W. Villiger

Resin development for electron microscopy and an analysis of embedding at low temperature

We have tested a wide range of acrylate and methacrylate resin formulations and have concluded that embedding resins can be developed which are usable within a broad range of environmental conditions. To demonstrate this versatility, we have designed two highly‐cross‐linked resins, one polar and the other nonpolar, that are usable to temperatures from 243 to 223 K. Both of these resins formulations, which are now commercially available, show that systematic experiments can be easily done to study the effects of environmental parameters, such as water content, temperature, or resin polarity, on biological material during embedding. Using these resins and aldehyde‐fixed protein crystals, it can be shown that low temperature minimizes the loss of molecular structure to an extent that is not often obtainable with conventional methods of dehydration and embedding. Embedded crystals of aspartate aminotransferase still retained molecular order to 0·6 nm. Embedded crystals of catalase show X‐ray diffraction maxima out to 0·8 nm. When sectioned, catalase revealed stain‐limited electron and optical diffraction patterns to 2·5 nm. Nevertheless, our work clearly demonstrates that low temperature embedding procedures are superior and that versatile, general purpose resins can be designed to take advantage of this fact.

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.

The role of the head and tail domain in lamin structure and assembly: Analysis of bacterially expressed chicken Lamin A and truncated B2 lamins

Nuclear lamins like cytoplasmic intermediate filament proteins exhibit a characteristic tripartite domain structure with a segmented alpha-helical rod domain flanked by an N-terminal head and a C-terminal tail domain. To examine the influence of the head and tail domains on the structure and assembly properties of nuclear lamins, we have engineered headless, tailless, and rod chicken lamin B2 cDNAs and expressed them in Escherichia coli. A full-length chicken lamin A cDNA was also expressed in E. coli, and the recombinant protein compared with the structure and assembly properties of full-length chicken lamin B2 (E. Heitlinger et al. (1991) J. Cell Biol. 113, 485-495). As with lamin B2, at their first level of structural organization, lamin A and the headless lamin B2 formed myosin-like dimers consisting of a 51- to 52-nm-long tail flanked by two globular heads at one end. Similarly, the tailless and rod lamin B2 fragments formed tropomyosin-like dimers consisting of a 51 to 52-nm-long rod. In contrast to the lateral mode of association of cytoplasmic IF dimers into four-chain tetramers, at their second level of structural organization, lamin A dimers, just as lamin B2 dimers (E. Heitlinger et al. (1991) J. Cell Biol. 113, 485-495), associated longitudinally to form polar head-to-tail polymers. Whereas dimers made of the truncated B2 headless and rod lamins had lost their propensity to associate head-to-tail, tailless lamin B2 dimers revealed an enhanced head-to-tail association. Finally, at their third level of structural organization, rather than assembling into stable 10-nm filaments, both lamin A and the three truncated B2 lamins formed paracrystalline arrays exhibiting distinct transverse banding patterns with axial repeats of either 24 or 48-49 nm depending on the species.

Developments of new Lowicryl® resins for embedding biological specimens at even lower temperatures

Two new Lowicryl resins have been developed for embedding biological materials at temperatures down to 210K (hydrophilic K11M) and to 190K (hydrophobic HM23). They have similar properties to Lowicryl K4M and HM20. The new resins were first tested for low temperature applications by the ‘progressive lowering of temperature’ procedure and this shows that the low viscosity of K11M and HM23 is favourable for the infiltration of biological specimens. Hardening is achieved through photo‐polymerization at these lower temperatures. These properties make K11M and HM23 suitable for cryosubstitution of rapidly frozen material and it is speculated that the preservation of antigenicity may be further improved.

Extraction of lipids during freeze-substitution of Acholeplasma laidlawii-cells for electron microscopy

Cells of the bacterium Acholeplasma laidlawii were rapidly frozen against a copper block cooled by liquid helium. The frozen cells were transferred to liquid nitrogen and subsequently to acetone or methanol at 183 K. After 24 h the cells were infiltrated at 203 K with a non‐polar methacrylate resin of the same type as Lowicryl HM20. The resin was cured at the same temperature. Acetone extracted approximately 5% of the lipid content of the cells, methanol 15–45% and the resin only negligible amounts. Similar results were obtained with A. laidlawii‐ghosts. The cells appeared well preserved when examined in the electron microscope.

Conjugational junctions: morphology of specific contacts in conjugating Escherichia coli bacteria.

F-plasmid-mediated bacterial conjugation was studied with hfr (traDts) and tra I mutant Escherichia coli donor strains. This allowed us to observe a statistically significant number of conjugation-specific contacts by video and electron microscopy. Single mating events between E. coli were observed in real time by video-enhanced light microscopy. Conjugation in vivo takes place by initial contact formation via pili, followed by direct and transient wall-to-wall contact, during which DNA is transferred and disaggregated. Electron microscopic observations of the contact zone between donor and recipient bacteria were made by thin sectioning of mating pairs that were arranged in monolayers. We defined the conjugation-specific contact found in stabilized mating pairs as the conjugational junction. Within this junction no specific substructure such as plasma bridges by fusion could be detected during transfer of DNA.

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.

New Electron Microscopic Data on the Structure of the Nucleoid and Their Functional Consequences

The DNA of the bacterial genome is organised quite differently from that of eukaryotic cells: Since only one linkage group exists, the “genome” of the bacterium is considered to be a single chromosome. A simple mechanism of division would allow an equipartition of the bacterial genome, in contrast to organisms with multiple chromosomes, where the chromosomes, condensed in metaphase, are presumably separated by the mitotic spindle fibers. In the condensed, compact form, the DNA is neither replicating nor transcriptionally active. In prokaryotes, replication and transcription are continuous when the cells are under optimal growth conditions (see Ingraham et al. 1983). Furthermore, Miller et al. (1970) showed that initiation of translation occurs as soon as a piece of the messenger RNA becomes available on the transcribing DNA.