LLSM

The LLSM was home-built in the Kiyosue laboratory at RIKEN Center for Biosystems Dynamics Research following the design of the Betzig laboratory (Chen et al., 2014) under a research license agreement from Howard Hughes Medical Institute. Electric wiring was performed at RIKEN Advanced Manufacturing Support Team. Metal parts were processed by Maeda Precision Manufacturing Ltd. and Zera Development Co. To create a lattice light sheet, a dithered square lattice was used through a spatial light modulator (Fourth Dimension Displays) in combination with an annular mask with 0.55 outer and 0.44 inner numerical apertures (Photo-Sciences) and a custom NA 0.65 excitation objective (Special Optics). Images were acquired through a CFI Apo LWD 25XW 1.1-NA detection objective (Nikon) and a scientific sCMOS camera, Orca Flash 4.0 v3 (Hamamatsu Photonics). Caco2 cells expressing LifeAct-RFP were seeded on a collagen-coated coverslip 5 d before imaging. During imaging, cells were maintained in L-15 medium (Thermo Fisher Scientific) supplemented with 10% serum at 25°C. For live imaging of LifeAct-RFP, a 560-nm laser (MPB Communications) and a longpass emission filter BLP02-561R-25 (Semrock) were used. Image stacks were collected with a 200-nm step size between planes with 20-ms per plane exposure times and 24.099-s intervals. After acquisition, images were deskewed and deconvolved using LLSpy (Lambert and Shao, 2019). After deskew processing, the voxel pitch was 0.104 × 0.104 × 0.103 µm. Images were represented in 3D using Imaris software (Bitplane).