Low-temperature electron microscopy for the study of polysaccharide ultrastructures in hydrogels. I. Theoretical and technical considerations.

Abstract

The high-pressure freezing (HPF) technique was applied to the cryo-immobilization of alginate gels and the quality of the freezing analyzed on a TEM by comparison of the segregation pattern of samples of decreasing thickness. Dynamic simulations of heat transfer within an idealized slab of pure water surrounded by two walls of aluminium were performed to illustrate the effect of the heat-transfer coefficient by convection on the cooling rate of the sample. Heat-transfer coefficients in liquid nitrogen and liquid propane at ambient pressure were measured using a carefully characterized thermocouple and the values incorporated as parameters in heat-transfer simulations to compare the efficiency of the plunge-freezing technique with the high-pressure freezing technique. Values of the heat-transfer coefficient in liquid nitrogen and liquid propane, calculated between 273 K and 173 K were 670 and 18420 W/m(2)/K, respectively. Based on TEM observations and the results of heat-transfer simulations, the HPF technique was adapted to the cryo-fixation of 50-microm-thick alginate gels. The occurrence of artifacts was rejected because no differences were observed in the pattern of cryo-fixed and freeze-substituted samples of various thickness, with and without ethanol as cryo-protectant. A sample thickness of 50 microm was found to ensure an adequate preservation of structures as small as a few nanometers, as verified by TEM and SEM observations. Finally, DSC measurements on alginate solutions and alginate beads revealed that under the experimental conditions (0-3%), alginate cannot be considered to be an efficient cryo-protectant.