Iragavarapu, A. G.; Artemchuk, O.; Bobe, D.; Ratliff, A.; Pavlov, E.; Aydin, H.

Mitochondria are dynamic signaling organelles that transduce metabolic and biochemical cues to facilitate cellular adaptation. Their complex structure and dynamics are essential for integrating metabolic pathways, responding to stressors, and communicating inter- and intra-cellular signals. While optimal mitochondrial activity is frequently linked to cellular and organismal health, influencing processes ranging from metabolism and regulated cell death to differentiation and growth, the mechanistic links between mitochondrial dysfunction and cellular defects leading to human disease remain incompletely understood. Understanding how mitochondrial shape and function are linked is crucial for deciphering the regulatory mechanisms of cell survival and fate. Here, we present a molecular resolution cryo-electron tomography (cryo-ET) imaging and image analysis platform to investigate the structure of isolated human mitochondria under different conditions. We describe optimized protocols for isolating mitochondria from human cells, vitrifying these samples with high-pressure freezing (HPF) using the waffle method, cryo-focused ion beam (cryo-FIB) milling to generate thin sections (lamellae), and imaging with cryo-transmission electron microscopy (cryo-TEM). This is complemented by a robust downstream processing pipeline for tilt-series alignment, tomogram reconstruction, and three-dimensional (3D) segmentation of tomograms using the latest state-of-the-art algorithms. With some variations, this versatile workflow is adaptable to other subcellular compartments for structural studies in isolation or within intact cells. Furthermore, our protocols provide a critical foundation for investigating the in-situ structure of protein machineries that govern key cellular processes.