Elyra 7 microscope

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 Halo-TOMM20 using FuGENE HD transfection reagent (Promega), according to manufacturer’s protocol, and incubated overnight. The following day, the cells were incubated with 600 μl of 2 μM SiR-CA in DMEM ph(–) for 1 h at 37 °C and CO₂ and washed three times with 600 μl of DMEM ph(–) supplemented with 10% FBS. Cells were visualized via Lattice SIM imaging on a Zeiss Elyra 7 microscope. The following settings were used: laser power of 0.4% (488 nm, estimated at 0.005 kW cm^–2), field of view of 80.14 μm × 80.14 μm, camera exposure time of 40 ms, acquisition of 13 phases per frame for SIM reconstruction, interval time of 100 ms between each frame, acquisition of 500 frames over 297 s, an LBF 405/488/561/642 beam splitter and an SBS LP 560 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. Fluorescence intensity was obtained from randomly picked 3 μm × 3 μm regions using ImageJ and plotted using Graphpad Prism 8.4.3. The signal intensity half-life was obtained by performing a nonlinear regression of the exponential decay function using OriginLab OriginPro 2022b.

Before seeding, four-well LabTek II (Nunc) chambers were treated by sonication for 15 min in 1 M KOH, rinsed three times with deionized water, sterilized with 100% ethanol and air dried overnight in a biological safety cabinet. HeLa cells were seeded onto pretreated plates at a density of 3.0 × 10⁴ cells per well and incubated overnight at 37 °C with 5% CO₂. The following day, the cells were labeled with 600 μl of 75 nM MAO-N₃ in DMEM ph(–) for 40 min at 37 °C and 5% CO₂, washed three times with 600 μl of DMEM ph(–), incubated with 600 μl of 0.6 μM HMDS₆₅₅-DBCO in DMEM ph(–) for 20 min at 37 °C and 5% CO₂ and washed again six times with DMEM ph(–) and 10% FBS for 10 min each at 37 °C and 5% CO₂. Finally, the medium was exchanged with 600 μl of DMEM ph(–) supplemented with 1% FBS, 100 μM Trolox and 500 μM sodium ascorbate. The sample was subsequently visualized via SMLM imaging on an inverted Zeiss Elyra 7 microscope equipped with a Plan-Apo ×63/1.4-NA oil immersion lens (40% laser power for the 642-nm laser, 60.57° TIRF mirror angle, TIRF-uHP mode, 2.5-ms exposure time for each frame and 6,000 frames obtained in 36 s).

The live-cell data were acquired from different systems, and the image’s effective pixel size was adjusted to ~100 nm. Specifically, the data shown in Figs. 4, 5, 7, and the corresponding supplementary figures were acquired from a commercial Zeiss Elyra 7 microscope in HILO mode with a 60×/1.46 oil objective. For a FOV size of 25.6 × 25.6 µm², we recorded dual-color live-cell images at 100 Hz with 15 W/cm² illuminance for 5000 time points (Figs. 4, 5, and 7, and the corresponding supplementary figures) except for the data in Fig. 7a which is recorded at 0.5 Hz for 200 time points and Supplementary Fig. 29 which is recorded at 1 Hz and for 250 time points. And for whole-cell imaging with a FOV size of 60 × 50 µm², due to the data transmission limitation of the system, we used a 20 Hz imaging speed for 5000 time point recordings (Fig. 5a); in this process, the illumination intensity was reduced to 3 W/cm². The data in Fig. 6 was acquired with a Zeiss SP8 confocal microscope at 3 W/cm² illuminance with a 63×/1.4 oil objective. We recorded 300 time points at 0.4 Hz for a FOV of 51.2 × 51.2 µm².

FLAG-ST-CCR5 expressing cells, transfected with βarr2-GFP, were plated on MatTek plates 72 hr before imaging. Cells were stained with the anti-FLAG M2-Cy3 (5 min) and incubated in the presence or absence of 3 nM PSC-RANTES in DMEM/1%BSA medium for the indicated time. Cells were put on ice and fixed with paraformaldehyde (PFA) 4% at 4 °C for 40 min before three washes in PBS. Experiments were performed using a Elyra 7 microscope (Carl Zeiss Gmbh) equipped with two sCMOS cameras PCO Edge 4.2, and using an alpha Plan Apo 63 x/1.46 oil objective, a 488 nm (500 mW) and a 561 nm (500 mW) laser line, and a quad band filter coupled to BP 495–550 or BP 570–620 filters. All TIRF images analyses were performed using ICY software and the spot detector and the colocalization studio plugins. The number of spot detected per cell was normalized to the size of the cell surface.

Images were acquired with an Elyra 7 microscope (Zeiss) with a Lattice Structured Illumination Microscopy (SIM²) module. Cell suspensions were spotted on a slide and covered with a 170 ± 5 µm high precision cover glass (Marienfeld). A Plan-apochromat 63×/1.4 DIC M27 objective lens was used. Chl and GFP were excited with a 488 nm laser with an exposure time of 100 ms. Fluorescence emission channels were separated with a 560 nm beam-splitter. Emission wavelengths were defined by a 495–550 nm bandpass filter for GFP and a 655 nm long-pass filter for Chl.

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.

To determine UPR^mt or UPR^ER activation, strains carrying hsp‐6p::GFP or hsp‐4p::GFP were utilized. Synchronized worms were visualized at of day 1 of adulthood. For the fluorescence imaging, worms were anesthetized with M9 buffer containing 20 mM sodium azide, and an Olympus BX63 microscope was used. Worm GFP intensity was then quantified with ImageJ software. In SAM, methionine, and homocysteine supplementation experiments, P₀ worms were grown on empty vector or RNAi bacteria from hatching. F₁ eggs were then transferred on plates seeded with UV‐killed bacteria and supplemented with the vehicle, 2 mM SAM (Sigma‐Aldrich, A2408), 2 mM L‐methionine (Sigma‐Aldrich, M5308), and 2 mM homocysteine (Sigma‐Aldrich, 69453). Day 1 adult F₁ worms were then scored for GFP intensity.
For monitoring the subcellular localization of SLC‐25A26::GFP or TRMT‐10C.2::GFP, worms were mounted on 2% agarose pads with 50 mM tetramisole (Sigma, T1512). Images were acquired using Zeiss Elyra 7 microscope and Zeiss LSM 900 laser scanning confocal microscope, respectively.
For Mitotracker Red staining, L4 worms carrying TRMT‐10C.2::GFP were cultured on UV‐killed bacteria with 2 μM Mitotracker (Cell Signaling Technology, 8778) for 24 h. Worms were subsequently transferred to stain‐free plates for 30 min to eliminate the excess stain in the gut lumen before proceeding with image analysis.