Brightlineff01 600 37

A custom, three-colour SIM microscope [99 (link)] was used to collect images. A ferroelectric binary Spatial Light Modulator (SLM) (SXGA-3DM, Forth Dimension Displays) was used to pattern the light with a grating structure (3 angles and 3 phases). Light was collected by a 60× water immersion objective with a NA of 1.2 (UPLSAPO60XW, Olympus) and a sCMOS camera (C11440, Hamamatsu). eYFP-ICP0 fluorescent emission was captured using a 488 nm laser (iBEAM-SMART-488, Toptica) and an BA510-550 (Olympus) emission filter. gC-mCherry fluorescence was captured using a 561 nm laser (OBIS561, Coherent) and a BrightLineFF01-600/37 filter (Semrock). AF647 fluorescence was captured using a 640 nm laser (MLD640, Cobolt) and a BrightLineFF01-676/29 filter (Semrock). Background-reduced and resolution-enhanced images were reconstructed from raw SIM data using FairSIM [100 (link)]. Deconvolution was performed alongside using a Richardson-Lucy algorithm with 5 iterations. 100 nm beads (TetraSpeck Microspheres, Thermo Fisher) were used to determine the shifts in X and Y for channel alignment. The channels were aligned using the TransformJ plugin in Fiji [101 (link)].

Structured illumination images were collected on a custom-built structured illumination microscopy (SIM) setup, which has been described in detail (86 (link)). A ×60/1.2 NA water immersion lens (UPLSAPO 60XW, Olympus) focused the structured illumination pattern onto the sample was used. This lens also captured the samples' fluorescence emission light before imaging onto a sCMOS camera (C11440, Hamamatsu). Laser excitation wavelengths used were 488 (iBEAM-SMART-488, Toptica), 561 (OBIS 561, Coherent), and 640 nm (MLD 640, Cobolt). Respective emission filters were BA 510-550 (Olympus), BrightLine FF01-600/37, and BrightLine FF01-676/29 (Semrock, New York, USA). Imaging was done on fixed or live cells, as indicated. Images were acquired using custom SIM software (HCImage, Mamamatsu Corporation, Sewickley, NJ, USA). Nine raw images were collected at each plane and each color. FairSIM plugin in FIJI was used to reconstruct images (87 (link)).