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

By A. Chaudhry, Rocky Shi, D. Luciani. bioRxiv, 2019

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Mitochondrial network morphology and dynamics are inextricably linked to cellular metabolism and health, and live-cell imaging of mitochondria can therefore provide vital insights into both physiology and pathophysiology. As a notable example, mitochondria are critical for nutrient-induced insulin secretion from pancreatic β-cells, and a better understanding of their dynamics and dysregulation could shed significant light on the pathogenesis of type 2 diabetes. However, without using super-resolution microscopy and commercial analysis software it can be exceedingly challenging to accurately extract mitochondrial features in cells with dense multi-layered networks, such as β-cells. Motivated by this, we developed an analysis pipeline and ImageJ plugin that enables 2D/3D quantification of mitochondrial network morphology and dynamics in β-cells and, by extension, in other similarly challenging cell-types. Importantly, this approach is based on standard confocal microscopy and shareware, making it widely accessible. The accuracy of mitochondrial identification was established by photo-labelling individual mitochondria, and we validated the ability to quantitatively categorize mitochondrial 2D/3D shape and network descriptors using unsupervised cluster analysis. We further demonstrate the capability for analyzing mitochondrial morphology and function on a per-mitochondrion basis, as well as 4D (xyzt) analysis of mitochondrial dynamics. The pipeline also offers a convenient framework for multiplexing of imaging-based readouts of mitochondrial state/function, which we propose could facilitate expansive identification of mitochondrial “morpho-functional” fingerprints associated with various physiological and pathophysiological states. Overall, this analysis pipeline permits robust multidimensional quantification of mitochondrial morphology and dynamics, and provides a valuable resource to accelerate research into the role of mitochondria in disease.

Subfields: Molecular Biology, Physiology

View Paper: https://www.semanticscholar.org/paper/8dd5489a2d4872fe41d3d7eb0dc64d2535e5b3cf