3D-structured illumination microscopy

Animals were mounted essentially as for standard confocal microscopy (above); in some experiments live animals were immobilized on No. 1.5 gamma-irradiated glass-bottomed dishes (MatTek, Ashland, MA, USA). 3D-SIM was performed on an OMX SR microscope setup (Cytiva, Marlborough, MA, USA) equipped with an Olympus PlanApo N 60X/1.42 oil objective. Using 100 nm multicolor TetraSpeck beads (ThermoFisher) the FWHM at 488 nm was measured to be between 90–100 nm in xy and 310 nm in z. Reconstruction of images was performed using in the OMX software package softWoRx. Reconstruction accuracy was verified using the SIMcheck plugin for ImageJ;⁷⁰ all reconstructions were classified as good to very good. The average modulation contrast to noise ratios (MCNRs) in the 3D-SIM reconstructions in Fig. 5 were >8, apart from the DPY-7 reconstructions which scored 4–7.3; an MCNR > 8 is considered good to excellent according to SIMcheck documentation. Visualization of the hollow centers of BLI-containing struts did not require further processing. To smoothen pixelated individual struts (e.g. Figure 5b), images were upscaled with bicubic interpolation (ImageJ). 3D rendering of struts was performed using the open-source software Icy⁷¹.