Glycol methacrylate embedding of bone and cartilage for light microscopic staining

A method is described for embedding bone and cartilage in glycol methacrylate (GMA) for light microscopy. Dehydration-infiltration of the hard tissue is with aqueous GMA solutions minimizing solvent and dehydration artefact, and polymerization is by UV light in the cold to minimize thermal damage. Over fifty stains, enzyme localizations and related histochemical methods for 0·5–3·0 μm thick sections of GMA embedded tissue are listed. The increased resolution plus the localization of cellular and extracellular chemical moieties is now easier and more accurate providing an improved method for the study of the musculo-skeletal system by light microscopic histochemistry.     

Quantitative studies on the preservation of choline and ethanolamine phosphatides during tissue preparation for electron microscopy. II. Other preparative methods

The loss of ¹⁴C-ethanolamine- and ³H-choline-labelled phospholipids from rat liver during preparation for electron microscopy by some less frequently used processing methods has been examined. Permanganate and formaldehyde-potassium dichromate fixation followed by Araldite embedding were investigated and five procedures involving embedding in water-miscible methacrylates (GMA). These procedures included a conventional method of dehydration and embedding in GMA, a low-temperature GMA embedding method, dehydration with ethylene glycol, freeze drying and freeze substitution. These results are compared with those obtained after conventional tissue preparation (presented previously, Cope & Williams, 1969). Formaldehyde-potassium dichromate compared favourably with the conventional procedures for the preservation of both phosphatides, especially phosphatidyl ethanolamine. Permanganate fixation was much less effective. Severe loss of both phosphatides occured after freeze drying and freeze substitution in glutaraldehyde-alcohol. GMA is shown to be a more potent phospholipid solvent than ethanol under the conditions employed. Low-temperature embedding reduced the loss of phosphatidyl choline during embedding. Results obtained by scintillation counting were confirmed by grain counts on thick-section autoradiographs. No direct relationship between extraction and the electron-microscopic appearance of membranes was discernible. It is believed that membrane prominence is largely dependent upon the electron density of the surrounding cytoplasm rather than on the degree of phospholipid extraction.     

Histologic investigation of glycol methacrylate embedded chick embryonic tissue.

Specific light microscopic investigations (i.e. histochemical) of early embryonic material have always been beset by difficulties in processing and obtaining tissue sections of good quality. The advent of glycol methacrylate (GMA) as an embedding medium now provides a means to overcome these inherent problems with this tissue. Investigations were carried out to assess the histological results produced by different fixatives and times of fixation of GMA embedded 5-day chick embryonic tissue. Optimum cellular preservation of all tissues occurred following fixation in a mixture of acetic acid, 95% ethanol and neutral buffered formalin (AAF). With the procedures described in this study, a new method is available for more comprehensive examination of all types of early embryonic material.     

Quantitation of shrinkage during preparation for scanning electron microscopy: Human lung

Human lung tissue is found to shrink considerably with preparation for SEM. Fifty-one blocks of glutar-aldehyde-fixed and inflated lung, approximately 2.5 cm × 2.5 cm × 1 cm, shrank a mean of 19% (± 4.0% SD) linear dimension through post fixation, dehydration and critical point drying. Shrinkage with fixation was not measured. Blocks of lung were observed to shrink equally in length (L) and width (W), L = 19.4% ± 2.7 SD, W = 19.0% ± 4.0 SD. Final shrinkage was the same whether samples were dehydrated in acetone or ethanol, although with acetone more of the shrinkage occurred during the dehydration process and less occurred during critical point drying.     

The biological reality of the interlacunar network in the embryonic,cartilaginous, skeleton: A thiazine dye/absolute ethanol/LR White resin protocol for visualizing the network with minimal tissue shrinkage

Third toe phalanges of chicks aged 8–13 days in ovo and 7-day post-natal rat femoral growth plate were examined to determine whether the interlacunar network (IN), a structure with no lipoprotein membrane component or cytoplasmic organelles, is a genuine component of young growth cartilage. In chick phalanges dehydrated by 70% (v/v) ethanol and LR White resin, variable metachromatic staining of the interlacunar network by toluidine blue and red staining by picro-Sirius red indicate the presence of glycosaminoglycans and collagen. The network in phalanges dehydrated by 80% (v/v) ethanol appears little different; however, the network is much less widely detectable in phalanges dehydrated by 90% (v/v) ethanol and, after dehydration by absolute ethanol, is almost completely undetectable. In contrast, when the young cartilage is permeated by a thiazine dye such as toluidine blue, using a solution of dye in the aldehyde fixative, the network is widely detectable, following dehydration by absolute ethanol, both in chick phalanges and in rat growth plate. Comparison of projected areas shows that the extent to which whole chick feet are found to have shrunk, by the time that they are photographed under LR White resin, is determined principally by the extent of dehydration, by 70% (v/v) or absolute ethanol; post-shrinkage areas are 33% or 35% of areas measured in buffer for 70% (v/v) ethanol/LR White resin and 71% or 75% for absolute ethanol/LR White resin (the higher value in each is for the toluidine blue treatment). The network is thus present in radically shrunk tissue, but, significantly, is also fully represented in tissue shrunk by only a conventional margin and is therefore not produced as an artefact by exceptional tissue shrinkage as has been suggested.     

