A1r hd microscope

For localization of TRMT1 tagged with GFP, 293T human cells were seeded onto coverslips in a six-well plate followed by transfection with the GFP plasmids noted above using Lipofectamine 3000 reagent (Thermo Fisher). For mitochondrial localization, the cells were infected with baculovirus expressing RFP targeted to mitochondria (CellLight Mitochondria-RFP, BacMam 2.0, Life Technologies). To visualize the nucleus, Hoechst dye was added to the media for 30 min before the cells were washed with PBS, fixed with 4% formaldehyde, and mounted in Aqua Poly/Mount (Polysciences Inc) followed by imaging on a Nikon A1R HD microscope.

Transfected cells had media replaced with prewarmed HBSS imaging buffer composed of (in mM): 5.33 KCl, 0.44 KH₂PO₄, 4.16 NaHCO₃, 137.9 NaCl, 0.33 Na₂HPO₄ supplemented with 20 HEPES and 3-5 glucose for at least 20 min prior to imaging. For some experiments, TMRM (25 nM) or Rhodamine123 (10 nM) was added to the imaging media. For respiratory toxin experiments, varying concentrations of rotenone (Sigma) and antimycin A (Sigma) were included in the imaging buffer. Cells treated with rotenone and antimycin A were incubated at 37 °C with 5% CO₂ for 30 and 60 min, respectively, prior to imaging.
Cells were imaged using an inverted Nikon A1R HD microscope with 405, 488, and 561 nm laser lines with a CFI Apochromat TIRF 60x oil objective using Nikon NIS-Elements AR version 5.42.01 build 1793. HyPer7 was imaged in dual excitation, single emission setup with 405 and 488 nm excitation unless otherwise stated. KillerRed was imaged using the lowest possible 561 nm excitation. Images taken using resonant scanning used at least 2x line averaging which was kept constant for each experiment. Galvano scanning was used for single mitochondrial imaging with 11.67x optical zoom (19.3 px/µm). To ensure minimal drift, samples were allowed to equilibrate on the microscope for 5–10 min prior to imaging onset.