Abstract
Mitochondria are double membrane bound organelles indispensable for biological processes such as apoptosis, cell signaling, and the production of many important metabolites, which includes ATP that is generated during the process known as oxidative phosphorylation (OXPHOS). The inner membrane contains folds called cristae, which increase the membrane surface and thus the amount of membrane-bound proteins necessary for the OXPHOS. These folds have been of great interest not only because of their importance for energy conversion, but also because changes in morphology have been linked to a broad range of diseases from cancer, diabetes, neurodegenerative diseases, to aging and infection. With a distance between opposing cristae membranes often below 100 nm, conventional fluorescence imaging cannot provide a resolution sufficient for resolving these structures. For this reason, various highly specialized super-resolution methods including dSTORM, PALM, STED, and SIM have been applied for cristae visualization. Expansion Microscopy (ExM) offers the possibility to perform super-resolution microscopy on conventional confocal microscopes by embedding the sample into a swellable hydrogel that is isotropically expanded by a factor of 4–4.5, improving the resolution to 60–70 nm on conventional confocal microscopes, which can be further increased to ∼ 30 nm laterally using SIM. Here, we demonstrate that the expression of the mitochondrial creatine kinase MtCK linked to marker protein GFP (MtCK-GFP), which localizes to the space between the outer and the inner mitochondrial membrane, can be used as a cristae marker. Applying ExM on mitochondria labeled with this construct enables visualization of morphological changes of cristae and localization studies of mitochondrial proteins relative to cristae without the need for specialized setups. For the first time we present the combination of specific mitochondrial intermembrane space labeling and ExM as a tool for studying internal structure of mitochondria.
Highlights
Super-resolution imaging has revolutionized fluorescence imaging by its capability to bypass the resolution limit of optical microscopy as defined by Abbe (1873)
As an example of the applicability of this technique, using the combined resolution power of Expansion microscopy (ExM) and structured illumination microscopy (SIM) we demonstrate that the mitochondrial transcription factor TFAM associates with cristae, and we observe changes in mitochondrial morphology after membrane potential dissipation by CCCP or knockdown of the member of the mitochondrial intermembrane space bridging complex (MIB), Sam50
This approximately fourfold expansion resulting in a resolution of ∼60 nm (Chen et al, 2015) appears to be a promising tool for mitochondrial research, especially for cristae, folds of the inner membrane, with a distance often below 100 nm (Jakobs and Wurm, 2014)
Summary
Super-resolution imaging has revolutionized fluorescence imaging by its capability to bypass the resolution limit of optical microscopy as defined by Abbe (1873). The most common methods, stimulated emission depletion (STED) microscopy (Hell, 2007), photoactivated localization microscopy (PALM) (Betzig et al, 2006) and (direct) stochastic optical reconstruction microscopy (d)STORM (Heilemann et al, 2008), were applied to countless biological specimens and can provide new insights into cellular structures and tissue in 2D and 3D (Xu et al, 2013; Michie et al, 2017). With an expansion of ∼4–4.5 times, ExM empowers scientists to resolve structures with a lateral resolution of ∼60– 70 nm on a confocal microscope and in combination with structured illumination microscopy (SIM) (Gustafsson, 2000) of even ∼30 nm, approaching the resolution of other conventional super-resolution methods (Wang et al, 2018)
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