The influence of high pressure freezing on mammalian nerve tissue

Abstract

Vitrification of biological specimens in liquid nitrogen can be achieved under high pressure (2,100 bars). This procedure obviates the use of aldehyde fixation and cryoprotection (glycerol). The present work demonstrates its applicability to the freeze-etching of mammalian brain tissue. Freeze-fracture repicas from rat cerebellar cortex and subfornical organ prepared by this method are compared to conventionally processed material using aldehyde fixation, glycerination and freezing with Freon. The formation of large ice crystals is prevented in tissue blocks up to 0.5 mm thick; deep etching is markedly enhanced. Cytoplasmic microstructures such as mitochondrial cristae, microtubules and microfilaments, are readily observable against a finely granulated cytosol matrix. An additional advantage is the combined application with freeze-substitution.

The combination of chemical fixation procedures with high pressure freezing and freeze substitution preserves highly labile tissue ultrastructure for electron tomography applications

“…After this time, morphological deterioration was observed and it was reported that the tissue resembled chemically fixed specimens. One of first descriptions of an HPF device was its application of cryofixation for freeze fracture electron microscopy of dissected rat cerebellar cortex and subfornical organs (Moor et al, 1980). Recent tomographic studies have shown the HPF of rat CA1 hippocampal slices produces 3D structures of synapses that were larger, had less densely packed synaptic vesicles and contained small filamentous linkers to groups of vesicles (Rostaing et al, 2006) with a network of synaptic vesicles linked together and forming a scaffold for the active zone (Siksou et al, 2007).…”

Electron microscopy of high pressure frozen samples: bridging the gap between cellular ultrastructure and atomic resolution

“…The Wrst possibility is to add cryoprotectants; as mentioned above this is only possible with deleterious eVects on ultrastructure. The alternative exploited Wrst by Moor and his student Riehle (Moor et al 1980;Riehle and Hoechli 1973) is to increase pressure to 210 MPa (2,048 bar) during cooling. Water has some very remarkable properties (see Chaplin: http://www.lsbu.ac.uk/water/).…”

“…The specimen is introduced into a small closed chamber that is Wrst pressurized to 210 MPa, and then immediately cooled from outside to ¡196°C by a double jet of liquid nitrogen. The other machines (Bal-Tec, Wohlwend), developed according to Moor et al (1980), pressurize liquid nitrogen to 210 MPa and then "shoot" it onto the sample holder to freeze the specimen. Thus, in this design the cryogen also acts as pressurizing agent.…”

“…Thus, although atomic resolution is possible with the electron microscope in theory (and in practice with inorganic samples; Batson et al 2002), preparation artifacts limit the eVective resolution for biological specimens in practice to about 2 nm (Hayat 2000). As outlined in the next paragraph, cryoWxation (Dubochet et al 1988;Moor et al 1980;Studer et al 1989) is the basis for pushing the electron microscopic resolution toward the imaging of macromolecular assemblies in intact cells and tissues.…”

Distribution and serotonin-induced activation of vacuolar-type H+-ATPase in the salivary glands of the blowflyCalliphora vicina

“…The samples were transferred to a high-pressure freezing apparatus (HPM 010, Balzers Union, Liechtenstein), frozen in liquid nitrogen at a hydrostatic pressure of 2×10 8 ·Pa (Moor et al, 1980) and freeze-substituted in dry acetone containing 2% OsO4 as described in detail previously (Klein and Zimmermann, 1991;Zimmermann, 2000). After being washed in dry acetone at room temperature, specimens were embedded in Epon resin.…”