The effects of glycol methacrylate as a dehydrating agent on the dimensional changes of liver tissue

The dimensional changes of liver sections during the course of processing with glycol methacrylate (GMA) or with ethanol are described. Tissue processing with ethanol served as a control. During prolonged processing steps (24 h each), linear shrinkage of tissue specimens dehydrated with GMA at room temperature was 13.2%. Subsequent infiltration with GMA resulted in trivial swelling, and polymerization in slight shrinkage (2.3%). In comparison, processing with cold GMA resulted in shrinkage during dehydration (about 10.8%), a slight swelling in pure GMA, followed by shrinkage during polymerization (2.2%). Short routine processing schedules resulted in similar shrinkage/swelling patterns, although precise values differed slightly. In all experiments, ethanolic dehydration resulted in smaller dimensional tissue changes than did GMA dehydration. The dimensional changes of tissue sections during stretching on water, mounting and drying compensated for the major part of the shrinkage manifested during processing.     

A procedure for obtaining thin sections of undercalcified bone biopsies embedded in methyl methacrylate.

A complex of techniques is described, including a specially designed mould and a method for flat mounting of the sections, enabling to obtain well-stretched and undamaged thin sections of undecalcified bone biopsies. Apart from a gain in polymerisation time, a clear advantage of the special mould is a more favorable temperature course in and around the tissue during the polymerisation process.     

Intercellular junctions in embryonic chick cardiac muscle revealed by rapid freezing and freeze-substitution.

Using the method of rapid freezing and freeze-substitution, the embryonic chick cardiac muscle was investigated by transmission electron microscopy. Initially, the intercellular junctional complexes (fasciae adherentes and desmosomes) were formed in close proximity to each other along a nearly straight line. Subsequently, the separation of fasciae from desmosomes took place to form intercalated discs. The cell membranes of fasciae adherentes were reinforced with highly interwoven fine fibrils at which myofibrils terminated. The intercellular space of fasciae was bridged with fine fibrillar structures seemingly connected by a thin line at their middle portions. In the intercellular space of desmosomes, central lamina and traversing filaments were clearly observed. The outer and inner leaflets of the desmosomal plasmalemma were asymmetrically differentiated; the outer leaflet was thinner than the inner leaflet. On the inner side of the cell membrane, an electron-lucent layer and a dense desmosomal plaque were observed. The latter structure had protrusions with less electron density towards the cytoplasmic side. Further inside, a meshwork of fine fibrils was seen along and toward which bundles of intermediate filaments ran. The results obtained with freeze-substitution appeared to provide more information than those with thin sections after conventional fixation or with replicas of chemically fixed/glycerinated or physically fixed/deep-etched materials.     

Microstructural investigation of colloidal silver embedded in glass

Nanoparticulate composites consisting of very small silver particles embedded in near-surface regions of glass were obtained by sodium–silver ion exchange. Colloidal silver is formed by reduction of silver ions and aggregation of silver atoms in the glass matrix at elevated temperatures. Owing to absorption bands in the visible region, the silver particles cause a coloration of the glass that depends on their size and depth distribution. From high-resolution electron microscopy imaging of lattice plane fringes of the silver particles, size-dependent lattice contractions are deduced that are larger than those reported in the literature for supported particles not interacting with a matrix. This effect is the more pronounced the higher the annealing temperature is during the particle formation. The increased lattice contraction is attributed to compressive stresses that arise during the ion exchange process as well as during cooling after annealing.     

A simple method of preparing 1–2 μm sections of large tissue blocks using glycol methacrylate

The method described allows the production of 1–2 μm sections of large tissue blocks. Sections can be obtained from conventional routine histological microtomes. The hydroxyethyl methacrylate is compatible with a wide range of fixatives and a variety of standard histological staining techniques may be applied. Small biopsies, e.g. renal, liver, jejunal etc., can be processed cut and stained within 24 h of receipt. Blocks and stained sections show no apparent deterioration over the 4 years in which the technique has been employed in this laboratory.     

