Three-dimensional Structured Illumination Microscopy

The microscopy samples were observed using an Elyra PS.1 SIM microscope (Zeiss) with objective lens alpha Plan-Apochromat 100x/1.46 oil (Immersol 518F/30°C, Zeiss), as described previously (Iwai et al., 2018 (link)). The fluorophores Alexa Fluor 488 and Alexa Fluor 546 were excited with a 488 nm laser and 561 nm laser, and the fluorescence was acquired through a 495–550 nm and 570–620 nm bandpass filters, respectively. Image acquisition was performed with ZEN software (Zeiss). Each focal plane for 3D-SIM image was captured sequentially by the excitation with the patterned light of 3 rotated angled, each of which contains five shifted phases. The optimal z-interval distance was set to 101 nm. Raw SIM images were processed to reconstruct 3D-SIM images using ZEN software. Extraction of the intensity data was done using the SIMcheck plugin for ImageJ software (Ball et al., 2015 (link)).