SUMMARYRoot tips from Sorghum and Dahlia were frozen without cryoprotection by dipping into nitrogen slush, rapid immersion in liquid propane and by the high‐pressure method. Structural preservation of the samples was studied using freeze‐fracture (FF) and freeze‐substitution (FS) techniques for electron microscopy. It was found that most of the organelles were disrupted by freezing in nitrogen slush and that only the boundary beween the cytoplasm and the vacuole remained visible. If the samples were frozen by rapid immersion in liquid propane, small membraneous organelles, such as dictyosomes, were preserved in peripheral regions of the rhizodermal cells up to 10 μm below the surface of the tissue. Specimens frozen by the high‐pressure freezing technique showed good ultrastructural preservation throughout the tissues up to a depth of more than 100 μm.

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.

During the last few years, several techniques for the rapid freezing of thin aqueous layers of cells and tissues have been developed and successfully applied to various biological questions (spray-freezing, propane-jet freezing, metalmirror-freezing, plunge-freezing, high pressure freezing). For practical work, techniques where the biological material (mainly suspensions) is sandwiched between two metal platelets are most favourable, because the frozen sandwiches are easily freeze-fractured, freeze-substituted or freeze-dried. In addition, the thickness that is sufficiently well frozen (vitrified or in the microcrystalline state) is greater in a sandwiched sample than in a specimen cooled only from one side (1). Heat can be extracted from the specimen sandwich either by controlled rapid plunging into a suitable coolant (2) or by shooting the coolant simultaneously from opposite directions onto the specimen surface (propane-jet) (3).

The 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.

In order to investigate the myelin and the glial cell membranes in the optic nerve of the mutant mouse "Jimpy," the method of freeze-etching was applied. The compact myelin lamellae and the first two glial membranes of the mutant, as compared to the normal mouse, show several abnormalities: absence of intramembraneous particles on the P-face, myelin lamellae separated by cytoplasmic layers, vesicular protrusions forming irregular invaginations, and elevations and tight junctions with a discontinuous, zigzag course. Some of these characteristics were found in the membrane of the oligodendroglia cell of the pathological animal, as well. The astrocytic membranes of both normal and Jimpy mice contain two types of gap junctions. The occurrence of one type seems to be increased in the mutant. Freeze-fractured internodal regions of the axolemma are not significantly different from those of normal animals.

Ever since the freeze-etching method became a routine laboratory technique, an increasing number of scientists has applied it in biological and especially in membrane research. The manifold details of membrane architecture gave rise to a multitude of questions as to the functional significance of these structures, the possible introduction of artificial alterations and the presumptive limits of the method.The limitations of freezing originate from segregation phenomena in the cytosol caused by rapidly growing ice crystals. Using the standard freezing technique (dipping specimens in liquid Freon or propane) segregation artifacts are avoided by the application of antifreeze agents. Mani structures are stable or can be stabilized against the action of the antifreeze. If this is not possible, very high freezing rates have to be achieved. These require special techniques using a propane jet or a jet of liquid nitrogen under high hydrostatic pressure (1).

Resting bodies of Bdellovibrio sp. strain W were produced following the infection of Rhodospirillum rubrum in liquid culture. Thin sections showed that young resting bodies possessed a narrow layer of amorphous material at their periphery, and storage granules in the region of the nucleoplasm. In mature resting bodies, the amorphous material (now called the outer layer) had thickened considerably to 30–40 nm, and the cell wall had differentiated into a folded, tripartite inner layer. Freeze-etched preparations of mature resting bodies showed a roughly particulate plasma membrane, a more finely particulate inner layer, and an outer layer having little structure.Bdellovibrio W did not produce resting bodies in a second host, Escherichia coli B. Also B. bacteriovorus strain 109 failed to form resting bodies in E. coli B, its usual host. It also failed to grow in cultures of R. rubrum. These restricted experiments suggest that the development of resting bodies may be specific for the Bdellovibrio W – R. rubrum parasite–host system.

