Freeze-fracture Technique Research Articles

Cryogenic-scanning electron microscopy as a technique to image sol-to-gel transformation in chelated alkoxide systems

Direct imaging of wet sol-to-gel microstructures in chelated alkoxide systems is reported for the first time by using fast-freeze and freeze-fracture techniques for sample fixing. The system chosen for microstructural imaging is that of a chelated alkoxide, in an effort to observe sol-to-gel transformation. The batch composition corresponds to a 1: 1 molar ratio of aluminum sec-butoxide and ethyl acetoacetate made using isopropyl alcohol as a mutual solvent. About 100 μl of samples in varying stages of sonogelation, at a water addition rate of 0.4 ml per hour per gram of alkoxide, were injected between two 300-mesh TEM grids mounted on 0.1 mm copper plates and plunged into melting Freon to bring about a rapid solidification. The sample was cryo-transferred into an SEM, equipped with a cold stage maintained at −170 °C, using liquid N 2. The specimen was fractured and imaged for microstructural detail under the electron beam. The direct imaging techniques developed here have been applied to chelated alkoxides for the first time, showing the morphology and microstructure in unhydrolysed as well as fully hydrolysed sols, which were found to indicate complex shapes that were not previously known to exist in such systems. Thus, more microstructural information could be directly gained as a result of this cryo-SEM technique.

A quick-frozen, freeze-fracture and deep-etched study of the cuticle of adult forms of Strongyloides venezuelensis (Nematoda).

The cuticle of adult forms of Strongyloides venezuelensis was studied by routine transmission electron microscopy, conventional freeze-fracture and also using quick-freeze and deep-etch techniques. In routine thin sections the cuticle of S. venezuelensis comprises 7 layers: epicuticle, outer cortical, inner cortical, external medial, internal medial, fibrous and basal. Observation of replicas of specimens fractured across the thickness of the body wall, revealed at the epicuticle an ordered array of particles accompanying the cuticular annulations. At the level of the cortical and medial layers we observed few scattered particles embedded in an amorphous matrix without a particular arrangement. The fibrous layer was represented by several parallel lines of ordered particles of similar size. In tangentially fractured specimens, the epicuticle cleaves readily exposing 2 faces, one exhibiting intramembranous particles without any particular arrangement, immersed in a smooth matrix (P face), and the other showing depressions and very few particles (E face). In replicas of fractures submitted to etching, we observed at the level of the cortical, medial fibrous and basal layers an interconnecting fibrous and globous structure which was organized in a different direction at the fibrous layer. The association of freeze-fracture to deep-etch technique revealed the internal structural organization of the cuticle layers showing details that were not seen before using conventional freeze-fracture technique.

SEM characterization of the resin-dentine interface produced in vivo

Objectives: A freeze-fracture technique with critical-point drying has been shown to preserve the structure of collagen in demineralized primed dentine in vitro (Titley K et al. Am J Dent 1994; 7: 22–26). This study utilized the technique to examine the attachment to dentine produced in vivo by All-Bond 2 (AB2), Scotchbond Multi-Purpose (SBMP) and Prisma Universal Bond 3 (PUB3). Methods: Eighteen medium-depth restorations were placed in buccal and/or lingual surfaces of premolar teeth ( n = 7) scheduled for extraction for orthodontic purposes. After extraction, the restored teeth were split transversely by freeze-fracture, critical-point dried and examined under SEM. Results: AB2 and SBMP produced significant gap-free attachment to dentine showing similar microscopic structural features. Resin interdiffusion with collagen fibrils of the intertubular matrix was clearly seen. Microspaces, observed at the periphery of the widened tubules and among the interdiffused collagen fibrils, suggested incomplete resin penetration. Fractures occurring parallel with the interface frequently showed detachment in the deepest layers of the hybrid zone. Adhesive gaps were seen at the resin-dentine interface, indicating some clinical technique failure. The PUB3 attachment was generally incomplete, with separation between the primed dentinal surface and the composite restoration. Where present, the bonded interface showed no evidence of true hybridization. Conclusion: Dentine conditioning, as a separate step, prior to priming, appears to be an important factor in successful resin-dentine attachment. Critical-point drying revealed variations in the degree of resin interdiffusion with intertubular collagen, suggesting potential sites for bond failure.

