Cryo-Expansion Microscopy of C. elegans and Tardigrades v1

Expansion microscopy (ExM) improves imaging resolution through sample-level physical expansion, complementing optical resolution improvements and enabling the two to compound (1). Beyond resolution gains, ExM sample preparation itself enhances antibody labeling, likely by unmasking epitopes through increased physical separation of biological targets (2,3,4). Replacing chemical fixation with cryo-fixation further boosts labeling quality — presumably by eliminating formaldehyde and glutaraldehyde from the workflow — to a degree that enables quantitative expansion microscopy (qExM): the estimation of endogenous protein complex abundances through a dual-antibody labeling paradigm (5,6). However, cryo-fixation for ExM has so far been limited to cultured cells and yeast (5,6,7,8). Extending this approach to intact invertebrate model organisms presents unique challenges due to sample size, cuticle permeability, and the need for whole-animal structural preservation. This protocol describes a pipeline for cryo-fixation-based expansion microscopy of whole Caenorhabditis elegans and wild-caught tardigrades. Specimens are cryo-fixed by high-pressure freezing (HPF), subjected to freeze substitution in pure acetone, and rehydrated through a graded ethanol series before undergoing standard ExM processing — including gelation, disruption, and fluorescent labeling. This approach preserves ultrastructural morphology while maintaining high antigenicity, enabling high-resolution fluorescence imaging of intact whole-animal specimens. Cryo-fixation followed by acetone freeze substitution offers several advantages over aldehyde-based chemical fixation. Unlike formaldehyde or glutaraldehyde, acetone does not covalently crosslink proteins, leaving protein-rich structures such as the cuticle more pliable and amenable to disruption during the expansion process — likely reducing distortions upon gel swelling. Consistent with this, the groundbreaking C. elegans ExM paper (ExCel) demonstrated, among many contributions including some of the earliest NHS ester labeling in expansion experiments, that chemically fixed specimens do not expand to the degree of the surrounding gel (9). There is the potential that with cryo-fixed specimens, that their expansion of the specimen will be closer to that of the hydrogel due to the absence of aldehyde based crosslinking resisting the expansion of the sample. This method also enabled antibody labeling of endogenous ATP synthase in C. elegans, yielding mitochondrial structures without any potentialcompromise from genetically encoded labels (Figure 1). In wild-caught tardigrades, NHS ester total protein staining captured the total protein content, enabling visualization of neurons, muscle cells, and storage cells in the full whole-animal context (Figure 2). Finally, synapse staining with anti-synapsin alongside DNA labeling in wild tardigrades provides a promising first step toward whole-invertebrate visual proteomics and, ultimately, quantitative expansion microscopy at the scale of the entire animal (Figure 3). By combining high-pressure freezing with freeze substitution for expansion of invertebrates, we extend the benefits of cryo-ExM to larger, intact organisms. The resulting increase in antigenicity allows for measurement of endogenous targets via antibody labeling. We anticipate that these advances in cryo-based sample preservation will compound with the broader trajectory of expansion microscopy, complementing qExM with fields such as parasitology and connectomics (10,11). References Chen F, Tillberg PW, Boyden ES. Expansion microscopy. Science. 2015;347(6221):543–548. doi: 10.1126/science.1260088 Sarkar D, et al. Revealing nanostructures in brain tissue via protein decrowding by iterative expansion microscopy. 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