Cryofixation and cryosubstitution: a useful alternative in the analyses of cellular fine structure.

A study of the fine structure of cultured mouse P815 cells as well as of mouse liver tissue after having undergone cryofixation and cryosubstitution is reported here. The P815 cells were cryofixed by a projection onto a liquid nitrogen (LN2), or liquid helium (LHe), cooled copper mirror: the liver tissue was processed (cryofixed) by high pressure freezing using a Balzers HPM 010 apparatus. No conventional chemical fixatives were used in the substitution medium which consisted of pure acetone. Embedding was carried out either in Lowicryl K11M, Lowicryl HM23, Epon or in LR White resins. The results of this study enabled us to conclude that a) one can obtain good preservation of cellular ultrastructure using different cryofixation methods and cryosubstitution without the use of chemical fixatives: b) different methods of embedding can be applied after various cryofixation techniques giving rise to slight differences in the well preserved fine structure; and c) high pressure freezing is to be recommended, in cryosubstitution studies, over that of slam freezing especially when cryofixing larger pieces of tissue which can result in a good morphology of up to 400 microns in depth.

Artefacts and morphological changes during chemical fixation.

The normally 'condensed' (darkly stained) chromosomes of dinoflagellates 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 coli 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 degrees C, but do not in the absence of lysozyme nor when fixed at room temperature. If OsO4 is added at concentrations < or = 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.

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.

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.

Coralline shape of the bacterial nucleoid after cryofixation.

A new procedure of immunostaining sections of cryofixed and freeze-substituted Escherichia coli shows that DNA extends from its bulk into small ribosome-free spaces throughout the cytoplasm, resulting in a coralline-shaped nucleoid. Low-resolution imaging of a bacterium reconstructed from serial sections demonstrated that the small excrescencies are not resolved. The resulting photograph shows the same features as phase-contrast light micrographs.

Freeze-substitution of gram-negative eubacteria: general cell morphology and envelope profiles.

Freeze-substitution was performed on strains of Escherichia coli, Pasteurella multocida, Campylobacter fetus, Vibrio cholerae, Pseudomonas aeruginosa, Pseudomonas putida, Aeromonas salmonicida, Proteus mirabilis, Haemophilus pleuropneumoniae, Caulobacter crescentus, and Leptothrix discophora with a substitution medium composed of 2% osmium tetroxide and 2% uranyl acetate in anhydrous acetone. A thick periplasmic gel ranging from 10.6 to 14.3 nm in width was displayed in E. coli K-12, K30, and His 1 (a K-12 derivative containing the K30 capsule genes), P. multocida, C. fetus, P. putida, A. salmonicida, H. pleuropneumoniae, and P. mirabilis. The other bacteria possessed translucent periplasms in which a thinner peptidoglycan layer was seen. Capsular polysaccharide, evident as electron-dense fibers radiating outward perpendicular to the cell surface, was observed on E. coli K30 and His 1 and P. mirabilis cells. A more random arrangement of fibers forming a netlike structure was apparent surrounding cells of H. pleuropneumoniae. For the first time a capsule, distinct from the sheath, was observed on L. discophora. In all instances, capsular polysaccharide was visualized in the absence of stabilizing agents such as homologous antisera or ruthenium red. Other distinct envelope structures were observed external to the outer membrane including the sheath of L. discophora and the S layers of A. salmonicida A450 and C. crescentus CB15A. We believe that the freeze-substitution technique presents a more accurate image of the structural organization of these cells and that it has revealed complex ultrastructural relationships between cell envelope constituents previously difficult to visualize by more conventional means of preparation.

Some applications of cryosubstitution in ultrastructural studies of the cell nucleus.

Cryofixation followed by cryosubstitution, without the use of any chemical fixatives, was carried out on cultured mouse P815 cells. The principal aim of our work, which was to show that these techniques provide excellent morphological preservation of cellular and in particular nuclear components, was demonstrated. All nuclear structural components, nucleolar or nucleoplasmic, were clearly revealed using this technology. The cells were cryofixed by impact freezing onto a copper mirror cooled with liquid nitrogen or helium, cryosubstituted in acetone and embedded in either Lowicryl K11M or 1R White acrylic resin. Ultrathin sections were contrasted using either the usual uranyl acetate-lead citrate double staining, a differential staining for nuclear nucleoprotein structures, or the silver staining revealing nucleolar organizer regions. In view of the absence of conventional fixatives, the specimens prepared in this way would offer to be material of choice for ultrastructural identification of intra-nuclear antigens, especially those sensitive to conventional fixatives such as, for example, aldehydes. Advantages and differences of these techniques with regard to more conventional electron microscopic procedures are discussed.

