A First Step for Expansion X-Ray Microscopy: Achieving Contrast in Expanded Tissues Sufficient to Reveal Cell Bodies

Abstract: Existing methods in nanoscale connectomics are at present too slow to map entire mammalian brains. As an emerging approach, expansion microscopy (ExM) has enormous promise, yet it still suffers from throughput limitations. Mapping the human brain and even mapping nonhuman primate brains therefore remain distant goals. While ExM increases effective resolution linearly, it enlarges tissue volume cubically, which dramatically increases imaging time. As a rapid tomographic technique, X-ray microscopy has potential for drastically speeding up large-volume connectomics. But to the best of my knowledge, no group has so far imaged cellular features within expanded tissue using X-ray microscopy. I herein present an early-stage report featuring the first demonstration of X-ray microscopy reconstruction of cell bodies within expanded tissue. This was achieved by combining a modified enzymatic Unclearing technique with a metallic gold stain and imaging using a laboratory X-ray microscope. I emphasize that a great deal of work remains to develop "expansion X-ray microscopy" (ExXRM) to the point where it can be useful for connectomics since the current iteration of ExXRM only resolves cell bodies and not neurites due to extensive off-target staining. Additionally, the current method must be modified to accommodate for the challenges of synchrotron X-ray microscopy, a vastly speedier approach than laboratory X-ray microscopy. Nonetheless, achieving X-ray contrast in expanded tissues represents a significant first step towards realizing ExXRM as a connectomics imaging modality.

• The paper demonstrates that enzymatic DAB deposition followed by gold enhancement enables sufficient X-ray contrast to reveal cell bodies in expanded tissues.
• It combines iterative expansion of mouse cortex tissue with lab-based X-ray microtomography, achieving an 8x enlargement and clear visualization of cellular structures.
• The study underlines the potential for high-throughput connectomic imaging while noting current limitations in resolving subcellular features and stain diffusion.

Expansion X-Ray Microscopy: Enabling Cellular Contrast in Expanded Brain Tissue

Introduction

Expansion microscopy (ExM) has emerged as a transformative technique for imaging biological tissues at nanoscale resolution by physically enlarging specimens using a swellable hydrogel matrix. This method enhances the effective resolution of conventional imaging modalities but increases imaging time due to the cubic amplification in tissue volume. In mammalian and primate brain connectomics, the throughput limitations of electron and fluorescence-based imaging preclude whole-brain mapping. X-ray microscopy (XRM), particularly synchrotron-based modalities, offers rapid tomographic imaging but has not been previously integrated with ExM for cellular feature reconstruction in expanded tissue.

This paper introduces a first demonstration of expansion X-ray microscopy (ExXRM) by achieving sufficient contrast to visualize cell bodies within expanded mouse cortex samples using laboratory X-ray microtomography. The methodology combines enzymatic DAB-based signal amplification and metallic gold staining to generate X-ray contrast in expanded gels. Although current results are limited to cell bodies without resolving finer neurites, the approach marks a foundational advance toward scalable, high-throughput connectomic imaging.

Technical Methodology

The key technical premise involves iterative ExM to enlarge mouse cortex tissue up to 18-fold using the pan-ExM protocol, which was further stabilized by re-embedding in a secondary hydrogel. Cellular resolution was enhanced by biotinylating primary amines followed by Streptavidin-HRP-mediated DAB polymer deposition (Unclearing technique), enabling subsequent gold deposition using the GoldEnhance™M LM reagent kit. Notably, gold ions were catalytically deposited on DAB polymer rather than the conventional colloidal nanogold substrate, motivated by prior enzymatic Unclearing work. This protocol targeted signal amplification to provide absorption contrast for XRM.

Brightfield microscopy confirmed cell body visibility in both DAB-only and DAB-gold samples, with the latter showing darker appearance due to gold but characterized by off-target staining and incomplete reagent diffusion. Samples were imaged using a Zeiss Xradia 620 Versa system at 2.95 μm isotropic voxel size, revealing expanded cell bodies as lower-intensity volumes within the tissue.

Results and Numerical Findings

The imaging successfully reconstructed cell bodies within expanded tissue, with diameters inflated from ~10 μm to ~80 μm due to physical enlargement ($\sim$8x final expansion during XRM acquisition). The cell bodies were distinctly visible due to differential staining efficiency in extracellular vs. intracellular compartments, although no subcellular neurite structures were decipherable. The contrast was obtained primarily as a negative-space effect where off-target gold staining underscored the cell bodies.

While laboratory XRM is more accessible than synchrotron XRM, it offers lower X-ray flux, affecting volume throughput and resolution. The present system's limits were dictated by the need to prevent gel desiccation and sample destruction from X-ray exposure. Imaging throughput can theoretically be increased via larger detectors, maintaining $\sim$0.3 μm voxel size, and accelerating mm$^3$-scale acquisition (see supplemental calculations), provided that optimal contrast agents and cryoprotective strategies are developed.

Implications and Challenges

This work provides the first explicit demonstration that XRM, combined with iterative ExM and metallic signal amplification, is capable of reconstructing cellular features in highly expanded soft tissue. The practical implications for connectomics are substantial, given XRM's capacity for volumetric, nondestructive tomography at unmatched speeds compared to EM or LSFM. However, several challenges remain before ExXRM becomes a viable tool for dense neuronal mapping:

• Signal Amplification Specificity: Current gold enhancement lacks sufficient targeting and diffusive penetration, limiting resolution to cell bodies and not revealing subcellular processes like dendrites or axons.
• Contrast Agent Optimization: Gold, while highly absorptive for X-rays, may cause heat damage in expanded hydrogel matrices, especially under synchrotron irradiation. Alternative contrast agents, or genetically encoded tags (e.g., SpyTag/SpyCatcher systems), may offer improved specificity and compatibility.
• Sample Stability: Synchrotron XRM requires mitigation of hydrogel decomposition and bubble formation. Approaches such as cryogenic conditions and radioprotective additives are imperative but remain to be validated.
• Penetration Depth of Stains: Techniques such as stochastic electrotransport or hydrogel chemistry optimization will be necessary to reliably deliver signal enhancers throughout large tissue volumes.

Theoretical and Practical Outlook

If signal amplification and sample stabilization challenges are resolved, ExXRM is poised to become a breakthrough modality for connectomic studies. The theoretical imaging rate can be quadratically scaled by detector size, making whole-brain mammalian maps plausible within practical timeframes previously inaccessible by nanoscale imaging. Metal nanoparticle stains, genetic targeting, and advanced diffusion techniques may enable visualization of synapses and fine neurites, facilitating accurate segmentation and connectivity mapping at scale.

Future research should focus on improving the signal-to-background ratio, expanding stain diffusion, optimizing compatibility with high-flux synchrotron sources, and validating the approach on intact brains and other tissue types. These advances will have broad implications not only for neuroscience but for the imaging of large, complex biological systems at nanometric resolution.

Conclusion

This study establishes the feasibility of achieving sufficient contrast for X-ray microtomographic imaging of expanded brain tissue, enabling visualization of cell bodies as a first step toward expansion X-ray microscopy for connectomics. While current techniques are limited in resolving fine neural features, the results demonstrate the utility of combining ExM with metallic signal amplification for large-volume, high-throughput tomography. Continued development in stain targeting, sample stability, and tomographic throughput will be required to realize ExXRM's full potential for rapid, nanoscale mapping of mammalian brains and beyond.

Reference: "A First Step for Expansion X-Ray Microscopy: Achieving Contrast in Expanded Tissues Sufficient to Reveal Cell Bodies" (2601.13370)