Lattice light sheet 7

Before imaging, cells were stained overnight using the JF646 HaloTag ligand according to the manufacturer’s instructions (Promega), then treated with 10 μM oligomycin and 4 μM antimycin A (OA) to depolarize mitochondria. Time-lapse live-cell data were acquired using a Lattice Light Sheet 7 (Zeiss, pre-serial). Light sheets (488 nm and 633 nm) of length 30 µm with a thickness of 1 μm were created at the sample plane using a ×13.3/0.44 NA objective. Fluorescence emission was collected via a ×44.83/1 NA detection objective. Aberration correction was set to a value of 182 to minimize aberrations as determined by imaging the Point Spread Function using 170 µm fluorescent microspheres at the coverslip of a glass-bottom chamber slide. Resolution was determined to be 454 nm (lateral) and 782 nm (axial). Data were collected with a range of frame rates of 16–20 ms and a z-step of 300 nm. Light was collected through a multi-band stop, LBF 405/488/561/633, filter. Images were collected at 37 °C and 5% CO₂.

CAAX-RFP hiPSCs were stained with 100 nM MitoTracker Green FM (Invitrogen, M7514) for 30 min prior to imaging. Cells were plated onto 25 mm MatTek dishes and imaged in phenol red-free mTeSR1 (Stemcell Technologies, 85850). Cells were kept under 5% CO₂ and 37 degrees C. For imaging, we used Zeiss Lattice Lightsheet 7 with 10× N.A. 0.4 illumination objective lens and 48× N.A. 1.0 detection objective lens. We acquired images in two channels: green channel with excitation at 488nm and emission at 512nm; red channel with excitation at 561nm and emission at 597nm. For both channels we used 18% laser power and 8ms exposure. The illumination light-sheet was the Sinc3 beam with length 15 μm, thickness 650 nm and no side lobes. The volume size was 2048 x 448 x 57 pixels or 296.94 x 64.96 x 8.12 μm with isotropic pixel size 145 nm after coverglass transformation. Images were saved with bit depth 16 bits. For each region, we imaged 93 frames with frame rate 3.26 s per volume for total 5 min. For LLSM data processing, we used the Lattice Lightsheet 7 Processing Module on ZEN Blue for deconvolution, deskew, cover glass transformation and bleach correction.

TIRFM images of live cells were acquired on a Nikon Ti Eclipse inverted microscope with a CFI Plan Apo Lambda 100x Oil (#MRD01905, Nikon) silicone objective and a sCMOS camera controlled by NIS-Elements (Nikon). Sample drift was reduced using an autofocus system (Perfect Focus, Nikon) for time-lapse imaging.
Confocal images of fixed cells were obtained with a UPLSAPO 60X S (NA 1.3; WD 0.3 mm) silicone objective on an Olympus FV3000 inverted microscope at the EMBL advanced light microscopy facility.
Epifluorescent and bright-field imaging of fixed cells was performed using a 40x objective (#MRD00405, Nikon), a SOLA SE II, and 100 W halogen lamps (Nikon) using appropriate filter sets.
Polarized TIRFM (pTIRFM) modality was implemented based on previous work^{63 (link)–68} . For imaging, dHL-60 cells were stained before plating with carbocyanine dye DiI (#D3911, ThermoFisher Scientific).
Lattice light sheet imaging of live cells was performed on a Zeiss Lattice Light sheet 7 (Zeiss, Oberkochen, Germany) using appropriate filter sets and a 10 × 550 μm base beam.