Membrane contact site (MCS)-mediated organelle interactions play essential roles in the cell. Quantitative analysis of MCSs reveals vital clues for cellular responses under various physiological and pathological conditions. However, an efficient tool is lacking. Here, we developed DeepContact, a deep-learning protocol for optimizing organelle segmentation and contact analysis based on label-free EM. DeepContact presents high efficiency and flexibility in interactive visualizations, accommodating new morphologies of organelles and recognizing contacts in versatile width ranges, which enables statistical analysis of various types of MCSs in multiple systems. DeepContact profiled previously unidentified coordinative rearrangements of MCS types in cultured cells with combined nutritional conditions. DeepContact also unveiled a subtle wave of ER–mitochondrial entanglement in Sertoli cells during the seminiferous epithelial cycle, indicating its potential in bridging MCS dynamics to physiological and pathological processes.

DeepContact is a deep learning software for high-throughput quantification of membrane contact site (MCS) in 2D electron microscopy images. This protocol will guide users through incorporating available DeepContact models in Amira's artificial intelligence module, thereby allowing invoking of DeepContact functions in organelle segmentation and quantifying of MCS with a user-friendly graphical user interface of Amira software. For complete details on the use and execution of this protocol, please refer to Liu et al. (2022).¹.