Eduard Kellenberger

The efficiency of immunolabel on Lowicryl sections compared to theoretical predictions.

The surface of thin sections of aldehyde-fixed biological material shows a specimen-related relief of 2-6 nm with Lowicryl. Epon sections are about three times smoother. The relief is the consequence of thin-sectioning being in reality a cleavage. Epitopes are supposed to be laid open (or set free) because cleavage follows the interfaces between protein and Lowicryl. We have developed a simple theory on this basis and have theoretically estimated the efficiency of on-section labeling and compared it with experimental data. For randomly dispersed proteins in cytoplasm, Lowicryl sections will yield significant label only when the concentration of the antigen is about 10 microM or more. The complex situation of more compact proteins, as represented by fibers, sheets, and biological membranes is discussed and the difficulty of significant calculations is explained. Pre-embedding labeling and melted cryosections should give 10-30 times more label. The possible reasons for the observed much smaller gain of not more than two to three times are discussed.

Specimen preparation for electron microscopy using low temperature embedding resins

Studies using polar and non‐polar methacrylate‐based resins (Lowicryl® K4M and HM20) suitable for low temperature embedding are described. We present the first applications of the system to various membrane structures in glutaraldehyde‐fixed, uranyl acetate‐stained thin sections of bacteria, mitochondria and cell‐cell contact regions.

Low temperature embedding with Lowicryl resins: two new formulations and some applications

Lowicryl K4M and HM20 are methacrylate/acrylate based low temperature embedding resins for biological material which can be used in conjunction with either the progressive lowering of temperature (PLT) technique or with freeze‐substitution. K4M and HM20 are applicable over a very extended temperature range, approximately 220 K to 340 K. With two new resins, K11M and HM23, one can reach even lower temperatures, c. 200 K. Freeze‐substitution combined with low temperature embedding allows for very mild or no chemical fixation which seems to increase the sensitivity of immunocytochemical localization of antigens on sections.

Organization of double-stranded DNA in bacteriophages: a study by cryo-electron microscopy of vitrified samples.

In this paper it is shown that conformation and packing of double‐stranded DNA within the head of bacteriophages lambda and T4 can be assessed by cryo‐electron microscopy of vitrified specimens. Electron diffraction patterns show that DNA within vitrified bacteriophages has a B conformation. Electron micrographs of vitrified bacteriophages show domains within the head formed by a approximately 2.5‐nm striation and arising from the DNA packing. The number of differently oriented domains seen within a vitrified bacteriophage depends upon the geometry of the DNA container: the bacteriophage capsid. The packing of DNA within bacteriophages seems then to be governed by at least two phenomena. The first is the tendency of DNA to form local alignments (nematic liquid crystals). The second is the orientation of these liquid crystals by the bacteriophage capsid. From these observations we propose a possible packaging mechanism: constrained nematic crystallization.

Dehydration and embedding temperatures affect the antigenic specificity of tubulin and immunolabeling by the protein A-colloidal gold technique.

Qualitative and quantitative tests were performed to determine whether the temperature at which dehydration and embedding occur affects the antigenic specificity of tubulin and the protein A-gold (pAg) immunolabeling technique. The analysis indicates that low temperature (-35 degrees C) treatment increased the specificity and density of pAg labeled anti-tubulin antibodies to Leishmania tropica subpellicular microtubules as compared to samples prepared at 0 degrees C or 20 degrees C.

Artefacts and morphological changes during chemical fixation.

The normally ‘condensed’ (darkly stained) chromosomes of dinofiagellates decondense by swelling. This occurs in an increasing number of cells when the concentration of added OsO4 is decreased. With different fixatives other types of disintegration can be observed, which vary with the concentration. With cryofixation and freeze‐substitution the chromosomes are most ‘condensed’. Escherichia colt infected with bacteriophage T4, with or without active lysozyme production, were studied by optical densitometry for partial lysis and by light and electron microscopy for observing swelling. When active lysozyme is present some of the acrolein (2.5%) ‐ glutaraldehyde (2%)‐fixed cells swell at 0°C, but do not in the absence of lysozyme nor when fixed at room temperature. If OsO4 is added at concentrations ≤0.5%, partial lysis occurs when lysozyme is present. The optical density decreases, the cells lose some matter and swell slightly. The corresponding electron micrographs show gap formation by curdling and/or a decreased concentration of the cytoplasm which reveals certain phage‐related particles.

