Originpro 2022b

Two days before imaging, HeLa cells were plated (2.5 × 10⁴ cells per well) in a four-well chamber and incubated overnight. After 16 h, the cells were transfected with 10 μl of Celllight BacMam 2.0 MitoGFP for 8 h, washed three times with 600 μl of DMEM ph(–) supplemented with 10% FBS and incubated overnight. The following day, the cells were labeled with 600 μl of 75 nM HAO-N₃ in DMEM ph(–) for 1 h at 37 °C with 5% CO₂, washed three times with 600 μl of DMEM ph(–), incubated with 600 μl of 0.6 μM SiR-DBCO in DMEM ph(–) for 1 h at 37 °C with 5% CO₂ and washed again six times with DMEM ph(–) and 10% FBS for 10 min each. The sample was subsequently visualized via Lattice SIM imaging. The following parameters were used: laser powers of 6% (642 nm, estimated at 0.04 kW cm^–2) and 3% (488 nm, estimated at 0.04 kW cm^–2), field of view of 80.14 μm × 80.14 μm, camera exposure time of 40 ms, acquisition of 13 phases for SIM reconstruction, an LBF 405/488/561/642 beam splitter and a BP 495–550 + LP 655 filter set. Cells were incubated with DMEM ph(–) supplemented with 10% FBS at 37 °C and 5% CO₂. SIM reconstruction was performed with Zeiss Zen Black software. Nonlinear regression for Gaussian and Lorentzian fitting of peaks from line profiles was performed using OriginLab OriginPro 2022b and plotted using Graphpad Prism 8.4.3.

Two days before imaging, HeLa cells were plated (2.5 × 10⁴ cells per well) in a four-well chamber and incubated overnight. After 16 h, the cells were transfected with plasmids encoding mEmerald–TOMM20 using FuGENE HD transfection reagent (Promega), according to manufacturer’s protocol, and incubated overnight. The following day, the cells were labeled with 600 μl of 75 nM MAO-N₃ in DMEM ph(–) for 1 h, washed three times with 600 μl of DMEM ph(–), incubated with 600 μl of 0.6 μM SiR-DBCO in DMEM ph(–) for 1 h and washed again (six times for 10 min each with DMEM ph(–) supplemented with 10% FBS). The sample was subsequently visualized via Lattice SIM imaging on a Zeiss Elyra 7 microscope. The following parameters were used: laser powers of 6% (642 nm, estimated at 0.04 kW cm^–2) and 3% (488 nm, estimated at 0.04 kW cm^–2), field of view of 80.14 μm × 80.14 μm, camera exposure time of 40 ms, acquisition of 13 phases for SIM reconstruction, an LBF 405/488/561/642 beam splitter and a BP 495–550 + LP 655 filter set. Cells were incubated with DMEM ph(–) supplemented with 10% FBS at 37 °C and 5% CO₂. SIM reconstruction was performed with Zeiss Zen Black software. Nonlinear regression for Gaussian and Lorentzian fitting of peaks from line profiles was performed using OriginLab OriginPro 2022b and plotted using Graphpad Prism 8.4.3.