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.

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 210 K (hydrophilic K11M) and to 190 K (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.

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.

Shape and fine structure of nucleoids observed on sections of ultrarapidly frozen and cryosubstituted bacteria.

Very rapidly frozen cells of Escherichia coli and Bacillus subtilis were substituted at low temperature into acetone with 1% OsO4 and embedded in Epon. They showed ribosome-free spaces filled with globular and fibrillar material of up to 15 nm. The sizes of structures seen do not exclude DNA superstructures such as supercoils, aggregates, and nucleosomes. With the Feulgen analog osmium-ammines stain, DNA was localized within the ribosome-free space. The bulk of DNA, the nucleoid, is therefore a major part of, or identical to, the main ribosome-free space. The ribosome-free space would correspond directly to the light microscopy phase-contrast image of nucleoids in living bacteria. The shape of the ribosome-free space does not reflect intracellular salt concentrations, nor do the Feulgen-positive areas. The previously observed dependency on the salt concentration of the growth medium seems to be due to permeabilization induced by the chemical fixative at room temperature. The ribosome-free space is more cleft in appearance than the nucleoid obtained by fixation with OsO4 but more confined than its very dispersed form found after aldehyde fixation.

Periplasmic gel: new concept resulting from the reinvestigation of bacterial cell envelope ultrastructure by new methods.

Bacterial cell envelope ultrastructure was investigated both by the progressive lowering of temperature embedding technique and freeze-substitution, using conventional and scanning transmission electron microscopy. Comparison with standard embedding procedures revealed a new aspect of cell envelope structure in specimens at low temperatures. The envelope was delimited by an electron-dark layer, beneath which was a uniform matter-containing layer lying between the outer and inner membranes. There was no empty periplasmic space. Buoyant densities of isolated peptidoglycan obtained in Percoll (1.02 to 1.07 g ml-1) and CsCl2 (1.44 g ml-1) led to a calculated hydration of the peptidoglycan which was more than was previously assumed. Peptidoglycan therefore possibly fills the entire space between the inner and outer membranes in the form of a periplasmic gel. The new model of cell envelope organization is discussed with respect to the current knowledge on bacterial cell wall structure and function.

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.

Perspectives for achieving improved information by the observation of thin sections in the electron microscope.

Obtaining biologically significant fine detailed information from sections is limited firstly by the unknown distribution of heavy metal stain and secondly by conformational changes induced by dehydration. Only afterwards we have to be concerned with beam induced alterations. By Z-imaging in a STEM, completely unstained material can now become imaged sharply with high contrast. By this the first limitation is eliminated and the second can become explored. For this purpose new resins designed for low temperature embedding might become important. First biological results are presented which illustrate the potential of these techniques: The transmembrane protein of the separate junction has been revealed as such and shown that only the hydrophilic part of the protein is stained by uranyl acetate. This type of staining therefore does not allow the detection of any transmembrane proteins in sections.

Enhancement of structural preservation and immunocytochemical staining in low temperature embedded pancreatic tissue.

The recently developed low temperature embedding procedure with the resin Lowicryl K4M (Carlemalm E, Garavito M, Villiger W: Proc 7th Eur Cong Electron Microsc, 1980, p 656; Garavito M, Carlemalm E, Villiger W: Proc 7th Eur Cong Electron Microsc, 1980, p 658) was tested for its suitability for embedding of glutaraldehyde-fixed rat pancreatic tissue and for postembedding staining of thin sections with the protein A-gold (pAg) technique (Roth J, Bendayan M, Orci L: J Histochem Cytochem 26:1074, 1978) for amylase. Compared to conventional Epon embedding of glutaraldehyde fixed tissue, the low temperature embedding method with Lowicryl K4M resulted in a superior preservation of the general cellular fine structure, particularly in the Golgi apparatus. For low temperature embedded tissue, the quantitative evaluation of the immunocytochemical labeling for amylase showed a more specific staining of the rough endoplasmic reticulum, the Golgi apparatus, and the zymogen granules. This was due to a significant lowering of the background staining over all cellular organelles. The use of Lowicryl K4M at low temperature, due to the superior preservation, yields improved resolution and specificity in immunocytochemical postembedding staining.