Comparative study of freeze‐substitution techniques for x‐ray microanalysis of biological tissue

Abstract

A comparative study of some aspects of freeze-substitution as a preparative method for X-ray microanalysis of diffusible ions in biological specimens was carried out. Four substitution fluids were compared with each other and with cryosections. As criteria, elemental ratios as determined by X-ray microanalysis of thin sections of mouse pancreas were used. Freeze-substitution for 3 weeks in diethyl ether or for 2 days in tetrahydrofurane gave results comparable to those obtained in cryosections. Slow warming to room temperature after freeze-substitution in tetrahydrofurane gave slightly better results than fast warming. Freeze-substitution in acetone resulted in noticeable loss and redistribution of ions and freeze-substitution in methanol gave very poor results.

Dispersed Polyphosphate in Fungal Vacuoles in Eucalyptus pilularis/Pisolithus tinctorius Ectomycorrhizas

“…The structural preservation of our freeze-substituted ectomycorrhizas is remarkably good, considering that plunge-freezing rather than high-pressure freezing was used for this large specimen (see Lepault et al, 1997). Although our dry-cut sections were out of necessity thick, the resolution was sufficient to show the vacuoles, and we know that the anhydrous tetrahydrofuran freeze-substitution protocol as we used it here retains ions in situ just as effectively as the anhydrous diethyl ether substitution method used in our earlier work Ashford, 1993, 1995;Pålsgård et al, 1994). The success of the procedure in avoiding significant ion redistribution was confirmed for these particular specimens by the distribution of La, which was concentrated in the apoplast as expected.…”

“…The distinct rounded profile at the vacuole boundary indicates that the vacuoles have not been disrupted by the freezing, and the high retention of K in the vacuoles with a sharp drop at this boundary is also an indication that significant redistribution of ions has not occurred, at least at the detection levels of X-ray microanalysis (cf. Pålsgård et al, 1994;Zierold et al, 1991). We know that vacuoles in the sheath and Hartig net are rich in polyphosphate, and so the results as a whole indicate that the polyphosphate in the tissues of ectomycorrhizas also ccurs in a dispersed form in the vacuoles as in growing hyphae.…”

“…Only one mycorrhiza was taken from each plant, to avoid pseudoreplication (Hurlbert, 1984) and wounding effects. Samples were freeze-substituted in tetrahydrofuran (Pålsgård et al, 1994) over a molecular sieve for 3 days at Ϫ80°C, gradually warmed to Ϫ30°C, left for 24 h, warmed to 6°C, left for a further 1 h, and then transferred to a glove box flushed with dry nitrogen gas (Fitzgerald and Allaway, 1991;Orlovich and Ashford, 1995) where they were infiltrated over a molecular sieve with Spurr's resin (Spurr, 1969) in steps of 30, 60, 90, and 100% for a day at each step at 23°C. Samples were flat embedded and polymerized at 60°C for 48 h, and the blocks stored in a desiccator until sectioning.…”

Electron and ion microprobe analysis of calcium distribution and transport in coral tissues

“…Araldite TM was the preferred embedding medium as it contains negligible levels of elements detectable by energy dispersive spectrometry (Pålsgård et al, 1994). All solutions were anhydrous, with processing conducted in a dry box at a relative humidity of 10%.…”

Multi-element bioimaging of Arabidopsis thaliana roots

“…In order to be able to prepare very thin sections (less than 5 mm) while still maintaining cell structures and element composition, freeze-substitution also has been employed (Siegele et al, 2008;Smart et al, 2010). This technique is based on ultra-rapid freezing followed by slow substitution of the ice with acetone, then chemical fixatives like osmium tetroxide (Smart et al, 2010) or tetrahydrofuran (Pålsgård et al, 1994). It has been shown, however, that the localization of highly diffusible ions, like K + and Na + , may be altered significantly during freeze substitution (Smart et al, 2010).…”