The Ultrastructure of the Connective Tissue Matrix is Significantly Improved Using Cryo-Fixation At High Pressure

Abstract

The investigation of connective tissue ultrastructure has historically relied on chemical fixation to stabilize micro architecture. These fixatives are not effective in retaining many matrix molecules, including proteoglycans, and allow precipitation of many other components in subsequent processing steps. The result is a preponderance of open space between matrix components. Cells shrink considerably and a precipitation of molecules in highly concentrated regions leads to artifacts including the lamina lucida and lamina densa of basement membranes. Cryo-fixation at high pressure followed by freeze substitution seeks to immobilize tissue components within vitreous (non-crystalline) ice, which is later substituted with acetone at temperatures below the recrystalization temperature of water. Formation of large ice crystals during any part of the preparation protocol significantly distorts structure and can be recognized in the compound microscope. Recognition of smaller freezing artifacts, including microcrystalline ice, is imperative for interpretation of resulting images.