Fast cryofixation technique for X‐ray microanalysis

Abstract

The proposed cryofixation technique uses a tubule-shaped needle chilled in liquid propane for simultaneous excision and freezing of a tissue specimen. Due to this simultaneity, ionic shifts created by traumatic influences are avoided even in the outermost cells of the specimen.Moreover, it is shown here that stopping the blood flow for more than about 10 s results in notable ionic shifts between cells and extracellular space in rat heart and liver. Such preparative ischaemic injury is minimized by the Fast Cryofixation Technique because it can be easily performed on organs within the circulatory system, whilst the heart of the animal is still beating.Intracellular concentrations of the monovalent ions in rat heart and liver, obtained by this method, tally well with recent results from different independent techniques reported in the literature.As demonstrated by cross-sectioning and freeze-fracturing, the structural preservation of the freezing technique is sufficient for X-ray microanalytical work.

Dynamic structure of glomerular capillary loop as revealed by an in vivo cryotechnique

“…The changes created by stopping blood circulation were avoided in the outermost tissues due to the simultaneity of cutting and freezing [34]. The time interval betweeen excising and freezing was dramatically shorter than that used in the conventional preparation methods.…”

“…It means quick resolution of dynamic morphology, sufficient to fix rapid ultrastructural changes [14,23]. It is accepted that cryofixation is the best way to preserve antigenecity and chemical composition of tissues [15,34] and morphological changes produced by chemical fixation and dehydration steps are avoided [4,5,10,16,23,24,33]. Over the last decade, it has become a popular tool for the ultrastructural study of biological specimens.…”

“…Quick-freezing with such specimens has some limitations, as it is well known that the ultrastructure of the kidneys is easily changed by stopping the blood supply [12]. As it is impossible to obtain natural morphology by using conventional cryotechniques there is a need for a new method capable of freezing tissues in vivo and obtaining acceptable morphology in fuctioning organs [6,34]. In this paper, we will describe the morphological aspects of functioning kidneys, whose ultrastructures are reassessed, with regard to the haemodynamic conditions operating at the time of cryofixation.…”

Low temperature techniques for X‐ray microanalysis in pathology: Alternatives to cryoultramicrotomy

“…Immediately after death, redistribution of mobile ions takes place, leading to an increase in the cellular concentrations of sodium, chloride, and calcium, and a decrease in magnesium and potassium (Kuypers and Roomans, 1980;Roomans and Wroblewski, 1985;von Zglinicki et al, 1985). In work with experimental animals, the tissue should therefore prefera- Fig.…”

A survey of ultra‐rapid cryofixation methods with particular emphasis on applications to freeze‐fracturing, freeze‐etching, and freeze‐substitution

“…Tissue is punched out of an organ by means of a propane, or nitrogen, chilled syringe (Von Zglinicki et al, 1986).…”