A Standardized Method to Quantify ER-Mitochondrial Interfaces in Electron Mircographs

Abstract

The interaction sites between endoplasmic reticulum (ER) and mitochondria are signaling hubs in the cell implicated in calcium transfer, lipid metabolism, autophagy, and cell death among other functions. Transmission electron microscopy (TEM) is commonly used to visualize the geometry of these interfaces, but metrics used to describe and compare between conditions, as well as the techniques of measurement are not standardized. Here we describe a pair of scripts for ImageJ that allow for rapid, reproducible and flexible quantification of interface geometries. One script is focused on measuring the physical apposition of the membranes while the other considers the potential exposure of the outer mitochondrial membrane (OMM) to calcium released from the ER. In both cases, the user simply traces the OMM and nearby ER membranes; the script bins the interface distances and returns the length of the OMM that participates in an interface of a given gap width, as well as the total OMM and ER lengths. A ‘score’ based on the inverse-square of the distance is also generated as an attempt to make a single value representing ‘interface-ness’. The additive nature of these measures allows cell-wise totals to be calculated for a given TEM section. While this approach standardizes the measurement technique for a number of parameters of ER-mitochondrial interfaces, it remains to be seen which parameters best correspond with physiological changes in different models.