Embedding of large specimens in glycol methacrylate: prerequisites for multi-signal detection and high-resolution imaging

Acrylic resin mixtures are commonly used to study microscopic sections of biological specimens, giving the advantage of good morphological preservation. Existing embedding protocols, however, are suitable for tissue blocks, not exceeding 1 mm in thickness. We have developed a protocol to embed larger specimens (up to 2 cm(3)) in Technovit 8100. This medium allowed us to perform classic histological (trichrome), silver, as well as immunohistochemical staining, needed for multi-signal detection at high-resolution imaging to reconstruct a three-dimensional interpretation of a serially sectioned muscle. The technique was applied to reconstruct the semitendinosus muscle of a fetal pig, 44 days post conception, featuring connective tissue, intramuscular nerves, blood vessels, and muscle fibre types. For the reconstruction, a technique was used that enabled us to insert high-resolution images of histological details into low-resolution images of the entire muscle.     

A comparative study of softeners and catalyst systems upon dimensional changes and sectioning quality of glycol methacrylate sections

It is shown that softeners and temperature have an important influence upon the stretching parameters of glycol methacrylate sections. Also it is demonstrated that the catalyst system used may significantly interfere with section stretching.     

Volume changes during preparation of mouse embryonic tissue for scanning electron microscopy

This paper describes the results of experiments in which the volume changes in mouse embryo limb samples were followed more or less continuously after fixation through dehydration and critical point drying, with in some instances data relating to post critical point drying shrinkage. 14 and 15 day p. c. mouse embryos were fixed in 3 % glutaraldehyde in cacodylate buffer and stored in this fixative until use. Single specimens were studied using a Quantimet image analysing computer to record the changes in projected area of the unmounted specimens as they were passed through the usual series of reagents according to various commonly used dehydration schedules. The area changes were converted to volume changes for the purposes of presentation in this paper. The Quantimet system could not be used to follow volume changes in the CPD bomb so that most experiments detail the volume in the intermediate fluid before CPD and the size of the specimen immediately after it was removed from the CPD bomb. A few experiments were conducted in which the specimens were measured whilst they were in the CPD bomb. The measurements relating to dehydration and CPD procedures were compared with measurements of air dried and freeze dried specimens. All three drying methods cause considerable shrinkage: freeze drying to 85 % of the glutaraldehyde fixed tissue volume; critical point drying to 41% (after 24 h); and air drying from a volatile solvent to about 18% of the fixed tissue volume. Air drying from water caused a shrinkage to about 12% of the original volume. There was no significant difference between the various commonly used CPD schedules or between GA only and GA + Os O₄ fixed tissue. CPD via cellosolve and CO₂ caused substantially more shrinkage than other methods. Dimensional changes during specimen preparation are probably associated with changes in shape and in relative relationships between organelles, cells and tissues having different compositions. This should be borne in mind by all those interpreting scanning electron micrographs of dried animal soft tissue specimens.     

The influence of tissue handling before fixation on the morphology of the chick blastoderm

The morphology of cells and tissues of chick blastoderms handled before fixation in different ways, was compared. Blastoderms were fixed in ovo or after mounting on a glass ring (New, 1955). Differences in the morphology were observed.     

Effects of porosity on the fatigue performance of polymethyl methacrylate bone cement: an analytical investigation

Porosity has been shown to affect the fatigue life of bone cements, but, although vacuum mixing is widely used to reduce porosity in the clinical setting, results have been mixed and the effects of porosity are not well understood. The aim of this study was to investigate the effects of porosity using stress analysis and fracture mechanics techniques. The stress concentrations arising at voids in test specimens were found using analytical solutions and boundary element methods. The fatigue life of specimens containing voids of various sizes was predicted using fracture mechanics techniques. For spherical voids that do not occupy a significant proportion of the cross-section, the resulting stress concentration is independent of void size and too small to account for the observed crack initiation. Cracks must therefore initiate at additional stress raisers such as radiopacifier particles or additional voids. For large voids, the stress increases as the remaining cross-section of the specimen decreases, and this may account for much of the observed reduction in fatigue strength in hand-mixed cement. Although crack initiation may be largely independent of void size, there is an effect on crack growth rate. Cracks are predicted to grow faster around larger voids, since they remain in the stress concentration around the void for longer. This effect may account for the relationship between porosity and fatigue life that has been observed in samples without large voids. Since porosity appears to affect crack growth more than initiation, it may be less damaging in high-cycle clinical fatigue, which may be predominantly initiation controlled, than in short laboratory tests.     

