A study by Graham Knott at EPFL has successfully used an innovative method, called 'cryofixation', to prevent brain shrinkage during the preparation for electron microscopy. To study the fine structure of the brain, including its connections between neurons, the synapses, scientists must use electron microscopes. However, the tissue must first be fixed to prepare it for this high magnification imaging method. This process causes the brain to shrink; as a result, microscope images can be distorted, e.g. showing neurons to be much closer than they actually are. Scientists have now solved the problem by using a technique that rapidly freezes the brain, preserving its true structure.
The next step is to embed the frozen tissue in resin. This requires removing the glass-water and replacing it first with acetone, which is still a liquid at the low temperatures of cryofixation, and then, over a period of days, with resin; allowing it to slowly and gently push out the glassified water from the brain. After the brain was cryofixed and embedded, it was observed and photographed in using 3D electron microscopy.
The researchers compared the cryofixed brain images to those taken from a brain fixed with an "only chemical" method. The analysis showed that the chemically fixed brain was much smaller in volume, showing a significant loss of extracellular space - the space around neurons.
In addition, supporting brain cells called 'astrocytes', seemed to be less connected with neurons and even blood vessels in the brain. And finally, the connections between neurons, the synapses, seemed significantly weaker in the chemically-fixed brain compared to the cryofixed one. The researchers then compared their measurements of the brain to those calculated in functional studies, studies that measure the time it takes for a molecule to travel across that brain region.
The story in published in the journal eLife.
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