High-pressure freezing and freeze-fracture replication of starch gels for high-resolution TEM

Abstract

Gelled food products with a high water content (∼80%) must be vacuum stabilized for electron microscopic studies of their ultrastructure. Conventional chemical fixation techniques produce unacceptable artifacts when applied to many gel or emulsion type samples. Rapid freezing is necessary in order to stabilize the matrix without appreciable ice crystal formation. High pressure freezing has been shown to produce vitrification of thick samples (up to .5 mm).Freeze-fracture/etch surface replication and TEM has been used successfully to visualize the spatial structure of aqueous carboxymethyl cellulose gels after ultra rapid freezing/Cryo-SEM has, been used to study starch gel and food emulsion structure directly. Cryo-SEM is generally limited in resolution and does not provide structural details in the 10-20 angstrom range. High pressure freezing and freeze-fracture replication is the preferred method for immobilizing and studying ultrastructural details at high resolution.