Exploring the unknown: The silent revolution of microbiology

Comparing shape and development has permitted biologists to classify all known animals and plants into more than one million and some half a million species, respectively. For micro‐organisms, however, such morphological criteria are much less suited. Complemented by biochemical criteria, taxonomists were at least able to order the micro‐organisms responsible for infectious diseases in humans, plants and animals. But only about 5000 prokaryotic organisms have so far been formally described, which represents barely one percent of the existing bacterial species (Pace, 1997). This situation will probably change as soon as it is accepted that micro‐organisms produce substances beneficial in the therapy of human diseases or for industrial applications. > The interest in microbiology will grow as soon as expectations become confirmed that micro‐organisms produce substances beneficial in the therapy of human diseases or in industrial applications The biomass of micro‐organisms on our planet is estimated to be much more important than that of plants. Prokaryotes are the basis of every food chain on this planet. They still dwell where higher organisms cannot survive, not only on the surface, but also deep in the oceans and in the earth. But even today, the popular view of micro‐organisms is focused nearly exclusively on those that cause disease. Few are aware of their additional benefits, namely in the fermentation of dairy products, sausages and beverages. But their indispensable presence and necessity in natural biotopes—which includes our digestive tract for instance—is still not commonly accepted. During an excursion to a Swiss lake, I recently heard a perfect exposition of aquatic food chains by a young biologist: to my great surprise he started with the plankton and completely omitted the important role of micro‐organisms in feeding and thus sustaining it. This narrow view has certainly something to do with the obsolete taxonomy, which, as we have …

Head structure of bacteriophages T2 and T4

The length-to-width ratios of bacteriophage T2 and T4 heads and stereometric angles specifying the prolate icosahedral T2 capsid were evaluated on electron micrographs recorded from samples prepared by a variety of methods. The copy numbers of the major capsid protein, gp23*, of T2 and T4 phages were compared by quantitative gel electrophoresis. Taken together, the resulting values are most compatible with triangulation numbers T = 13 and Q = 21 for both T2 and T4, thus confirming the previously proposed capsid architecture of T4 revealed by indirect measurements and thereby eliminating the repeatedly reported discrepancy between T2 and T4 in favor of a common Q number of 21 corresponding to 960 copies of gp23*.

The influence of the surface relief of thin sections of embedded, unstained biological material on image quality

Abstract Sections of resin embedded, aldehyde fixed T4 bacteriophages, adsorbed on bacterial envelopes, are observed unstained with different imaging modes and their surface reliefs analysed by heavy metal shadowing. It is found that details seen in bright-field and dark-field imaging reflect strongly the features of the relief. Most biological materials situated in the interior of the slice are not visible in bright-field; illustrating the few exceptions, the compact DNA of a bacterial virus that spans the entire section is visible by virtue of its density difference. By using resins with a chemically incorporated heavy metal the contrast is increased, as is the visibility of nearest neighbour distortions of the resin around the biological structures. The distortions studied with such a resin easily explain the blur observed with dark-field imaging in STEM of unstained sections with normal resin. It is confirmed that this blurring effect is eliminated either by ratio-contrast imaging that is less sensitive to thickness variations than the conventional modes or, obviously, by the abundant heavy metal stain commonly used. We then compared the experimental results with contrast calculations made previously and obtained excellent agreement between theory and experiment.

Storage, ultrastructural targeting and function of toposomes and hyalin in sea urchin embryogenesis☆

This study compares by immunogold labeling the ultrastructural localization of a hexameric 22S glycoprotein, called toposome, with that of hyalin in unfertilized eggs and cells of hatched sea urchin blastulae. Nearly all hyalin is present in the electron translucent compartment of the cortical granules and in the translucent non-cortical pigment granules. In the blastula both of these intracellular stores have vanished and hyalin now forms a broad band below the apical lamina. By contrast, in the egg toposomes are present on the surface, as well as stored in yolk granules and in the electron dense lamellar compartment of the cortical granules. In the hatched blastula, toposomes that have been modified by limited proteolysis in the yolk granules, are associated with the plasma membranes of all newly formed cells, while the toposomes originating from the cortical granules have been incorporated as unmodified 160 kDa polypeptides into an extracellular double layer enveloping the embryo on the outside of the hyaline layer. From evidence discussed in detail, we conclude that the extracellular toposomes rivet the apical lamina to the surface and underlying cytoskeleton of the microvilli, while the modified toposomes from the yolk granules are responsible for position specific intercellular adhesion as they are released to the surface of newly formed cells. We propose that all the material stored in yolk granules is utilized for the assembly of new membranes.