Scanning electron microscopy of the digestive tract of larval Simulium ornatum Meigen (Complex) (Diptera: Simuliidae) and its associated microbial flora

The structure and microbial flora of the digestive tract of larval Simulium ornatum were investigated using scanning electron microscopy. Direct observations of the microbial communities associated with the endoperitrophic surface of the peritrophic membrane and the endocuticular surface of the hindgut are presented. The endoperitrophic surface was frequently devoid of bacteria, although the fungus Harpella melusinae (Harpellales, Trichomycetes) was commonly attached. Rarely, spirochaetes and coccoid bacteria were attached to the endoperitrophic surface. In contrast, the endocuticular surface was regularly colonized by a diverse microflora composed of rod-shaped, coccoid, spiral, and filamentous bacteria and two species of Harpellales (Trichomycetes). A freeze-fracture technique is compared with a dissection technique for exposing the endoperitrophic and endocuticular surfaces of the digestive tract.

Freeze-fracture study of Bodo sp. (Kinetoplastida: Bodonidae).

Freeze-fracture technique was used to analyse the structure of conventionally fixed and quickly frozen Bodo sp., a free-living kinetoplastid. In the former method, chemically fixed and cryopreserved cells presented a corrugated membrane pattern in the flagella and cell body surfaces. In the latter, however, replicas from quickly frozen unfixed flagellates showed membranes with a smoother aspect, allowing the observation of intramembranous particles (IMPs) on the fracture faces, hardly detectable in previously fixed samples. The IMPs were randomly distributed throughout the cell surface, except in the sparsely seen short IMP rows.

Biogenic structure of early sediment fabric visualized by low-temperature scanning electron microscopy

The accurate visualization of early biogenic sediment fabric is important in the search for modern analogues to relict fossil structure and, therefore, for the interpretation of past environmental conditions. Water may have a significant structural role within hydrated material and sample dehydration may be detrimental to the structural interpretation of natural sediments and their associated microbiota. Water is present in sediments as free water or in the form of bound or hydration water. Extracellular polymeric substances associated with microbial assemblages have a high content of hydration water and are distorted by dehydration. The use of low-temperature scanning electron microscopy for the visualization of water-saturated sediments and associated biogenic structures is examined. Low-temperature SEM allows samples to be examined without the removal of water that is normally associated with the preparatory procedures for traditional ambient SEM. Samples for low-temperature SEM are first frozen (cryofixed) and then examined, fully-hydrated and still frozen, at low temperature (cf.–180°C) on the stage of a specially adapted SEM. The process of low-temperature SEM for the examination of hydrated sediments is described and several examples of cohesive and mixed-flat sediments and their associated biogenic fabrics are given. Detail of the internal fabric and associated microbiota can be examined by freeze-fracture techniques to preserve the natural condition of the sediments.

XPS Studies of the Interface Properties of Conductive Adhesive/Eutectic Solder

ABSTRACTThe continuing shrinkage of feature size and environmental concerns regarding the use of lead containing solder are major driving forces in the search for alternative technologies for metal interconnects in electronic packaging. Electrically conductive adhesives are considered a potential substitute for the lead-tin solder. This study concentrated on interfacial properties of eutectic solder and silver-filled epoxy adhesives. A freeze fracture technique was employed in order to preserve interfacial information. The chemical composition of the fractured faces varied; the adhesive side was rich in tin, whereas the eutectic solder side showed lead-rich characteristics.

