Expansion Microscopy of Lipid Membranes

Abstract

Lipids are fundamental building blocks of cells and their organelles, yet nanoscale resolution imaging of lipids has been largely limited to electron microscopy techniques. We introduce and validate a chemical tag that enables lipid membranes to be imaged optically at nanoscale resolution via a lipid-optimized form of expansion microscopy, which we call membrane expansion microscopy (mExM). mExM, via a novel post-expansion antibody labeling protocol, enables protein-lipid relationships to be imaged in organelles such as mitochondria, the endoplasmic reticulum, the nuclear membrane, and the Golgi apparatus. mExM may be of use in a variety of biological contexts, including the study of cell-cell interactions, intracellular transport, and neural connectomics. MainExpansion microscopy (ExM) physically magnifies biological specimens by covalently anchoring biomolecules or labels to a swellable polymer network (typically sodium polyacrylate) synthesized in situ throughout the specimen 1-4 . Following tissue softening and solvent exchange, the hydrogel-specimen composite expands isotropically, typically to a physical magnification of ~4.5x in linear dimension. The net result is that biomolecules or labels that are initially localized within the diffraction limit of a traditional optical microscope can now be separated in space to distances far enough that they can be resolved on ordinary microscopes. Expansion microscopy protocols 5 for the visualization of proteins 3,6,7 and nucleic acids 4 are in increasingly widespread use, raising the question of whether other biological molecule classes, such as lipids, can also be visualized by ExM. We here report an expansion microscopy-compatible lipid stain, as well as a

Expansion Microscopy provides new insights into the cytoskeleton of malaria parasites including the conservation of a conoid

“…Such approaches might pave the way for further investigation of the Plasmodium cytoskeleton and its molecular organization. Finally, the recently developed membrane-based expansion microscopy protocols 38,39 could also provide other exciting insights into the membrane biology of Plasmodium . In summary, the use of expansion microscopy can nicely complement electron and super resolution microscopy as it provides molecular details to the available structural information without the need for specialised microscopes.…”

Light microscopy of proteins in their ultrastructural context

“…In the other extreme: lipid droplets, having a lipid core, are dissolved by the used detergents, leaving only a shadow image of their former shape. Future systematic studies, refinements to the protocols and engineered probes, such as cross-linkable lipid labels [16] and reversible protein crosslinking reagents, will answer these questions and promise further improvements.…”

Visualizing cellular and tissue ultrastructure using Ten-fold Robust Expansion Microscopy (TREx)

“…We next explored the use of TREx for high-resolution imaging of specific proteins, NHS stains and lipid membranes in cultured cells. For membranes, a custom-synthesized membrane probe compatible with the ExM process has been shown to visualize membrane surfaces in fixed brain tissue (Karagiannis et al ., 2019). This probe relies on a peptide-modified lipid tail that intercalates in target membranes and provides opportunities for anchoring to the gel through D-lysines its peptide sequence.…”