A pipeline for multidimensional confocal analysis of mitochondrial morphology, function, and dynamics in pancreatic β-cells

TLDR

Live‑cell imaging of mitochondrial function and dynamics yields insights into physiology and disease, yet dense multilayered networks in insulin‑secreting β‑cells are difficult to analyze without super‑resolution microscopy and commercial software. To address this, we created a comprehensive pipeline and ImageJ plugin that quantifies mitochondrial network morphology and dynamics in 2D/3D for mouse β‑cells and other challenging cell types. The approach relies on standard confocal microscopy and shareware, was validated with mitochondrial photolabeling and unsupervised cluster analysis, and enables per‑organelle morphological and functional analysis in 4D (xyzt). This tool provides a powerful framework for multiplexed mitochondrial state and function analysis, accelerating research in health and disease.

Abstract

Live-cell imaging of mitochondrial function and dynamics can provide vital insights into both physiology and pathophysiology, including of metabolic diseases like type 2 diabetes. However, without super-resolution microscopy and commercial analysis software, it is challenging to accurately extract features from dense multilayered mitochondrial networks, such as those in insulin-secreting pancreatic β-cells. Motivated by this, we developed a comprehensive pipeline and associated ImageJ plugin that enables 2D/3D quantification of mitochondrial network morphology and dynamics in mouse β-cells and by extension other similarly challenging cell types. The approach is based on standard confocal microscopy and shareware, making it widely accessible. The pipeline was validated using mitochondrial photolabeling and unsupervised cluster analysis and is capable of morphological and functional analyses on a per-organelle basis, including in 4D ( xyzt). Overall, this tool offers a powerful framework for multiplexed analysis of mitochondrial state/function and provides a valuable resource to accelerate mitochondrial research in health and disease.