Chemical fixation · Lobeworks/17

Chemical fixation is the preservation of tissue by chemicals that cross-link its molecules into a rigid mesh, so that its structure survives the staining, dehydration and cutting that microscopy needs; it is the first step of every connectome, and the step that ends everything the tissue was doing.

Chemical fixation. Chemical fixation is the preservation of tissue by chemicals that cross-link its molecules into a rigid mesh, so that its structure survives the staining, dehydration and cutting that microscopy needs; it is the first step of every connectome, and the step that ends everything the tissue was doing.

Aldehydes do the cross-linking. Glutaraldehyde has a reactive group at each end and hooks one protein to its neighbour wherever they touch, until the whole cell is one mesh; formaldehyde is smaller, enters faster and links more loosely. For electron microscopy the tissue is then treated with osmium tetroxide, which binds the lipids of every membrane and leaves a heavy metal atom there, so membranes show dark under the beam, and finally the water is replaced by resin that hardens into a block. The fly brain of 2018 was fixed this way in a few minutes after dissection.

It destroys state. The ion pumps stop, the voltage across every membrane runs down, and the chemistry that carried the cell's activity and the animal's condition is frozen or washed out. What survives is structure.

Large organs fix slowly. The fixative has to diffuse into the tissue, so the middle of a big brain decays before it arrives unless the fixative is pumped through the blood vessels, which is one reason human tissue for connectomics comes from surgery.

The alternative is cold. Freezing a thin sample under high pressure stops it in milliseconds without ice crystals and keeps molecules in place better, and it still keeps a single instant, never the dynamics.

Fixation keeps the shape of a brain and erases what it was doing.

Every claim drawn from a fixed brain is a claim about structure, plus whatever other experiments on living animals can lend it.

Questions: What does fixation destroy that a connectome would need? Everything that is a process rather than a shape. Cross-linking stops the ion pumps, the voltage across every membrane runs down, and the chemistry carrying activity, modulators and the animal's condition is frozen or washed out. Structure survives, which is why a connectome records wiring and not state, and why projects such as MICrONS record activity in the living animal before fixing it. Why does osmium make membranes visible to an electron microscope? Because it binds the lipids of membranes and leaves heavy atoms there. Electrons passing through a section are scattered by heavy nuclei and pass almost straight through light ones, so wherever osmium sits the image is dark. Without it the membranes, which are the outlines every segmentation follows, would be nearly invisible.