Tamada, H., Blanc, J., Korogod, N., Petersen, C. C., & Knott, G. W. (2020).
Ultrastructural comparison of dendritic spine morphology preserved with cryo and chemical
fixation. Elife, 9, e56384. https://elifesciences.org/articles/56384
Fresh mouse cortex tissue was cut to 200 micron thickness and high-pressure
frozen (90 s from moment of decapitation), then processed for electron microscopy (EM) using
freeze substitution. This ‘cryo-fixation’ technique is understood to give the most genuine view of
in vivo ultrastructure. Authors compared EM (TEM and FIB-SEM) images of cryo-fixed tissue to
chemically-fixed (transcardially perfused 2.5% glutaraldehyde and 2.0% paraformaldehyde)
tissue.
Mouse cortex
Cryo-fixation succeeds in preserving the native ultrastructure of dendritic spines, but
the physics of heat conduction limits its use to tiny volumes (~200 microns thick). Chemical
fixation has no such limit, therefore almost all connectomics research uses chemically-fixed
tissue, but the chemical fixation process results in distortions of native ultrastructure and
extracellular space. This study provides a direct comparison between the two in order to
understand what measurements can be ‘trusted’ in chemically-fixed tissue. Key findings: The
volume of spine heads are unaltered by chemical fixation, but the diameter of the spine necks is
changed.