Abstract
Intracellular heterogeneity contributes significantly to cellular physiology and, in a number of debilitating diseases, cellular pathophysiology. This is greatly influenced by distinct organelle populations and to understand the aetiology of disease, it is important to have tools able to isolate and differentially analyse organelles from precise location within tissues. Here, we report the development of a subcellular biopsy technology that facilitates the isolation of organelles, such as mitochondria, from human tissue. We compared the subcellular biopsy technology to laser capture microdissection (LCM) that is the state-of-the-art technique for the isolation of cells from their surrounding tissues. We demonstrate an operational limit of >20 µm for LCM and then, for the first time in human tissue, show that subcellular biopsy can be used to isolate mitochondria beyond this limit. Graphical abstract
Highlights
Whilst low level heteroplasmy is well tolerated, the accumulation and spread of mutant mtDNA molecules in excess of a threshold level can lead to impaired oxidative phosphorylation that often culminates in mitochondrial disease [16, 17]
Using laser capture microdissection (LCM), the most commonly utilised approach for studying single cells from tissue samples [8, 10, 44–46, 65], as a comparator, we show that an adaptation of nanobiopsy, Fig. 1 (a) Subcellular biopsy in skeletal muscle
Having demonstrated the working limit of LCM, we investigated if an adaption of the nanobiopsy technology based on a scanning ion conductance microscope (SICM) could be implemented to enable subcellular sampling from human tissue sections with a spatial resolution surpassing the limits of LCM
Summary
Inter-tissue and inter-cellular heterogeneity is a known contributor to a number of human diseases including cancer [1–3]; cardiovascular disease [4, 5]; metabolic disease [6–9]; and neurodegeneration, neurodevelopmental disorders, and pathological ageing [10–13]. Evaluating heterogeneity at the tissue and cellular level can often mask subtle subcellular and organelle heterogeneity [14]. Whilst low level heteroplasmy is well tolerated, the accumulation and spread of mutant mtDNA molecules in excess of a threshold level can lead to impaired oxidative phosphorylation that often culminates in mitochondrial disease [16, 17]. The mechanism behind this process, termed clonal expansion, is not fully understood. Investigating clonal expansion at the subcellular level may advance our understanding of the mechanisms behind it and help improve characterisation of mitochondrial disease [18, 19].
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