Organometallic and organometalloid compounds as standards for microprobe analysis of epoxy resin embedded tissue

X-ray microanalysis of phosphorus, transition elements and heavy metals in biological tissue is frequently carried out on thin sections of specimens embedded in epoxy resin. A logical choice for the quantitive microprobe analysis of these specimens is a standard, consisting of a homogeneous solution of the elements of interest in the epoxy resin. Four kinds of compounds were found suitable for this purpose: (1) phenyl compounds containing group Vb elements, (2) cyclopentadienyl-derivatives, (3) a pentanedione derivative (acetylacetonate) and (4) complexes of metals with dialkyldithiocarbamates. In the latter case, the standard also contains sulphur. Standards for P, Sb (1) Mn, Fe (2) Ni (3) Cu, Zn, Cd, Hg, Pb, Bi (4) were prepared in Epon 812 or Spurr epoxy resin. The compounds were mixed with the resin (without accelerator) to which some propylene oxide may be added, and dissolved immediately or after short heating. The maximal concentration of metal was in the order of magnitude of several promilles to 1%. Solubility in the Spurr resin was better than in Epon 812. After addition of the accelerator, polymerization was carried out as usual. The compounds used are commercially available at low cost or can be easily prepared.     

Visualization of proteoglycans and link protein in embryonic chick limb cartilage via cryofixation, freeze-substitution and immunochemical techniques

Chick embryo limb bud cartilage contains a family of proteoglycans, a few of which have been identified ultrastructurally by antibody labelling. Limb bud cartilage from stage 30–34 chick embryos was high-pressure frozen, freeze-substituted and embedded in Lowicryl resin. Sections were treated with polyclonal antibodies for core protein and monoclonal antibodies for chondroitin-6-sulphate and link protein. Label for core protein was demonstrated on both structural matrix and free within the compartmental space. Quantitative analysis indicates that core protein is preferentially localized on electron-dense structural matrix, and that this distribution is uniform between stages 30 and 34. The association of protein epitopes on electron-dense lattice is strongly influenced, rather than a chance observation. Significant quantities of core protein are also located in the free compartments of the cartilaginous lattice. Chondroitin-6-sulphate and link protein were localized predominantly within the compartments of the embryonic lattice. Our data provide convincing evidence that the proteoglycans were immobilized within a microcrystalline matrix of the embryonic compartments. A role for core protein as a stabilizer within the lattice and in the free space where it serves to aggregate polymeric proteoglycans is suggested from our results.     

A new,less toxic polymerization system for the embedding of soft tissues in glycol methacrylate and subsequent preparing of serial sections

The paper describes a new polymerization system for embedding soft tissues in glycol methacrylate (GMA). The polymerization of GMA is initiated by means of a barbituric acid derivative in combination with chloride ions and dibenzoyl peroxide. The catalyst system contains no aromatic amines which constitutes a toxicological advantage over the commonly employed system of peroxide/aromatic amine. Clear blocks are obtained from which 1–2 μm sections are easy to cut. In combination with an appropriate softener, polyethylene glycol 400 , serial sectioning may be practised.     

Three-dimensional reconstruction of single particles embedded in ice.

Single particles embedded in ice pose new challenges for image processing because of the intrinsically low signal-to-noise ratio of such particles in electron micrographs. We have developed new techniques that address some of these problems and have applied these techniques to electron micrographs of the Escherichia coli ribosome. Data collection and reconstruction follow the protocol of the random-conical technique of Radermacher et al. [J. Microscopy 146 (1987) 113]. A reference-free alignment algorithm has been developed to overcome the propensity of reference-based algorithms to reinforce the reference motif in very noisy situations. In addition, an iterative 3D reconstruction method based on a chi-square minimization constraint has been developed and tested. This algorithm tends to reduce the effects of the missing angular range on the reconstruction, thereby facilitating the merging of random-conical data sets obtained from differently oriented particles.     

