N sim super resolution system

NSC‐34 cells plated on glass coverslips were fixed with 3% paraformaldehyde and 0.1% glutaraldehyde in phosphate‐buffered saline (PBS) for 10 min at 20°C. After washing with PBS, samples were quenched by incubation with 50 mM NaBH₄ in PBS for 7 min, washed in PBS and then permeabilized for 30 min in PBS containing 0.2% Triton X‐100 and 3% bovine serum albumin (BSA). Following blocking with 3% BSA in PBS, samples were incubated with primary antibodies diluted in blocking solution, washed with PBS and incubated with goat anti‐rabbit or anti‐mouse secondary Igs conjugated to AlexaFluor 546 or AlexaFluor 633 (1:1,000) (Invitrogen). Following final washings in PBS, the samples were mounted in Mowiol‐DABCO mounting medium containing 10% (w/v) Mowiol 4‐88 (Calbiochem), 25% (w/v) glycerol and 2.5% (w/v) DABCO (1,4‐diazobicyclo[2.2.2]octane) in 100 mM Tris–HCl pH 8.5. Samples were analysed on a Nikon Eclipse Ti‐E inverted microscope equipped with a Nikon N‐SIM Super Resolution System. Confocal images were captured using a CFI Plan Apo IR SR 60X water immersion objective and then reconstructed using Nikon Imaging Software Elements AR with N‐SIM module. ER–mitochondria interactions were quantified by Mander's coefficient using ImageJ software. Co‐localized signals were displayed using the ImageJ 1.44p RG2B co‐localization plug‐in to determine co‐localized pixels.

Live imaging was performed on an N-SIM super-resolution system (Nikon, Japan) equipped with 100× Plan Apo TIRF lens (NA=1.49) and iXon 897 EM-CCD camera (effective pixel size 63 nm) (Andor, Ireland) in 3D-SIM mode (excitation laser line 488 nm, 120 nm Z-steps) under control of NIS-Elements 4.6 software. Raw image stacks (3 grating angles×5 phase shifts) were analyzed for image quality with the SIMcheck module of ImageJ software and processed using SIM module of NIS-Elements using parameters selected on the basis of Fourier transform analysis. Reconstructed stacks were further deconvolved using the Richardson-Lucy algorithm built into NIS-Elements.
Images for colocalization were processed using FIJI (Schindelin et al., 2012 (link); Schneider et al., 2012 (link)), including the Enhance Contrast feature to correct for bleaching and the Maximum Instensity projection feature for presentation in the paper.