A quantitative study of the preparation of rabbit aortic endothelial cells for scanning electron microscopy

Quantitative studies were done with the scanning electron microscope (SEM) on aortic endothelial cells from ten rabbits. Of these, five were plastic casts and five were dehydrated with three different, but standard, techniques. The results indicated that all forms of dehydration caused significant shrinkage artefacts and that these were different in different directions in both the thoracic and abdominal aorta. The greatest shrinkage was found with the critical point drying technique, 45% in the abdominal aorta and 31% in the thoracic aorta. In the abdominal aorta this shrinkage was mainly due to a shrinkage in length (36%) rather than a shrinkage in width (15%). In comparison, in the thoracic aorta critical point drying resulted in a 15% shrinkage in length and a 19% shrinkage in width. Air drying and alcohol dehydration caused considerable shrinkage (29% and 18% respectively in the thoracic aorta, 29% and 36% respectively in the abdominal aorta). Directional differences were also found with these techniques, for instance alcohol dehydration in the thoracic aorta resulted in 0% shrinkage in length and 18% shrinkage in width.     

A synchrotron X-ray study of the changes occurring in the corneal stroma during processing for electron microscopy

Using a high-intensity synchrotron X-ray source, the structural changes occurring in the corneal stroma were monitored during each stage of several different processing runs for the transmission electron microscope (TEM) and scanning electron microscope (SEM). The parameters studied were interfibrillar spacing, intermolecular spacing, D-periodicity and fibril diameter. The processing schedule that produced the least changes in spacings for TEM specimens involved extended fixation in glutaraldehyde followed by low-temperature embedding in Lowicryl K4M resin. However, interfibrillar material was better preserved after embedding in LR White resin or Nanoplast. Almost every processing stage for electron microscopy produced significant changes in one or more structural parameters in the cornea. Glutaraldehyde fixation significantly increased the intermolecular spacings, while resin infiltration and resin polymerization each resulted in shrinkage of all the spacings monitored. Critical-point drying for SEM specimens resulted in considerable shrinkage in all three spacings, but was still preferable to air drying, which caused reduction in the order of the fibril packing, resulting in loss of the interfibrillar X-ray pattern. Perhaps the most drastic effect was caused by post-fixation in osmium tetroxide, which resulted in loss of the intermolecular pattern, and also increased the amount of shrinkage in the interfibrillar spacings and the D-periodicity which occurred during later stages of processing.     

Staining sections of water-miscible resins

Penetration of hydrophilic acid and basic dyes into sections cut from glycol methacrylate (GMA)-embedded tissues was studied; as were the effects on such staining of superficial coatings of thin layers of GMA. Dye size was a major factor in controlling penetration of resin and staining of tissues. ‘Large’ dyes (>1000 Da) entered GMA very slowly, and only stained those tissue components poorly infiltrated by resin. ‘Small’ dyes (< 550 Da) penetrated GMA readily, and stained tissue components whether or not they were resin-infiltrated. Dyes of intermediate size penetrated the resin, but the staining of resin-infiltrated tissue elements was slow. Background staining of resin also varied with dye size. Large dyes gave no staining of GMA. Small dyes did, but were readily removed by water washing. Dyes of intermediate size penetrated resin slowly, and once inside were lost slowly. This gave background staining which required use of the plasticizing solvent ethanol for its removal. Increases in resin cross-linking also reduced staining rates. As a consequence, it is possible to predict the probable suitability, or otherwise, of various staining reagents proposed for use with GMA sections; and also the probable influences of histoprocessing on stain penetration. In particular it is suggested that penetration of colloidal metals and macromolecular reagents (e.g. labelled antibodies and lectins) will be limited to resin-free structures, and to the surface of resin sections. The use of superficial GMA coatings as convenient semipermeable membranes for enzyme histochemistry is also noted.     

Improved method of producing satisfactory sections of whole eyeball by routine histology

To overcome the loss of structural integrity when eyeball sections are prepared by wax embedding, we experimentally modified the routine histological procedure and report satisfactorily well‐preserved antero‐posterior sections of whole eyeballs for teaching/learning purposes. Presently histological sections of whole eyeballs are not readily available because substantial structural distortions attributable to variable consistency of tissue components (and their undesired differential shrinkage) result from routine processing. Notably, at the dehydration stage of processing, the soft, gel‐like vitreous humor considerably shrinks relative to the tough fibrous sclera causing collapse of the ocular globe. Additionally, the combined effects of fixation, dehydration, and embedding at 60°C renders the eye lens too hard for microtome slicing at thicknesses suitable for light microscopy. We satisfactorily preserved intact antero‐posterior sections of eyeballs via routine paraffin wax processing procedure entailing two main modifications; (i) careful needle aspiration of vitreous humor and replacement with molten wax prior to wax infiltration; (ii) softening of lens in trimmed wax block by placing a drop of concentrated liquid phenol on it for 3 h during microtomy. These variations of the routine histological method produced intact whole eyeball sections with retinal detachment as the only structural distortion. Intact sections of the eyeball obtained compares well with the laborious, expensive, and 8‐week long celloidin method. Our method has wider potential usability than costly freeze drying method which requires special skills and equipment (cryotome) and does not produce whole eyeball sections. Microsc. Res. Tech. 77:138–142, 2014. © 2013 Wiley Periodicals, Inc.     