An electron microscopical investigation of synchronously dividing yeast cells (S. cerevisiae) prepared by freeze-etching revealed that ER is inducing bud formation. In the first step, ER elements join and form a nearly-closed bag-like envelope which surrounds the nucleus and vacuoles. From the small opening of the ER-envelope, vesicles are produced by a splitting or proliferation of the ER-membranes. The vesicles fuse with the plasmalemma and release their content into the cell wall. In this limited area, bud formation starts explosively by a local evagination of the cell wall. The ER-derived vesicles are concluded to contain proteindisulfide-reductase. The limited introduction of the enzyme into the cell wall explains bud formation to be initiated by a local increase of wall plasticity caused by the reduction of disulfide bonds between cell wall proteins. The wall is forced to extrude by the internal pressure (turgor) of the cell.

Im Zellkern der Bäckerhefe wunden beim Übergang von anaeroben zu aeroben Kulturbedingungen drei verschiedene Sorten vom Mikrotubuli beobachtet. Die Durchmesser der Röhrchen betragen 210, 224 und 250 Å, die Lumina 60, 75 und 105 Å. Der Grundbaustein dieser Mikrotubuli ist ein 80 Å großes, in erster Näherung globuläres Teilchen, in dem höchst wahrscheinlich acht Untereinheiten mit einem Durchmesser von 40 Å enthalten sind. Das Bauprinzip der Mikrotubuli ist eine eingängige Schraube, die von einer Perlenkette der Grundbausteine aufgebaut wird. Die drei verschiedenen Tubuli-Sorten unterscheiden sich darin, daß bei der einen 5, bei der anderen 6 und bei der drittem 7 Perlen (80 Å-Einheiten) pro Umgang der Schraube zu finden sind. Wenn man die 40 Å großen Untereinheiten berücksichtigt, so besteht die Tubulus-Wand aus einer doppelten Lage dieser Teilchen mit 10, 12 oder 14 Einheiten pro Schraubenumgang. Die Übereinstimmung vieler Daten aus der Literatur mit diesem Modell läßt den gleichartigen Aufbau aller Mikrotubuli und Spindelfasern als wahrscheinlich erschieinen.

The present study has shown that the thylakoid membrane consists of a central layer, probably lipid, covered on both sides with protein particles. The thickness of the middle layer reaches 40 Å, and the diameter of the attached globules 60 Å. The particles are partially embedded to a depth of about 20 Å in the central layer. If these globules are removed, the lipid layer appears perforated, indicating that the protein molecules are in direct contact through the lipid layer. On the outer side of the thylakoid membrane the particles are grouped in fours, forming a multienzyme complex of 120 Å diameter and 60 Å thickness. No such aggregates have been observed on the inner side.

Zotten 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.

Conventional freeze-etching is carried out in a vacuum of -10 -~ torr and at a specimen temperature of -100~ The relatively poor topographic resolution of most freeze-etch replicas, and the lack of complementarity of morphological details in double replicas have been thought to be caused by structural distortions during fracturing, and radiation damage during replication. Both phenomena can be reduced by lowering the specimen temperature. To prevent condensation of residual gases (especially H20) on the fracture faces at lower specimen tempera- ture, an improved vacuum is required. Therefore, an ultrahigh vacuum freeze- fracture apparatus has been developed which allows fracturing and Pt/C-shadow- ing of specimens at -196~ while maintaining a vacuum of 10 -9 torr. It consists of a modified Balzers BA 350 ultrahigh vacuum (UHV) unit, equipped with an aidock which enables the input of nonhoar-frosted specimens directly into the evacuated bell jar. A comparison of the paracrystalline plasmalemma structure in yeast cells portrayed by the conventional technique and by UHV-freeze-fracturing at -196~ shows the improved topographic resolution which has been achieved with the new technique. The improvement is explained by less structural distortions during fracturing at lower temperatures. The particles of the paracrystalline regions on the P face are more regularly arranged and exhibit a craterlike substructure which corresponds with a ringlike depression in the E face. The optical diffraction patterns of these paracrystalline regions demonstrate the improvement of the structural record by showing well-defined third- and fourth- order spots.