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

Abstract

Expansion microscopy (ExM) is a powerful technique to overcome the diffraction limit of light microscopy that can be applied in both tissues and cells. In ExM, samples are embedded in a swellable polymer gel to physically expand the sample and isotropically increase resolution in x, y and z. The maximum resolution increase is limited by the expansion factor of the polymer gel, which is four-fold for the original ExM protocol. Variations on the original ExM method have been reported that allow for greater expansion factors, for example using iterative expansion, but at the cost of ease of adoption or versatility. Here, we systematically explore the ExM recipe space and present a novel method termed Ten-fold Robust Expansion Microscopy (TREx) that, like the original ExM method, requires no specialized equipment or procedures to carry out. We demonstrate that TREx gels expand ten-fold, can be handled easily, and can be applied to both thick tissue sections and cells enabling high-resolution subcellular imaging in a single expansion step. We show that applying TREx on antibody-stained samples can be combined with off-the-shelf small molecule stains for both total protein and membranes to provide ultrastructural context to subcellular protein localization.

Optical nanoscopy reveals SARS-CoV-2-induced remodeling of human airway cells

“…The resolution increase is partly limited by the expansion factor of the gel, which is 4.5x for the original ExM protocol [51]. Recently, we co-developed TREx microscopy: a variation on the original ExM protocol that provides tenfold expansion and can be readily combined with small molecule stains to directly label lipid membranes or provide a total protein stain, thereby offering ultrastructural context (Figure 3A) [28]. With TREx, we set out to expand HNEC cultures directly from Transwell filters to visualize cellular morphology in 3D.…”

“…To label cellular membranes, we stained HNECs with mCLING, a palmitoylated small peptide that intercalates into the lipid bilayer and is compatible with TREx chemistry [28, 52], and combined this with a fluorophore-labeled N-hydroxysuccinimide (NHS) ester or maleimide that covalently binds to lysines or cysteines, respectively, and thereby provides density-based ultrastructural context [29]. Using glutaraldehyde-based fixation optimized to preserve membrane morphology, followed by gel polymerization inside the Transwell chamber and subsequent expansion, we visualized motile cilia (captured mid-beat) extending from the apical surface (Figure 3B and movie S1).…”

“…Using STimulated Emission Depletion (STED) microscopy, we studied the spatial organization of viral structural proteins, non-structural proteins and dsRNA at <100 nm resolution. In addition, for a comprehensive understanding of the morphological changes of airway epithelium induced by SARS-CoV-2 infection, we used a novel type of Expansion Microscopy (Tenfold Robust Expansion (TREx) Microscopy [28]) in combination with both selective and non-selective protein stains [28, 29]. This enabled the volumetric visualization of epithelial ultrastructure with specific labeling of viral proteins and revealed extensive intracellular reorganization, including Golgi fragmentation, the emergence of large multivesicular bodies containing spike-positive vesicles (MVBs), clustering of cilia, and apical membrane remodeling.…”

Dynein self-organizes while translocating the centrosome in T-cells

“…This method does not induce chemotaxis in the T-cells, so the kinapse form of the T-cell is not guaranteed before activation. To clearly visualize the MT network in these cells, we used tenfold robust expansion microscopy (TREx), where samples are physically expanded before imaging to better resolve the fine structure ( Damstra et al. , 2021 ).…”

Mapping densely packed GPIIb/IIIa receptors in murine blood platelets with expansion microscopy

“…To address the issue of signal loss and dilution, several modifications of the anchoring chemistry and homogenization procedure have been reported to allow post expansion labeling. 31–34…”