Freeze-fracture autoradiography: The in-vacuo coating technique

Summary Freeze-fracture autoradiography (FFA) was introduced in 1976 as a new method for electron microscopic autoradiography of diffusible compounds (Fisher and Branton, Rix et al.). With the original technique, the film monolayer was applied to the cold specimen in a cryostat at atmospheric pressure. Coating under these conditions did not exclude the risk of artifacts, mainly due to uncontrolled ice contamination of the cold specimen surface.A new method has been developed for coating the frozen specimen, immediately after replication, in the maintained vacuum of the freeze-fracture unit. Two main components of the new technique are described in detail, a specially designed coating device, and the use of spreading substances promoting adhesion of the film in vacuo. Using this technique artifacts so far inherent in the FFA method can be eliminated.Supported by the Deutsche Forschungsgemeinschaft     

Fundamental Technical Elements of Freeze-fracture/Freeze-etch in Biological Electron Microscopy

Freeze-fracture/freeze-etch describes a process whereby specimens, typically biological or nanomaterial in nature, are frozen, fractured, and replicated to generate a carbon/platinum “cast” intended for examination by transmission electron microscopy. Specimens are subjected to ultrarapid freezing rates, often in the presence of cryoprotective agents to limit ice crystal formation, with subsequent fracturing of the specimen at liquid nitrogen cooled temperatures under high vacuum. The resultant fractured surface is replicated and stabilized by evaporation of carbon and platinum from an angle that confers surface three-dimensional detail to the cast. This technique has proved particularly enlightening for the investigation of cell membranes and their specializations and has contributed considerably to the understanding of cellular form to related cell function. In this report, we survey the instrument requirements and technical protocol for performing freeze-fracture, the associated nomenclature and characteristics of fracture planes, variations on the conventional procedure, and criteria for interpretation of freeze-fracture images. This technique has been widely used for ultrastructural investigation in many areas of cell biology and holds promise as an emerging imaging technique for molecular, nanotechnology, and materials science studies.     

Freeze-fracturing and deep-etching with the volatile cryoprotectant ethanol reveals true membrane surfaces of kidney structures

Summary With the conventional freeze-fracture technique applied to biological specimens, cell membranes split along an interior plane and two membrane faces are produced. True membrane surfaces remain hidden and can only be uncovered by deep-etching. To date, deep-etching could not be satisfactorily performed in the presence of cryoprotective agents since conventional cryoprotectants do not sublime due to their low vapour pressure. This lack of suitable volatile cryoprotectants has limited deep-etching so far to very small objects which can be cryofixed without cryoprotectants. As a consequence, our freeze-fracture knowledge of cell surfaces is still poor.The present study shows that ethanol is a suitable volatile cryoprotectant for the freeze-fracture technique, and provides a novel approach to the routine deep-etching of freeze-fracture specimens without the need for special equipment. With ethanol deep-etching, true outer cell-surfaces are demonstrated within the kidneys of rat and Psammomys.     

Detection of surface-bound ligands by freeze-fracture autoradiography

This article describes a new freeze-fracture autoradiographic technique for the detection of radioactive ligands associated with the surface of cells in monolayer or suspension culture. Since freeze-fracture replicas are produced in the conventional way, all membrane features normally seen in freeze-fracture are retained, and autoradiographic grains produced by the labeled ligands are seen superimposed on unaltered exoplasmic membrane fracture faces. To assess the feasibility and resolution of this technique, we compared the surface distribution of alpha 2-macroglobulin and cholera toxin, labeled either with 125I or with colloidal gold, on 3T3-L1 fibroblasts. Both by autoradiography and cytochemical gold labeling, alpha 2-macroglobulin was associated specifically with coated pits, whereas cholera toxin was preferentially found over smaller, apparently non-coated membrane invaginations. Together with data on the surface localization of 125I-transferrin on HL-60 myelomonocytic cells, these results demonstrate the application of this technique for the accurate determination of ligand distribution over large areas of plasma membrane. The simplicity and reproducibility of the method should now allow freeze-fracture autoradiography to become a standard technique for investigating the distribution of both endogenous and exogenous cell surface-associated molecules, as well as the redistribution of such molecules under different experimental conditions.     

