Quantifying ER-Mitochondria Contact Sites

Cells were generally imaged 48–72 h after transfection with a Leica TSC SP5 inverted confocal microscope, using either a HCX PL APO 63X/numerical aperture 1.40–0.60 or a HCX PL APO ×100/numerical aperture 1.4 oil-immersion objective. Images were acquired by using the Leica AS software. To count ER–mitochondria contacts, a complete z-stack of the cell was acquired every 0.29 µm. Z-stacks were processed using Fiji [64 (link)]: images were first convolved, and then filtered using the Gaussian Blur filter. A 3D reconstruction of the resulting image was obtained using the Volume J plugin (http://bij.isi.uu.nl/vr.htm). A selected face of the 3D rendering was then thresholded and used to count ER–mitochondria contact sites.