Hans Moor

Theory and Practice of High Pressure Freezing

Pressure-freezing has often been regarded as a method for highly technical specialists. At the beginning of its development, this may have been true: it was introduced in 1968 by Moor and Riehle at the European conference on electron microscopy in Rome. The interest of the audience was not overwhelming, because everybody thought that this approach is oversophisticated and in principle unnecessary. In the following decade, many technically less pretentious freezing methods have been developed, which work in the absence of pressure. All of them became standardized and their methodology has been described in numerous reviews and textbooks (e.g. Rash 1983; Gilkey and Staehlin 1986; see also Sitte et al., Chap. 4, this Vol.). The compiled experience shows the manifold profits of applying impact-, plunge-, jet- and spray-freezing. In one aspect, however, all of these techniques are inadequate: namely they only enable satisfactory cryofixation of objects or superficial layers, which are not thicker than 10–20 μm. This limitation is caused by the physical properties of aqueous systems and it indicates that thicker specimens can be well cryofixed only if these properties are altered.

Die Gefrier-Fixation lebender Zellen und ihre Anwendung in der Elektronenmikroskopie

ZusammenfassungDer Gefriervorgang in den Zellen hängt in erster Linie ab von der Gefriergeschwindigkeit, der Frosthärte des Objektes und von der Konzentration eines Frostschutzmittels (Glyzerin) im Zytoplasma. Für die meisten Untersuchungen wurde Preßhefe als Testobjekt verwendet. Der Einfluß der Gefriergeschwindigkeit äußert sich auf drei verschiedene Weisen; das Zellwasser kristallisiert entweder extra oder intrazellulär oder es wird amorph verfestigt (Vitrifikation). Die Bestimmung von Gefrierpunkt, Unterkühlbarkeit und Rekristallisationspunkt ermöglicht eine Erklärung dieser drei Wirkungsweisen und führt zu einem physikalischen Verständnis des Phänomens der Frosthärte. Physikalische Untersuchungen zeigen, wie das Frostschutzmittel eine Erhöhung der Frosthärte bewirkt; physiologische Experimente veranschaulichen einige Nebenwirkungen des Glyzerins.Die Verwirklichung des Gefrierens lebender Zellen hängt in erster Linie von der Wahl geeigneter Gefriergeschwindigkeiten und Frostschutzmitteln ab. Die Endtemperatur des Gefriervorganges muß, je nach der Frosthärte des Objektes, d. h. je nach dem tiefsten in den Zellen auftretenden Rekristallisationspunkt, unter −50 bis −70° C liegen.Das Anwendungsgebiet des Gefrierens lebender Zellen ist sowohl auf biologischem wie auch auf medizinischem Gebiete sehr groß, sei es als reine Gefrierkonservierung oder in der Gefrier-Trocknung oder -Substitution. Mit Hilfe der Gefier-Ätzung können hochauflösende, elektronenmikroskopische Bilder der gefrorenen Objekte hergestellt werden, die vollkommen artefaktfrei sind, insbesondere frei von den durch die üblichen Präparationsmethoden eingeführten Veränderungen.Einige Beispiele illustrieren die Anwendung des Gefrierens lebender Zellen in der Elektronenmikroskopie. Die Methode der Gefrier-Ätzung ist besonders geeignet für die Darstellung der auf den Zytomembranen lokalisierten Partikel; z. B. Fibrillen synthetisierende Partikel in der Plasmamembran, Ribosomen auf einer Vakuolenmembran, Elementarpartikel auf den Cristae mitochondriales und Quantasomen auf den Granalamellen eines Chloroplasten. Die vielfältige Anwendbarkeit der Gefrier-Ätzung wird aufgezeigt an Hand von Mikroorganismen (Hefe), pflanzlichen (Wurzelspitze) und tierischen Zellen (Dünndarmepithel).

Elektronenmikroskopische Darstellung tierischer Zellen mit der Gefrierätztechnik

ZusammenfassungZotten des Dünndarms der Maus und Trabekel aus dem Herzen der Maus werden nach dem Gefrierätzverfahren präpariert. Die Abdrücke der unfixierten Gewebestücke geben alle vom Fixierungsbild bekannten Feinstrukturen wieder. Auf besondere Vorteile der neuen Technik und auf einige Fragen der Bild-interpretation wird hingewiesen.

