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

TL;DR

This paper demonstrates the first successful imaging of cell bodies in expanded tissues using X-ray microscopy, combining a modified staining technique with laboratory X-ray imaging, paving the way for faster large-volume brain mapping.

Contribution

It introduces the concept of expansion X-ray microscopy (ExXRM) and provides an initial demonstration of imaging cell bodies in expanded tissues with X-ray microscopy.

Findings

First X-ray microscopy reconstruction of cell bodies in expanded tissue.

Combined enzymatic clearing and gold staining enabled contrast in X-ray imaging.

Highlights the need for further development to image neurites and adapt for synchrotron X-ray microscopy.

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.