Diamonds are a cryosectioner's best friend

Abstract

K E Y w O R D s. High-pressure freezing, cryosectioning, cutting artefacts, plant leaves, diamond knives, ionization electrode, low-temperature microscopy, energy filter.High-pressure frozen Golden Delicious apple leaves were cryosectioned at low temperature with diamond knives. Good cryosections were obtained by optimizing the cutting parameters, i.e. sectioning temperature, mechanical stability of the sample, and sectioning velocity. Cutting artefacts were minimized by reducing the electrostatic interactions between the knife surface and the cryosection. This was accomplished by sectioning the sample in the presence of an ionization electrode. The ionization device, with a primary voltage of 7-8 kV, produces positively and negatively charged nitrogen ions which neutralize the surface charges of the knife and the section. This minimizes the friction on the knife surface and results in ultrathin sections without crevasses or knife marks. Compression of the sections could be minimized, but not avoided, by reducing the knife angle to 30". Improved contrast of the frozen-hydrated sections was obtained with the Zeiss E M 902 energy-filter microscope operated in the zero-loss mode.

Activity-Dependent Calcium Sequestration in Dendrites of Hippocampal Neurons in Brain Slices

“…Cryosectioning was performed as described previously (Michel et al, 1992;Buchanan et al, 1993). In brief, cryosections were cut en face from the well frozen surface (Ͻ20 m deep) of previously marked regions of CA3 st. lucidum, trimmed to a 0.25 ϫ 0.25 mm block face.…”

“…These slice cultures are also well suited for en face cryosectioning, which, using newer sectioning techniques (Michel et al, 1992;Buchanan et al, 1993) produces uniform, truly ultrathin cryosections in which even small organelles such as cisterns of ER are unambiguously recognizable (Fig. 2).…”

Correlated measurements of free and total intracellular calcium concentration in central nervous system neurons

“…Over the last decade, however, several major problems have been largely ameliorated by significant advances in instrumentation and techniques. Among the most important of these are: (1) the ability to prepare high-quality truly ultrathin cryosections of unfixed, rapidly frozen tissues that have structural detail comparable to conventional preparations Michel et al, 1992), while still retaining native distributions of diffusible elements; and (2) improved detectors and analytical microscopes (e.g., Leapman et al, 1993) that can achieve, in a clean environment, the high sensitivity and resolution necessary to characterize biologically relevant changes in cellular [Ca].…”

Electron‐beam‐induced amorphization of ice III or IX obtained by high‐pressure freezing

“…The observation by cryo-electron microscopy of thin cryosections of vitrified specimens makes it possible to observe samples in their native hydrated state without the usual artefacts of chemical fixation, dehydration and staining (Dubochet et al, 1988;Michel et al, 1991Michel et al, , 1992Sartori & Salamin Michel, 1994). The basic requirement for this method is that the specimen must be vitrified, i.e.…”