Nanobiopsy: a tool for mitochondrial research

Abstract

Understanding functional differences between mitochondrial subpopulations will help improve our understanding of mitochondrial disease. Mitochondrial DNA heteroplasmy contributes to mitochondrial heterogeneity through the co-existence of mutant and wild type mitochondrial DNA populations. However, current technologies do not permit the sampling of mitochondria from within cells without losing relevant information about the cellular context of isolated organelles and risking potential damage to cells and isolated mitochondria. Nanobiopsy comprises a glass pipette, coupled with a scanning ion conductance microscope (SICM), which facilitates high-precision sampling of biological material from within cells. This technique has enabled isolation of mitochondria from within cultured fibroblasts, whilst maintaining cell viability, but has yet to be shown in human tissue samples. This PhD encompasses three key aims: First, to demonstrate the ability to successfully isolate mitochondria from skeletal muscle fibres. Second, to define the relative application of nanobiopsy to sample subcellular mitochondrial subpopulations compared with laser-capture microdissection (LCM). Finally, to develop nanobiopsy to isolate mitochondria from distinct skeletal muscle foci to elucidate the role of mitochondrial heterogeneity in mitochondrial disease pathology. To enable subcellular isolation, a range of complimentary techniques required optimisation alongside nanobiopsy. Immunohistochemistry and immunofluorescent microscopy aided the successful capture of mitochondria and the success of nanobiopsy from skeletal muscle tissue was verified using qPCR. The operational range of nanobiopsy surpassed that of LCM and allowed isolation of mitochondria from distinct subcellular foci. The key outcomes of this project include the first demonstration of subcellular mitochondrial isolation from human tissue and the accompanying down-stream genomic analysis. This has ramifications beyond investigating subcellular mitochondrial disease mechanisms, such as clonal expansion, with the potential to sample other organelles and subcellular structures with nanoscale precision. This represents a paradigm shift, facilitating the means to move from single-cell omics to subcellular-omics.