The control of freeze-drying with deuterium oxide (D2O)

The course of freeze-drying under high vacuum conditions can easily be followed if the specimen water is exchanged with heavy water. Mass spectrometric analysis of D 2 O provides direct information about the drying procedure and the end point of drying. These parameters were measured at two frequently used drying temperatures (−80 and −35°C). A special magnetic cold table as support for electron microscopic grids has been developed and two test objects as examples for typical freeze-drying results are presented.

Complementary structures of membrane fracture faces obtained by ultrahigh vacuum freeze-fracturing at -196 °C and digital image processing

Conventional freeze-etch replicas of the cytoplasmic fracture face (PF) of the yeast plasmalemma membrane show hexagonally ordered regions. Complementary features on the extraplasmic face (EF) could not be identified. Replicas with improved topographical resolution were obtained for both fracture faces by ultrahigh vacuum freeze-fracturing at -196 degrees C. The hexagonally ordered structure on the PF is seen to consist of volcano-like particles with a crater of 5 nm diameter. The lattice constant is 16.5 nm. On the EF, ring-like depressions corresponding to particles on the PF can occasionally be detected; the existence of ordered regions can be established by optical diffraction. Complementarity of periodic features on the PF and EF is demonstrated by digital image filtration. A main structure, coarse features of which appear on conventional PF replicas is shown to have perfect complementarity at a resolution level of 2 nm. On the EF an additional substructure, completely obscured on normal replicas, is revealed. Its complementarity remains tentative as shadows cast by the main structure impair identification of substructural features on the PF.

Cytoplasmic actomyosin fibrils after preservation with high pressure freezing.

SummaryThe fine structure of the actomyosin system of Physarum polycephalum was investigated in vitrified specimens after applying a pressure of >2.1 kbar and freezing rates of 500 to 5,000° C/s. The frozen specimens were either freeze-substituted or freeze-fractured and compared with material processed according to conventional methods of freeze-etching preparation.Artifactual alterations, as seen in the form of destroyed areas of the cytoplasm after chemical fixation, were not observed after freeze-substitution. However, small ice crystals formed by recrystallization within most of the cytoplasmic actomyosin fibrils prevented a fine structural analysis.Such a destruction of the fibrillar fine structure was not found after freezeetching. In replicas of deep-etched objects 10 nm-thick filaments were localized, which could be conclusively identified as F-actin. The actin filaments are located randomly in the peripheral cytoplasm forming the cell cortex. By the process of parallel aggregation, the filaments can be differentiated to fibrils. Thick myosin filaments were not observed. However, structures resembling cross bridges between single actin filaments suggest the existence of oligomeric myosin.The present investigation shows that, in addition to biomembranes, other cytoplasmic differentiations such as components of the groundplasm can be successfully demonstrated employing the deep-etching technique when the freezing methods are improved by avoiding freeze-protection pretreatments.

Comparative studies of very thin shadowing films produced by atom beam sputtering and electron beam evaporation

Abstract Shadow-casting using thermal evaporation of heavy metals to achieve contrast enhancement of biological surface structures is widely applied in electron microscopy. Much less data are available about shadowing films produced under high vacuum conditions by ion or atom beam sputtering. Our experience shows that condensation of metal films proceeds differently according to whether sputtering or evaporation is used. With the same amount of material much thinner continuous layers can be produced with sputtering. However, very thin, non-continuous sputtered and evaporated films show a similar granularity. We used a periodically arranged biological test specimen for estimating the resolution of shadowing films. After electron beam shadowing onto this periodic structure the light-optical diffractograms show higher diffraction orders and the filtered images are richer in details compared to atom beam sputtering. The advantage of atom beam sputtering is the tendency of sputtered material to shadow in a purely geometrical fashion, whereas evaporated material tends to decorate.

Electron microscopy of polyheads of bacteriophage T4 prepared by freeze-etching

Intracellular and extracellular polyheads prepared by freeze-etching were observed on electron micrographs directly and by optical diffraction. Extra-cellular, multilayered polyheads showed fine structure on the capsoids, and in several cases two of the layers were simultaneously uncovered. Intracellular polyheads revealed fine structure neither on the capsoids nor in the core. The diffraction study suggests that the inner parts of the subunits of the still tubular capsoid are strongly bonded and probably maintain the local p6 symmetry, whereas the parts protruding outside, their summits covered by the shadowing material, produce a lattice constant of 110 A in the near axial direction and of 130–150 A in the near equatorial direction. In flat, negatively stained or shadowed polyheads the lattice constant had previously been found to be 110 A, independent of direction.