Recent advances in freeze-fracture electron microscop: the replica immunolabeling technique

Freeze-fracture electron microscopy is a technique for examining the ultrastructure of rapidly frozen biological samples by transmission electron microscopy. Of a range of approaches to freeze-fracture cytochemistry that have been developed and tried the most successful is the technique termed freeze-fracture replica immunogold labeling (FRIL). In this technique samples are frozen fractured and replicated with platinum-carbon as in standard freeze fracture and then carefully treated with sodium dodecylsulphate to remove all the biological material except a fine layer of molecules attached to the replica itself. Immunogold labeling of these molecules permits their distribution to be seen superimposed upon high resolution planar views of membrane structure. Examples of how this technique has contributed to our understanding of lipid droplet biogenesis and function are discussed.     

Studies on the orientation of brush-border membrane vesicles.

Orientation of rat renal and intestinal brush-border membrane vesicles was studied with two independent methods: electron-microscopic freeze-fracture technique and immunological methods. With the freeze-fracture technique a distinct asymmetric distribution of particles on the two membrane fracture faces was demonstrated; this was used as a criterion for orientation of the isolated membrane vesicles. For the immunological approach the accessibility or inaccessibility of aminopeptidase M localized on the outer surface of the cell membrane to antibodies was used. With both methods we showed that the brush-border membrane vesicles isolated from rat kidney cortex and from rat small intestine for transport studies are predominantly orientated right-side out.     

Freeze-fracture autoradiography: feasibility

We have shown that the combination of freeze-fracture with electron microscope autoradiography can be developed into a technique for correlating the molecular structure of the biological membrane with its chemical and functional characteristics. Within the limits of electron microscope autoradiographic resolution, FARG has the potential to detect the relative distribution of molecules in each half of the membrane and within the plane of the membrane. The use of radioisotopic labels in combination with freezing techniques requires minimal perturbation of the system being studied and may be suitable for the examination of substances which would be extracted or would diffuse during the normal fixation and embedding procedures used in standard electron microscope autoradiography.     

Improvement of freeze-fracture autoradiography for localization of soluble substances in tissue samples

Freeze-fracture autoradiography is accepted as an adequate technique for localization studies of soluble substances at the electron microscopical level. The method, however, involves many critical preparation steps, among them a protective carbon coating of the developed nuclear emulsion adhering to the replica. We demonstrate here that this additional carbon coating may be omitted. This simplification leads to a significant improvement of the sample yield as compared with the previously described procedures.     

Label-fracture: a method for high resolution labeling of cell surfaces

We introduce here a technique, "label-fracture," that allows the observation of the distribution of a cytochemical label on a cell surface. Cell surfaces labeled with an electron-dense marker (colloidal gold) are freeze-fractured and the fracture faces are replicated by plantinum/carbon evaporation. The exoplasmic halves of the membrane, apparently stabilized by the deposition of the Pt/C replica, are washed in distilled water. The new method reveals the surface distribution of the label coincident with the Pt/C replica of the exoplasmic fracture face. Initial applications indicate high resolution (less than or equal to 15 nm) and exceedingly low background. "Label-fracture" provides extensive views of the distribution of the label on membrane surfaces while preserving cell shape and relating to the freeze-fracture morphology of exoplasmic fracture faces. The regionalization of wheat germ agglutinin receptors on the plasma membranes of boar sperm cells is illustrated. The method and the interpretation of its results are straightforward. Label-fracture is appropriate for routine use as a surface labeling technique.     

Improvement of freeze-fracture autoradiography for localization of soluble substances in tissue samples

Summary Freeze-fracture autoradiography is accepted as an adequate technique for localization studies of soluble substances at the electron microscopical level. The method, however, involves many critical preparation steps, among them a protective carbon coating of the developed nuclear emulsion adhering to the replica. We demonstrate here that this additional carbon coating may be omitted. This simplification leads to a significant improvement of the sample yield as compared with the previously described procedures.These studies were supported by the Deutsche Forschungsgemeinschaft     

Improved coating and fixation methods for scanning electron microscope autoradiography

A simple apparatus for emulsion coating is described. The apparatus is inexpensive and easily assembled in a standard glass shop. Emulsion coating for scanning electron microscope autoradiography with this apparatus consistently yields uniform layers. When used in conjunction with newly described fixation methods, this new approach produces reliable autoradiographs of undamaged specimens.     