Photoelectron emission microscopy of biological tissue; the influence of glutaraldehyde and heavy or biligrafin on the emission image of chicken liver in methacrylate.

The effect of various preparations on the emission image was studied taking into account the influence of UV wave length, section thickness and specimen support. By suitable selection of these parameters, images with sufficient contrast can be obtained of unstained specimens which are only fixed with glutaraldehyde. With glutaraldehyde fixation intravitally injected Biligrafin used as a stain in cholecystography is recognized in the emission image of liver tissue labeling the intravital transport path from the blood capillaries to the bile ducts. Hypotheses for the image forming process are discussed.     

Immunofluorescence and protein A-gold techniques in localization of plant pathogen antigens in Lowicryl K4M embedded tissue

This paper investigates the use of Lowicryl K4M in the embedding of apple tissue for immunocytochemistry. The localization of the extracellular protease of the apple pathogen, Nectria galligena Bres., in infected apple tissue by immunofluorescence and protein A-gold immunoelectron microscopy is also described. Infected apple tissue was fixed in 0.5% glutaraldehyde and 4% paraformaldehyde and embedded in Lowicryl K4M at 313 K. The protease was isolated and purified from rotted apple tissue by gel filtration and ion-exchange chromatography. Monospecific antibodies against the protease were raised in rabbits and purified by protein A-affinity chromatography. Incubation of apple tissue sections, infected with N. galligena, with the mono-specific antibody and tetramethylrhodamine isothiocyanate (TRITC)-conjugate, resulted in specific fluorescence of fungal hyphae and cytoplasm of apple cells. Similar localization of colloidal gold particles over hyphae and host cell cytoplasm was demonstrated employing protein A-gold immunochemistry. The low temperature embedding resin Lowicryl K4M appears to provide adequate morphological preservation of apple tissue and excellent retention of antigenicity. TRITC conjugates and protein A-gold may prove useful in immunocytochemical investigations of plant-pathogen interactions.     

In situ hybridization and immunofluorescence on resin‐embedded tissue to identify the components of Nissl substance

It is not clear whether the Nissl substance is present at the axon hillock. To clarify this gap in knowledge, we conducted in situ hybridization (ISH) on mouse brain tissue using 30‐μm cryostat and 1–3‐μm acrylic resin sections. Cryostat and rehydrated resin sections were exposed to digoxygenin‐labeled glutamic acid decarboxylase 1 sense and antisense riboprobes. Consecutive sections from tissue embedded in resin were subjected to the ribosomal protein L26 primary antibody to determine the distribution of the ribo/polysomes. ISH results from the antisense riboprobe in both cryostat and resin‐embedded tissue sections demonstrated an abundance of message in the neurons from the substantia nigra pars reticulate. In addition, the resin sections demonstrated hybridization signal in the axon hillock of some neurons. Immunofluorescence labeling of consecutive sections using an antibody to the most abundant ribosomal protein L26 confirmed their distribution in the cell body and the axon hillock of similar neurons. Compared with the 30‐μm cryostat sections, the most striking feature of ISH in the thinner resin (2–3 μm) sections was that there was a phenomenal improvement in the overall clarity and spatial resolution. Reexamination of the axon hillock when continuous with the cell body in cryostat sections revealed that the same message was also present, except it was overlooked initially because of overlapping cell populations in thick tissue slices. As ribosomes are a component of Nissl substance, we propose that the axon hillock, like other parts of the neuron, does contain Nissl substance. Microsc. Res. Tech. 2010. © 2009 Wiley‐Liss, Inc.     

Ultrasonic investigation of blood flow.

Ultrasound and the Doppler effect are used to measure blood velocity non-invasively in order to diagnose blood vessel disease. Intrusive lesions on arterial walls give rise to an alteration of the time-varying blood velocity waveform and local blood flow disturbance which are detected and measured using the envelope and width of the Doppler signal spectrogram respectively. Flow may also be imaged in colour superimposed on a grey-scale anatomical image allowing vessel narrowing and the accompanying flow disturbance to be visualized. Developments in three-dimensional imaging, angle tolerant velocity measurements and increased sensitivity using second harmonic backscatter from encapsulated-bubble contrast media ensure increasing use of this modality.