Freeze‐fracture study of cell junctions in the epididymis and vas deferens of a seasonal breeder: The mink (Mustela vison)

The present study used the freeze-fracture technique and vascular infusion of horseradish peroxidase (HRP) as a junction permeability tracer, visible in thin sections, to investigate potential seasonal variations in the mink epididymis and vas deferens cell junctions. The junctions were studied in kits during the neonatal period, during and after puberty, and during adulthood monthly throughout the annual reproductive cycle. Results showed the existence, at the time of birth, of a junctional complex joining ductal cells that reached the lumen of the epididymis and vas deferens. The lumenal impermeable segment of the junctional complex was characterized by the presence of an occluding zonule made up of continuous tight junctional ridges extending around the perimeter of the cell. The basal permeable segment of the junctional complex contained mainly discontinuous ridges with frequent forming gap and tight junctions next to adhering junctions. Receding annular junctions were present in the apical and lateral cytoplasm of principal and clear cells. The membrane domain apical to the occluding zonule bore 30-35 nm exo/endocytosis blebs and 40-60 crenations associated with deforming tight and gap junctions made up of randomly scattered particles. Patterns of junctional strands varied greatly from one principal and/or clear cell to another. However, cell junctions did not significantly vary from the neonatal period to adulthood nor from breeding to testicular regression. Anatomical subdivisions of the epididymis with fewer junctional strands per zonule and high diversity in their patterns exhibit no permeability differences to HRP when compared with subdivisions containing more numerous strands. The junctions were impermeable during the neonatal period and throughout the annual reproductive cycle. The following conclusions were reached: 1) a competent occluding zonule developed in the mink epididymis and vas deferens before it did in the testis; 2) the number of strands and the diversity of patterns did not correlate with permeability differences; 3) after the development of a lumen in the testicular excurrent duct, neither receding cellular changes caused by testicular regression nor seasonal passage of a bolus of sperm through the duct modified the occluding zonules; and 4) in the testicular excurrent duct, junction modulation (i.e., formation and deformation) paralleled that in the testis and followed the direction of the synthesis-transport-secretion process.

Quantification of tight junction complexity by means of fractal analysis

The concept of fractal geometry provides an elegant tool for the quantitative and objective structural description of various objects, the fractal analysis. Fractal analysis quantifies the structural complexity of objects by a characteristic singular value, the fractal dimension (FD). It can be estimated, e.g. by the box-counting method and provides a highly integrated measure in the range 1 < FD < 2 for curves extending within a plane. In this study, fractal analysis is used for the first time to evaluate the complexity of the tight junction network between adjoining cells. Bovine brain endothelial cells were cultured under various experimental conditions and the tight junctions were drawn to scale as visualized by the freeze fracture technique. These drawings were analyzed by fractal analysis, and by two other methods commonly used in this field, viz. the strand counting (SC) and complexity index (CI) methods. In contrast to the latter methods, the FD shows no directional preference and therefore no assumptions on the dynamic properties of the network's complexity are required. Thus, FD is demonstrated to provide the most sensitive, reliable and complete measure of tight junction complexity. In combination with SC and CI, additional information can be achieved concerning the directionality of the altered arrangement of tight junctional strands. Our analysis allows for the following conclusions. (1) Defined experimental influences can modify the complexity of tight junctions that are formed between endothelial cells in vitro, and (2) these structural modifications of the tight junctions are mainly due to an altered strand branching pattern.

Quantitative evaluations of gap junctions in old rat brown adipose tissue after cold acclimation: A freeze-fracture and ultra-structural study

The morphological and functional modifications of brown adipose tissue (BAT), the tissue responsible for non-shivering thermogenesis, are well established during the phases of active stimulation (i.e. neonatal period and cold acclimation) in young animals. The ‘active’ brown adipocytes are filled with numerous small lipid vacuoles and large mitochondria packed with cristae rich in the protonophore uncoupling protein (UCP), whereas the ‘quiescent’ cell shows larger, confluent vacuoles and smaller mitochondria with rarefied cristae poor of the uncoupling protein. It is well known from literature that also gap junctions (gjs), responsible for the electrical coupling among adjacent adipocytes, modify their size following the physiological stimulus in young animals. This is in agreement with the morphology of the functionally active brown adipocyte, i.e. the multilocular, UCP-positive cell. Although the presence of the BAT in old animals is well documented, less is known about its reactivity to physiological stimuli. The present work demonstrates that after cold acclimation brown adipocytes of old rats (2 years) change their ultrastructure in a similar way as in young rats. A quantitative analysis of gap junction areas on replicas obtained by the freeze fracture technique, showed that gj increase in size (mean area 53.2 vs. 110.4 × 10 −3 μm 2, p = 0.003). All these morphological modifications are quite similar to those observed in BAT of young and young adult rats, supporting the hypothesis of a physiological role of brown adipose tissue at every age.

