Lipid Expansion Microscopy

Abstract

Strategies to visualize cellular membranes with light microscopy are restricted by the diffraction limit of light, which far exceeds the dimensions of lipid bilayers. Here, we describe a method for super-resolution imaging of metabolically labeled phospholipids within cellular membranes. Guided by the principles of expansion microscopy, we develop an all-small molecule approach that enables direct chemical anchoring of bioorthogonally labeled phospholipids into a hydrogel network and is capable of super-resolution imaging of cellular membranes. We apply this method, termed lipid expansion microscopy (LExM), to visualize organelle membranes with precision, including a unique class of membrane-bound structures known as nuclear invaginations. Compatible with standard confocal microscopes, LExM will be widely applicable for super-resolution imaging of phospholipids and cellular membranes in numerous physiological contexts.

Expansion Microscopy: Super-Resolution Imaging with Hydrogels

“…In other words: a technique such as click-ExM may be able to label any biomolecule in expansion microscopy, but not to differentiate them when combined in the same sample�something FLARE is specifically designed to accomplish. It should also be noted that the intermediary trifunctional linkers used by click-ExM, 49 TRITON, 48 and LExM 46 are likely to introduce a larger linkage error than the direct conjugation approach of FLARE. 110 This might particularly impact click-ExM, with its relatively large streptavidin intermediary.…”

“…Largely omitted from the discussions above were a few recent developments that aim to (or are conceptually capable of) making any biomolecule accessible to expansion microscopy. ,, The click-ExM, TRITON, and LExM approaches all developed strategies for anchoring and visualizing biomolecules that can be universalized to be applied to any class of biomolecule. For TRITON and LExM, this strategy is based in trifunctional linkers with the by now familiar modalities: (a) bioconjugation to the target, (b) integration into the hydrogel polymer, and (c) readout via an integrated fluorophore. For click-ExM, this strategy is based on a modular conjugation and staining approach: (a) biomolecules are labeled with a click chemistry-reactive group, (b) a biotin is reacted to that group via a click reaction, and (c) the biotin is detected via a fluorophore-conjugated streptavidin that can be linked to the hydrogel via familiar protein-retention strategies. , …”

“…The universal aspect of these approaches lies in the fluorophore conjugation strategy, which is the same no matter what biomolecule is targeted: click chemistry − for click-ExM and LExM, and tetrafluorophenyl chemistry for TRITON . LExM does not immediately universalize its strategy to biomolecules other than lipids, but it should be noted that it seems conceptually just as universally applicable as click-ExM …”

Imaging and Editing the Phospholipidome

“…Key to this approach, termed lipid expansion microscopy (LExM), are trifunctional detection probes equipped with an azide for tagging alkynyl PCs, a fluorophore for visualization, and a methacrylamide group for covalent incorporation into hydrogels that are ultimately expanded after sample clearing (Figure 6A). 51 A defining feature of LExM is the preservation of signal due to cross-linking of tagged lipids to the hydrogel prior to detergent-assisted permeabilization, which is a required step in expansion microscopy and otherwise rinses away the lipids. Using either metabolic labeling of PC with an alkynyl choline analogue 47 or IMPACT with an alkynyl alcohol, we demonstrated that LExM can enable visualization of very narrow membrane-containing structures, such as nuclearmembrane-derived channels whose dimensions are below the diffraction limit, using widely available confocal microscopes.…”

“…To better visualize narrow membranous structures and membrane contact sites, we developed a lipid-compatible version of expansion microscopy to enable super-resolution imaging of PC-containing membranes. Key to this approach, termed lipid expansion microscopy (LExM), are trifunctional detection probes equipped with an azide for tagging alkynyl PCs, a fluorophore for visualization, and a methacrylamide group for covalent incorporation into hydrogels that are ultimately expanded after sample clearing (Figure A) . A defining feature of LExM is the preservation of signal due to cross-linking of tagged lipids to the hydrogel prior to detergent-assisted permeabilization, which is a required step in expansion microscopy and otherwise rinses away the lipids.…”