Organelle Proximity Analysis for Enhanced Quantification of Mitochondria–Endoplasmic Reticulum Interactions in Single Cells via Super-Resolution Microscopy

Mitochondria (MT) and the endoplasmic reticulum (ER) maintain lipid and calcium homeostasis through membrane contacts, particularly MT–ER contacts (MERCs), spanning distances from 10 to 50 nm. However, the variation of different distance ranges and the metabolic factors influencing this variation remain poorly understood. This study employed microfluidic chip-based super-resolution microscopy in conjunction with a Moore-Neighbor tracing-incorporated organelle proximity analysis algorithm. This approach enabled precise three-dimensional localization of single-fluorescence protein molecules within narrow and irregular membrane proximities. It achieved lateral localization precision of less than 20 nm, resulting in a minimum MERC distance of approximately 8 nm in spatial and mean distances across multiple threshold ranges. Additionally, we demonstrated that the MERC distance variation was correlated with MT size rather than ER width. The proportion of each distance range varied significantly after the stimuli. Free cholesterol showed a negative correlation with various distances, while distances of 10–30 nm were associated with glucose, glutamine, and pyruvic acid. Furthermore, the 30–40 nm range was influenced by citric acid. These results underscore the role of advanced subcellular organelle analysis in elucidating the single-molecule behavior and organelle morphology in single-cell studies.