Fine structures of interphase nuclei : III. Replication site analysis of DNA during the S period of Crepis capillaris

The replication sites and morphological steps of chromosomal condensation during S period in the nuclei of Crepis capillaris root tip cells have been studied with light and electron microscopic autoradiography. From light microscopic autoradiographic observations, the S period can be divided with three portions, early S, mid S, and late S period. Labelled nuclei for each portion of the S period have also been found by using electron microscopic autoradiography. With electron microscopic autoradiography it has been found that in early, mid, and late S period, the replication sites are distributed in the electron transparent regions, interspersed with dense chromatin masses of variable size which are distributed throughout the nucleus. The time-dependent behavior of the label indicates that when compared with either mid or early replicated DNA, a majority of this chromatin, which contains predominantly late replicated DNA, is the earliest chromatin to be organized into the condensed chromatin. They are organized into the condensed chromatin within 15 min after the termination of replication.     

A method for detecting protein-DNA interactions at sites of chromatin replication

Two versions of an approach to identify DNA-protein interactions at sites of DNA replication in HeLa cell nuclei are described. In this procedure, newly replicated DNA chains are first labeled and photosensitized in vitro by the incorporation of [α-³²P]dCTP and bromodeoxyuridine triphosphate, respectively. Irradiation with ultraviolet light is then used to covalently crosslink the proteins that are adjacent to the photosensitized and isotopically labeled strands of newly replicated DNA. After the bulk of the DNA is digested with nucleases, the crosslinked proteins—marked by short covalently linked radioactive DNA tags—are fractionated by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels and detected by autoradiography. With this technology, certain proteins have been shown to associate selectively with newly replicated DNA. The method appears adaptable for application to a variety of problems involving DNA-protein association.     

Astrocyte membrane structure: changes after circulatory arrest

Membranes of the astrocytic processes investing small blood vessels and the surface of the brain contain numerous arrays of orthogonally packed particles as revealed by the freeze-fracture technique. The structure of these particle arrays, which we have termed "assemblies," is the same whether tissue is prepared for freeze-fracture by conventional fixation or by quick excision and rapid freezing. However, assemblies are progressively replaced by amorphous clumps and then disappear as the interval between decapitation and rapid freezing increases. Nearly normal numbers of assemblies may be maintained in cerebellar slices in vitro, but there too they disappear at low PO2 or in the presence of dinitrophenol. No other neuronal or glial membrane specialization exhibits a comparable lability.     

Scanning electron microscopy of surface and internal features of developing perithecia of Neurospora crassa.

Stages in the development of perithecia of Neurospora crassa, designated by the time elapsed after crossing, were investigated with the scanning electron microscope, from protoperithecia through perithecia. The usual examination of external features of whole specimens with this instrument was augmented by a freeze-fracture technique which allowed the viewing of development internally as well. Rapid increases in perithecial size soon after crossing were followed by the appearance, in section, of a centrum, at first undifferentiated but subsequently developing ascogenous hyphae. The perithecial beak appeared as a compact mass easily distinguishable in whole specimens from the surrounding hyphae by means of texture as well as shape. Two ascospores were photographed during emergence from an ostiole, but ostioles were found more frequently closed than open.     

Electron-microscopic study of the collagen fibrils of the rat tail tendon as revealed by freeze-fracture and freeze-etching techniques

Summary The ultrastructure of the collagen of rat tail tendon was investigated by the freeze-fracture technique. Collagen fibers were pretreated with the digestive enzymes, -amylase, elastase and collagenase to remove matrix substances. Some of the samples were etched for 20 min. Fibrils had an average diameter of 318±12 nm and a banded structure with a mean periodicity of 64.2±0.9 mm; the banding was most marked in -amylase/elastase-treated specimens, although the periodicity was independent of pretreatment. Microfibrils were well-displayed following -amylase/elastase and collagenase pretreatments. A difference in the diameters of microfibrils was, however, observed between etched specimens (8.3±0.3 nm) and those prepared by other experimental methods (11.4±0.5 nm). In replicas of collagenase-treated and etched specimens, the interconnecting filaments in the interfibrillar region formed a network that was continuous with the microfibrils of collagen fibrils. The diameter of the interconnecting filaments was the same as that of microfibrils. Microfibrillar bundles were observed in the interfibrillar region.     

