Using Expansion Microscopy to visualize and characterize the morphology of mitochondrial cristae

Abstract

15Mitochondria are double membrane bound organelles indispensable for biological processes such as 16 apoptosis, cell signalling, and the production of many important metabolites, which includes ATP that 17 is generated during the process known as oxidative phosphorylation (OXPHOS). The inner membrane 18 contains folds called cristae, which increase the membrane surface and thus the amount of membrane-19 bound proteins necessary for the OXPHOS. These folds have been of great interest not only because 20 of their importance for energy conversion, but also because changes in morphology have been linked 21 to a broad range of diseases from cancer, diabetes, neurodegenerative diseases, to ageing and 22 infection. With a distance between opposing cristae membranes often below 100 nm, conventional 23 fluorescence imaging cannot provide a resolution sufficient for resolving these structures. For this 24 reason, various highly specialized super-resolution methods including dSTORM, PALM, STED and SIM 25 have been applied for cristae visualisation. 26Expansion Microscopy (ExM) offers the possibility to perform super-resolution microscopy on 27 conventional confocal microscopes by embedding the sample into a swellable hydrogel that is 28 isotropically expanded by a factor of 4-4.5, improving the resolution to 60-70 nm on conventional 29 confocal microscopes, which can be further increased to ~ 30 nm laterally using SIM. Here, we 30 demonstrate that the expression of the mitochondrial creatine kinase MtCK linked to marker protein 31 GFP (MtCK-GFP), which localizes to the space between the outer and the inner mitochondrial 32 membrane, can be used as a cristae marker. Applying ExM on mitochondria labelled with this construct 33 enables visualization of morphological changes of cristae and localization studies of mitochondrial 34 proteins relative to cristae without the need for specialized setups. For the first time we present the 35 combination of specific mitochondrial intermembrane space labelling and ExM as a tool for studying 36 internal structure of mitochondria. 37

Visualizing subcellular structures in neuronal tissue with expansion microscopy

“…Several groups have optimized expansion protocols to visualize subcellular organelles across different sample types, including various cell lines 1,5,8,13–16 , rat liver 17 , clinical specimens 18 , fungi 6,19 , songbird 20 and drosophila 21,22 . Others have used ExM to visualize subcellular structures, including mitochondria 1,2,23–25 and/or spines 2,20,23 in brain tissue, but few have systematically analyzed how fluorescence intensities and expansion factors compare across protocols or with unexpanded measurements.…”