (g,h,k,l). (m) Long-term viability of STED-irradiated and control cells. Cells are categorized in alive, dead and indeterminable (labeled as “?”; see Supplementary Methods) after 24 h. Statistical information (N = total number of cells; M = number of independent experiments): (a) HeLa: N=17, M=3; COS7: N=18, M=4; (b) HeLa: N=15, M=3; COS7: N=15, M=3; (c) HeLa: N=30, M=3; COS7: N=30, M=4; (d) HeLa: N=32, M=4; COS7: N=30, M=5; (e-l) N=10, M=2; (m) HeLa: N=15, M=3; COS7: N=15, M=3; control HeLa: N=20, M=3; COS7: N=28, M=4.