Expansion tomography for large volume tissue imaging with nanoscale resolution

Abstract

Expansion microscopy enables conventional diffraction limit microscopy to achieve super-resolution imaging. However, the enlarged tissue lacks an objective lens with sufficient working distance that can image tissues with whole-brain-scale coverage. Here, we present expansion tomography (ExT) to solve this problem. We have established a modified super-absorbent hydrogel (ExT gel) that possesses high mechanical strength and enables serial sectioning. ExT gel enables tissue and cell imaging and is compatible with various fluorescent labeling strategies. Combining with the high-throughput light-sheet tomography (HLTP) system, we have shown the capability of large volume imaging with nanoscale resolution of mouse brain intact neuronal circuits. The ExT method would allow image samples to support super-resolution imaging of intact tissues with virtually unlimited axial extensions.

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

“…Successful application of TREx to filter-cultured Caco-2 cells further demonstrates the robustness of TREx, because we had repeatedly failed to cleanly recover epithelial cultures using standard ExM. The robustness of TREx has been further demonstrated by its adoption in other biological systems (Gros, Damstra, Kapitein, Akhmanova, & Berger, 2021) including in cultured neurons (Özkan et al, 2021) and primary cultured human cells (Nijenhuis et al, 2021), and by its superior mechanical properties as measured by traditional materials characterization methods (R. Chen et al, 2021).…”

Expansion Microscopy: Super-Resolution Imaging with Hydrogels

“…As pointed out above (see Basics of Hydrogel Chemistry for Expansion Microscopy), reducing gel elasticity in this way is expected to limit the expansion factor. The developers of ExT seem to have successfully compensated for this by almost doubling the concentration of ExT’s ionic hydrogel component AMPS-Na relative to ExM’s ionic component sodium acrylate in the classical recipe, resulting in an overall expansion factor of 4- to 4.5-fold . The authors propose that the increased mechanical stability is achieved via hydrogen bond formation between AMPS-Na components in the polymer .…”

“…Unfortunately, the outcome of optimizing expansion hydrogel chemistries is not fully predictablepartially because the fields of polymer chemistry and hydrogel material science are far from complete and many open questions on the molecular mechanisms remain, − ,, and partially because employing these chemistries to generate polymer hybrids with biological specimens introduces further uncertainties. Developing new hydrogel chemistries fit for purpose in expansion microscopy has thus often relied on painstaking trial and error and serendipity as much as on rational design. ,− ,, Still, are there any “rules of thumb” we can derive from a comparative analysis? What are the different approaches to controlling the expansion factor, and how do these impact other gel parameters?…”

GelMap: Intrinsic calibration and deformation mapping for expansion microscopy

“…One of the key advantages of ExM is that it can be used to directly expand tissues and organisms (Chen et al, 2015(Chen et al, , 2021Ku et al, 2016;Sim et al, 2022;Yu et al, 2020). We therefore tested whether GelMap could be applied to non-adherent biological samples by incorporating the grid at the stage of sample preparation.…”

“…In recent years, various specialized ExM variants have been described, focusing on improving preservation of specific structures (Gambarotto et al, 2019), expanding tissues and entire organisms (Chen et al, 2021; Ku et al, 2016; Sim et al, 2022; Yu et al, 2020) including human biopsy tissues (Valdes et al, 2021; Zhao et al, 2017), and compatibility with other super-resolution modalities (Gao et al, 2020; Halpern et al, 2017; Shaib et al, 2022; Xu et al, 2019). Since the effective resolution of ExM is in part limited by the expansion factor, there has also been a push to develop higher expanding methods compared to the original ExM method either by varying the crosslinking concentration (Chen et al, 2015; Damstra et al, 2022), using alternative crosslinking chemistry (Li et al, 2022; Truckenbrodt et al, 2019), or using iterative approaches (Chang et al, 2017; M’Saad and Bewersdorf, 2020).…”