Freeze–fracture analysis of muscle plasma membrane in Becker's muscular dystrophy

The intramembranous particle (IMP), orthogonal array (OA) and orthogonal array subunit particle (OASP) densities in skeletal muscle plasma membranes from eight patients with Becker's muscular dystrophy (BMD) were analysed by the freeze-fracture technique. The results showed almost normal IMP density with the significant decrease of OA and OASP densities in BMD. The group mean densities +/- SE of IMPs on the protoplasmic faces with and without OASPs, and on extracellular faces/microns 2 were 2137 +/- 207, 1839 +/- 68 and 895 +/- 108, respectively in controls; whereas those of BMD were 1989 +/- 259, 1837 +/- 203 and 900 +/- 239, respectively (P > 0.1 by two-tailed t-test). The group median density of OAs and their pits/microns 2 was 4.89 with mid-ranges (25-75% values of the counts) of 2.66-10.18 in controls; whereas that in BMD was 2.15 with mid-ranges of 1.14-4.31 (P < 0.01 by Wilcoxon rank-sum test). The group mean density +/- SE of OASPs in controls was 15.99 +/- 1.83; whereas that in BMD was 13.47 +/- 1.07 (P < 0.01 by two-tailed t-test). However, the diminution of OA and OASP densities in BMD muscle plasma membranes was not as severe as in Duchenne's muscular dystrophy. There was a relationship between OA density and clinical severity in BMD patients; the decrease of OA density in a severe BMD patient was more marked than that in mildly affected BMD patients. Therefore, it seems that marked depletion of OA density may lead to the severe disability in muscular dystrophies.

Tight Junctions of the Human Ciliary Epithelium: Regional Morphology and Implications on Transepithelial Resistance

The tight junctions of the human ciliary epithelium have been studied using the freeze-fracture technique with particular regard to regional differences in the tight junction networks and implications on the transepithelial resistance. The tight junctions of the non-pigmented epithelium always form continuous networks and consist of from two to more than 20 superimposed strands (mean 3 to 4), suggesting that relatively tight and leaky sites of the paracellular pathway coexist within the same cell perimeter. The tight junction morphology is more complex in the anterior pars plicata than in the posterior pars plicata, but its complexity increases again towards the pars plana. The application of an electrical circuit analysis to morphometric freeze-fracture data suggests that the transepithelial resistance decreases from the anterior pars plicata (32 Ω cm 2) towards the posterior pars plicata (26 Ω cm 2) and becomes lowest in the pars plana (19 Ω cm 2) and that the transepithelial resistance of the native epithelium is lower than most reported calculations based on electrophysiological measurements. We conclude that the human ciliary epithelium is a leaky layer, but that the anterior ciliary processes are less leaky than the posterior processes and the pars plana despite the occurrence of the breakdown of the blood-aqueous barrier predominantly in the anterior pars plicata.

A freeze-fracture study of mesophyll cell membranes in the embryonic Phaseolus leaf, in the dry seed and during the initial stages of germination

Using rapid-freezing and freeze-fracturing techniques, we have examined cellular organisation within mesophyll cells in primary leaves of Phaseolus, in the dry seed and over a period of 48 hr from the onset of germination. The rapidly-frozen, unetched image reveals a dynamic cytoplasm with membrane shape and role able to change and fluctuate with the increase in cell metabolism. The ER in this tissue is highly mobile, becoming progressively associated with protein and lipid bodies, then plastids, mitochondria and nuclei as cytoplasmic requirements change. The ER thus provides an efficient transport of substrates to metabolic sites within the cytoplasm. Nuclei and plastics change shape in early germination and the protein body membrane changes in role and character to that of a vacuolar tonoplast. The freeze-fractured image emphasizes the considerable movement that occurs within the cytoplasm during germination, with flexible and mobile membranes of the various organelles able to associate and interact with others. We have also measured the density of intramembrane particles on most of the membranes within the mesophyll parenchyma cell, during this period of early germination. Using our method of rapid-freezing, we find consistently higher IMP density on the protoplasmic face of each membrane. The counts provide a set of comparative values for membranes of a specific tissue, and for a specific cryofixation technique.

