A fast method to study the secretory activity of neuroendocrine cells at the ultrastructural level

Cryo field emission scanning electron microscopy (cryo-FE-SEM) is a versatile technique that allows the investigation of the three-dimensional organization of cells at the ultrastructural level over a wide range of magnifications. Unfortunately, cryopreparation of the specimens for this technique remains cumbersome, in particular because ice crystal formation must be prevented during freezing. Here we report that a light prefixation with glutaraldehyde and incubation in glycerol as cryoprotectant or a high-pressure freezing approach are both excellent procedures for cryopreparation of animal cells to be used in combination with cryo-FE-SEM. Using the proopiomelanocortin-producing intermediate pituitary melanotrope cells of Xenopus laevis as a physiologically inducible neuroendocrine system, we compared the ultrastructural characteristics of inactive and hyperactive neuroendocrine cells. The overall quality of the ultrastructural images was comparable for the two cryopreparation procedures, although some fine structures were better conserved using high-pressure freezing. Melanotrope cells in a secretory inactive state contained numerous storage granules and a poorly developed endoplasmic reticulum (ER), while large amounts of rough ER were present in hyperactive cells. Thus, the cryo-FE-SEM approach described here allows a fast ultrastructural study on the secretory activity of neuroendocrine cells.     

Elemental concentrations in air-exposed and vacuum-stored cryosections of rat liver cells

Elemental concentrations in different compartments of cryosections of isolated rat liver cells cryotransferred and freeze-dried were compared with those obtained after storage under vacuum for 12 or 60 h and after exposure to room air for 2 min. Poorer image contrast and segregation artefacts are frequently found in air-exposed sections, together with a slight but significant decrease of the K concentration in the cytoplasm and an increase of the S concentration in the liver cell nuclei and the extracellular medium. Extreme distortions of both ultrastructure and elemental distributions are observed if the sections are even slightly colder than the surrounding atmosphere. While storage of frozen-dried cryosections under vacuum for less than 12 h does not lead to alterations in the sections, gross changes are found both in morphology and elemental distribution in sections stored under vacuum for about 60 h. Long-time vacuum storage of frozen-dried cryosections is, therefore, not recommended.     

Effect of specimen preparation and section transfer techniques on the preservation of ultrastructure,lipids and elements in cryosections

Cryofixation, cryoultramicrotomy, and proper transfer of the cryosections into the electron microscope are important for the preservation of good ultrastructure and the measurement of subcellular elemental distributions. These techniques are applicable to tissue systems which can be rapidly frozen so that minimal to no ice damage occurs during the cryofixation step. For the transfer step we have compared the cryotransfer of hydrated sections and subsequent freeze-drying in the electron microscope with the transfer of sections into an external freeze-dryer, followed by exposure to room temperature and humidity before introduction into the electron microscope. The use of a cryotransfer stage for section transfer from the cryoultramicrotome to the electron microscope and low temperature observation of the thin sections avoids the potential problem of rehydration damage to freeze-dried sections as well as provides protection from the possibility of melting of the lipids in the sections. Both of these problems may lead to loss of in situ elemental distribution and morphology. In this report, observations are presented which show the damaging effects of temperatures above 273 K on ultrastructure due to lipid melting in tissues with high lipid content and the redistribution of elements which can be encountered when thin sections become inadvertantly rehydrated.