Sim system

Structured illumination microscopy (SIM) was done on a Nikon SIM system with a 100 × 1.49 objective and images were assessed as in [15 (link)].
Briefly, randomly selected cells were imaged at laser excitation of 405 nm (nuclei), 488 nm (SIGMA 1-R-GFP), 561 nm (ER or SEPN1), and 640 nm (mitochondria) with a 3D-SIM acquisition protocol. SIM images were quantified with ImageJ. Co-localization between SIGMA 1-R-GFP (channel 1) and SEPN1 (channel 2) was analyzed using the Jacop ImageJ plugin and expressed as Manders 2 index. After background normalization, the ER and mitochondria signals were segmentated to calculate the area of the ER and that of the overlapping ER and mitochondria, both expressed as fraction of total cell area. The segmentated mitochondrial signal was followed by the skeletonize function of ImageJ to perform mitochondria network analysis. We then applied the Analyze Skeleton 2D/3D plugin of ImageJ to measure each cell mean branch length, expressed as μm.

The SIM on brain sections was done with a Nikon SIM system with a 100 × 1.49 NA oil immersion objective, managed by NIS elements software. Tissues were imaged at laser excitation of 488 (for Tmem119) and 640 nm (for Iba1) with a 3D-SIM acquisition protocol. Fourteen-bit images sized 1024 × 1024 pixels, with a single-pixel of 0.030 μm, were acquired in a gray level range of 0–4,000 to exploit the linear range of the camera (iXon ultra DU-897U, Andor) and to avoid saturation. Raw and reconstructed images were verified by the SIMcheck ImageJ plugin (Ball et al., 2015 (link)).