The Human Adenoid: A Morphologic Study

We examined the route by which antigen on the surface of the adenoid may be brought into contact with the lymphoid follicles in the submucosa of the adenoid. We studied under light and electron microscopy 13 adenoids from children undergoing elective surgery. Portions of all of the specimens were fixed in formalin and embedded in paraffin and plastic for hematoxylin-eosin and periodic acid-Schiff staining. Portions of four adenoids were fixed in glutaraldehyde for electron microscopy. Two major types of epithelium were evident by light microscopy: a ciliated or squamous epithelium containing few lymphocytes and a nonciliated-flat epithelium with a heavy infiltration of lymphocytes ("lymphoepithelium"). Scanning microscopy showed the surface of this lymphoepithelium to be composed largely of cells with multiple microfolds known as M-cells. Freeze-fracture technique showed many lymphocytes under the M-cells. Transmission electron microscopy showed the lymphocytes to be located in compartments formed by the epithelial cells. Light microscopy study of 50 serial sections embedded in plastic suggested that the compartments communicated to form intraepithelial channels for the lymphocytes. The epithelium of the adenoid has areas with ciliated epithelium adjacent to areas with epithelium containing M-cells and intraepithelial lymphatic channels. The lymphoepithelium of the adenoid is a mechanism for transporting antigen via the M-cells to the underlying lymphoid follicles.

On the nature of linkage between retinal rod outer-segment disk and cellular membrane

In the retina of most vertebrates the cone outer segment disk are in continuity with the cell membrane, thus intradisk space is part of the extracellular space. The rod disk membranes, except for some basal ones, appear to be separated from the cell membrane. On the other hand it has been shown the existence of filament-like structures connecting retinal rod disks to one another and with the cell membrane. However due to the lack of observation of either electron-dense or fluorescent extracellular tracer penetration into rod outer segments, it is believed that rod intradisk space is isolated from the extracellular space. This investigation elucidates upon the nature of filament-like linkage between retinal rod outer segment disk and cell membrane. An incubation solution with 10 times lower than normal osmolarity was used to osmotically explode isolated dark adapted frog retina.Membranes under this condition were observed by transmission electron microscopy (TEM) using the freeze-fracture replica technique. Regularly spaced filaments were observed that were continuous between outer segment disks and the cell membrane in the retinal rods (Fig. 1). Probably these connections have a supporting function providing mechanical stability of the rod outer segment structure.

Freeze-fracture characterization of the cuticle of adult and dauer forms of Caenorhabditis elegans.

At the ultrastructural level, a trilaminate structure, designated as the epicuticle, is always present on the outermost surface of the nematode Caenorhabditis elegans. The freeze-fracture technique revealed the existence of two fracture faces: an inner face that showed homogeneously distributed particles and an outer fracture face that was almost completely smooth in adult nematodes but appeared delicately granular in the dauer forms.

A freeze-fracture study of the cuticle of adult Nippostrongylus brasiliensis (Nematoda)

The surface of the cuticle of adult Nippostrongylus brasiliensis has been studied by means of the freeze-fracture technique and by transmission electron microscopy. Some of the surface coat appears to have been shed from the surface of the cuticle of adults fixed in situ in the intestine of its host and from the surface of individuals removed from the intestine and freeze-fractured. Freeze-fracturing the cuticle of individuals removed from the host has shown that this surface coat varies in thickness from 30 to 90 nm. The epicuticle is about 20 nm thick and cleaves readily to expose E- and P-faces. The P-face of the epicuticle possesses a small number of particles, similar to intra-membranous particles, whilst the E-face possesses a few, widely scattered depressions. Despite the presence of these particles the epicuticle is not considered to be a true membrane. Freeze-fracturing the remainder of the cuticle has confirmed its structure as described by conventional transmission electron microscopy. Clusters of particles on the P-face of the outer epidermal (hypodermal) membrane and corresponding depressions on the E-face of the membrane are though to be associated with points of attachment of the cuticle to the epidermis (hypodermis). No differences in appearance of the cuticle and its surface layers were observed in individuals taken from 7-, 10-, 13- and 15-day infections.