Effects of pH during recombination of human erythrocyte membrane apoprotein and lipid.

The recombinates from human red cell membrane proteins and lipids resulting from dialysis of the components in 2-chloroethanol against aqueous buffers from pH2-12 have been studied by density gradient centrifugation, polyacrylamide gel electrophoresis and freeze-fracture electron microscopy. Between pH 4 and 10 most of the proteins were found in the recombinates whereas below pH 4 and above pH 10 only part of them were recovered in the lipoprotein band after density gradient centrifugation. At low pH, increasing incorporation of the "major glycoprotein" into the recombinates was detected by gel electrophoresis and in parallel increasing amounts of particles were found in the freeze-fracture membrane faces. The necessity of working at low pH values from pH 2-4, however, and a critical evaluation of all the data presently available leads to the conclusion that the 2-choloroethanol technique is not adequate for recombination studies tending to membrane reconsitution.     

An innovative coating technique for light and electron microscopic autoradiography.

We describe a modified nuclear emulsion coating technique for both electron and light microscopic autoradiography. We propose that by reversing the application of formvar film so that it adheres to and covers thin sections placed on grids, we have developed a technically accessible methodology that produces optimal conditions for the tracing of specific nuclear activity. A smooth, continuous base is formed over the sections on which a monolayer of evenly packed silver halide crystals can be applied by dip-coating. The same principle is applied to pre-stained 1-micron plastic sections of glass slides. We suggest that the application of formvar film over thin sections does not impede or interfere with the exposure of the emulsion by the labeled tissue. On the contrary, it virtually eliminates contamination and background radiation, enhancing the specificity and quality of resolution at even low magnifications. This technical modification, which facilitates the application of the emulsion, could render electron microscopic autoradiography a routine laboratory procedure, allowing for easily reproducible results and quantitative evaluation. At the light microscopic level, this technique prevents chemical fogging caused by certain stains, and thus allows routine pre-staining before coating with emulsion.     

Low-vacuum electron microscopy of carabid chemoreceptors: a new tool for the identification of live and valuable museum specimens

High-vacuum scanning electron microscopy, following coating of specimens with gold, produces high quality images that have proved invaluable for the study of insect sensilla. Unfortunately, the technique is essentially destructive, and cannot be used on live or valuable museum specimens. In particular, high-vacuum scanning usually causes the collapse of the tips of the palps, interfering with any examination of the sensilla in this area. A new low-vacuum technique is described that avoids these problems. Insect cuticle does not need to be coated with gold, thus avoiding damage to important specimens. Examples are given of scans of the palp tips of live carabid beetles, anaesthetised with CO₂. It was shown that the technique could consistently display these tips in their natural convex state. In all, four types of sensilla were identified by low-vacuum scans of the maxillary palps, and four further types on the terminal segment of the antennae, plus glandular openings. The antennae revealed a type of sensilla that has not previously been described on carabids. These sensilla showed clear structural differences between the two species studied, Pterostichus melanarius Illiger and P. niger Schaller (Coleoptera: Carabidae), and can be used as a diagnostic character for both fresh and dried specimens. The low-vacuum technique can be recommended for examining valuable type specimens without risk of damage.     

Identification of proteins interacting with newly replicated DNA in SV40-infected cells by UV-induced DNA-protein cross-linking

Protein species interacting with newly replicated DNA were analyzed using a photo cross-linking technique. Nascent DNA was labeled in vitro with [alpha-32P]dCTP and BrdUTP in SV40-infected CV-1 cells made permeable with saponin. The labeled cells were then irradiated with UV light (254 nm) and were treated extensively with DNase I. Proteins with radioactive DNA tags were separated by SDS-PAGE and visualized by autoradiography. Among 10-15 proteins which were cross-linked, the proteins with apparent molecular weights of 16.5 K, 44 K, 82 K and those in the 94-140 K region appeared to be associated with newly replicated SV40 DNA. A pulse-chase experiment showed that the 82 K and 94-140 K proteins interacted with new DNA in a relatively localized region close to the replication fork. The 44 K protein was identified as the major viral capsid protein, VP1, using antiserum to SV40 capsid proteins. It was suggested that VP1 binds to nascent DNA shortly after DNA synthesis and migrates into chromatin maturation regions.