N sim s system

Structured illumination microscopy (SIM) was performed on a Nikon N-SIM-S system (Ti-2 stand; Hamamatsu ORCA Flash 4.0 scientific CMOS dual cameras with Cairn splitter system; Nikon Perfect Focus; Chroma ET525/50m, ET595/50m and ET 700/75m emission filters; and Nikon laser bed with 405 nm/488 nm/561 nm/640 nm laser lines). A Nikon 100×1.49 NA TIRF oil objective was used. NIS Elements v5 software (Nikon) was used to control the system and acquire pre-expansion 3D-SIM images. Expanded samples were imaged on an ASI RAMM microscope frame. Widefield imaging was performed using a Nikon 100× TIRF (NA 1.45) objective and an Evolve Delta EM-CCD camera, via a quad-band emission filter (Semrock, 432/515/595/730 nm). iSPIM was performed using twin Nikon 40× (NA 0.8) water-dipping objectives, a similar quad-band emission filter (Semrock, 432/515/595/730 nm) and a Hamamatsu ORCA Flash 4.0 scientific CMOS camera. Illumination for both setups was from a Cairn Research laser bank containing 100 mW 405 nm, 150 mW 488 nm, 50 mW 561 nm and 100 mW OBIS 640 nm continuous wave (CW) lasers. Light was directed to the sample via a quad-band dichroic mirror (Semrock, 405/488/561/635 nm). Micro-Manager (https://micro-manager.org/) was used to control the system and scan the sample.