THE SPEED OF DIATOM MIGRATION THROUGH NATURAL AND ARTIFICIAL SUBSTRATA

The speed of diatom locomotion during cell migration was investigated in laboratory experiments using natural assemblages of intertidal diatoms inhabiting the upper intertidal mudflat at Portishead on the Severn Estuary in winter. Persistent vertical migration rhythms were verified for the epipelic diatom assemblages in the field and the migratory behaviour was successfully maintained in a tidal laboratory experimental system. No migratory behaviour was apparent for salt marsh assemblages at the same site and a brief investigation indicated that this behaviour was not overtly related to the water content of the sediments. The dominant diatom on the intertidal mudflat at the time of the investigation was Gyrosigma spencerii (W. Smith) Cleve which was used for the investigation of locomotion through sediments. Initial measurements of surface progression were made which compared well with values taken from the literature. G. spencerii was found to move over an artificial sediment (kaolin) at an average speed of 4.7 μms−1. Migration speed through, rather than over, kaolin and natural sediment was examined by deposition of kaolin and sediment, free from diatoms, onto the surface of diatom assemblages inhabiting cores of natural sediment maintained in the laboratory. Determination of the thickness of the deposited layer by freeze-fracture techniques allowed the calculation of the rate of upward movement. The results suggest much lower locomotion speeds for diatoms moving through substrata than on horizontal surfaces. Vertical migration of G. spencerii was found to be 25 times slower than surface speeds over kaolin and natural sediment with average values of 0.17 and 0.19 μms−1, respectively. Diatom species showed a synchronous emergence at the surface of the sediments with G. spencerii first to appear. The significance of these findings to diatom microspatial vertical distribution within sediment, endogenous rhythmic behaviour, frustule streamlining and energy budgets are discussed.

STM of freeze-fracture replicas: Problems and promises

The Scanning Tunneling Microscope (STM) offers exciting new ways of imaging surfaces of biological or organic materials with resolution to the sub-molecular scale. Rigid, conductive surfaces can readily be imaged with the STM with atomic resolution. Unfortunately, organic surfaces are neither sufficiently conductive or rigid enough to be examined directly with the STM. At present, nonconductive surfaces can be examined in two ways: 1) Using the AFM, which measures the deflection of a weak spring as it is dragged across the surface, or 2) coating or replicating non-conductive surfaces with metal layers so as to make them conductive, then imaging with the STM. However, we have found that the conventional freeze-fracture technique, while extremely useful for imaging bulk organic materials with STM, must be modified considerably for optimal use in the STM.

Seasonal changes of the blood‐testis barrier in viscacha (Lagostomus maximus maximus): A freeze‐fracture and lanthanum tracer study

Adult male viscachas (Lagostomus maximus maximus) were gathered from their natural habitat during the period of complete spermatogenesis (June) and during the month of maximum testicular regression (August). The testes were processed by conventional electron microscopic technique using lanthanum nitrate (electron-dense intercellular tracer) to define the intercellular spaces below the inter-Sertoli tight junctions and by freeze-fracture techniques. During complete spermatogenesis the tracer surrounds spermatogonia, preleptotene, and leptotene spermatocytes and stops at the level of the inter-Sertoli tight junctions below all germ cells displaying synaptonemal complexes (zygotene-pachytene spermatocytes) and germ cells in more advanced stages of differentiation. Conversely, during testicular regression the tracer percolates all intercellular spaces between Sertoli cells and the remaining germ cells (spermatogonia and few preleptotene and leptotene spermatocytes). During complete spermatogenesis, freeze-fracture replicas exhibit numerous inter-Sertoli tight junction strands parallel to each other and to the basal lamina. During spermatogenesis decay, the inter-Sertoli tight junctions are found to be short, tortuous, frequently interrupted, and often associated with extented membranous areas of gap junctions.