STED Imaging of Mitochondria and DNA

Imaging was performed with a Leica TCS SP8 gated STED (gSTED) microscope, with a white light laser and a 93× objective lens (HC PL APO CS2 93× GLYC, NA 1.30). For confocal images of mitochondria (TOM20) and DNA, Z-stacks were taken by exciting the fluorophores at 488 and 594 nm, respectively, and Hybrid detectors collected fluorescent signals. STED images of DNA channel were obtained with a 775-nm depletion laser. 2D confocal and gSTED images were acquired sequentially with the optical zoom set to obtain a voxel size of 17 × 17 nm. Excitation was provided at 594 nm and Hybrid detectors collected signal. Gating between 0.3 and 6 ns was applied. Performance of the microscope and optimal depletion laser power were tested as previously described (Nicholls et al, 2018 (link)).