In Situ X-ray Assisted Electron Microscopy Staining for Large Biological Samples

As the connectomics field seeks to increase the volume of neural tissue for reconstruction, traditional protocols for heavy metal staining are no longer sufficient. We developed a technique that uses a Zeiss Xradia lab source microCT to directly observe the effects of staining chemicals like osmium tetroxide on tissue. We measured the accumulation of osmium at different depths of a cylinder of brain tissue and found that diffusion followed a quadratic relationship. Accumulation of osmium also continued well beyond the longest time described in traditional protocols. Binding osmium tetroxide to phospholipids and other organic molecules requires an initial fast reaction to available binding sites followed by a slower unmasking of binding sites. We also found that osmium accumulation decreased in some contrast enhancers. The use of in situ x-ray assisted staining is advancing our understanding of chemical reactions of heavy metal stains for electron microscopy in large volume tissue samples.

Key Learnings:

• X-ray tomography is a valuable tool for assessing the progressive uptake of stain into biological specimens
• Using this live assessment approach osmium accumulation was found to continue far beyond the longest time described in traditional protocols
• Understanding of chemical reactions of heavy metal stains for electron microscopy is greatly enhanced using in situ x-ray assisted staining