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.     

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.     

Lipid losses during processing of cardiac muscle for electron microscopy

The lipid retaining properties of several methods of processing tissue for electron microscopy (EM) have been assessed quantitatively. Guinea-pig hearts were perfused in vitro at 37°C with ³H-oleic acid bound to albumin. The hearts were fixed by perfusion with 4% glutaraldehyde in 0.1 M cacodylate buffer. Pieces of left ventricle and interventricular septum were removed., weighed and processed for EM. The fluids used at each stage of processing were monitored for loss of radioactive lipid by scintillation counting. Lipids were extracted from the processed tissue immediately before the embedding stage using a mixture of chloroform: methanol (2:1 v/v). Counts from processed tissue were compared with counts from tissue extracted directly after perfusion fixation in order to monitor subsequent losses during processing. A modified version of Epon processing, omitting 100% ethanol, acetone or propylene oxide, gave a lipid retention of only 20.6%. The addition of para-phenylenediamine to the procedure did not improve the retention although this has been shown to be a useful stain for intracellular lipid. Water soluble Durcupan which does not involve ethanol or acetone dehydration has an average retention of 63% with 100% recovery while the lesser known polymer GACH, a mixture of glutaraldehyde and carbohydrazide used both for dehydration and embedding showed a lipid retention of 82% of the counts recovered although recovery was only 69%. An attempt was made to determine which classes of lipids were present in the tissue after perfusion fixation using thin layer chromatography. It was found that the presence of any of the processing fluids affected the polarity of the lipids and their rates of migration on thin layer plates.     

Floating on a water bath and mounting glycol methacrylate and hydroxypropyl methacrylate sections influence final dimensions

This paper reports the dimensional changes occurring in the different steps of the histoprocessing of tissues for light microscopy. Two water-miscible methacrylates used for embedding, namely 2-hydroxyethyl methacrylate and 2-hydroxypropyl methacrylate, were investigated. It was found that during stretching on the water bath and in the mounting step considerable size changes occur, which are of the same magnitude as during the dehydration step of histoprocessing. The final dimensions of the sections and of microscopic images are dependent on the response to surface tension at the water surface and mounting of the glycol and hydroxypropyl methacrylate sections, respectively. Between the two resins under study, significant differences in the size of the resin sections, with and without embedded liver tissue, were found. It is shown that the temperature at which the sections are mounted is of great importance. These observations indicate the importance of standardizing histotechniques if morphometry is to be applied.     

Scanning electron and light microscope correlation of individual human bone marrow cells before and after culture in nutrient agar.

This study was undertaken with the aim of identifying the different cell types found in human bone marrow by examining their surface morphology. In an attempt to obtain a homogeneous cell population, cells were both fractionated by discontinuous albumin density gradient centrifugation (DADGC) and selectively grown in nutrient agar. Both cell preparations underwent the critical point drying technique before examination under both the scanning electron microscope (SEM) and subsequently the light microscope (LM). When the SEM image of individual cells was compared with the corresponding LM image, it was not easy to identify the different cell types, because of the shrinkage and distortion that occurred during their preparation. The shrinkage observed under the SEM amounted to a 45% reduction in mean cell diameter. This shrinkage was confirmed by comparing the SEM and LM images of the same cell. Although shrinkage occurred throughout the dehydration sequence, critical point drying was responsible for a 25% reduction in mean cell diameter. Furthermore, direct observation under LM of fixed cells drying in air from ethanol, revealed visible contraction of the cell and distortion of the cell membrane. We assume that a similar morphological change occurred during critical point drying. We conclude that the shrinkage and distortion, caused by the dehydration process involved in SEM preparation, severely limit the value of a study of surface morphology by SEM in the identification of the different cell types found in human bone marrow.     

Ultrastructural effects of perfomic acid oxidation of epoxy-embedded tissue with and without early uranyl staining.

Epoxy-embedded blocks of glutaradehyde and OSO4 fixed adenohypophyseal tissue were immersed in performic acid, rinsed and sectioned for electron microscopy. Sections exhibited a general image intensity equivalent to control sections but their stainability with both uranyl acetate and lead citrate stains was increased. It was concluded that osmium was not removed by performic acid oxidation. Membranes of endoplasmic reticulum and the Golgi apparatus were not visible, and other components (acidophil granules, in particular) were markedly distored in shape after oxidation. Such changes, however, were not evident in specimens previously exposed to uranyl acetate during ethanol dehydration.     

Cell and organelle shrinkage during preparation for scanning electron microscopy: effects of fixation, dehydration and critical point drying.

The critical point drying method of preparing samples for scanning electron microscopy is associated with a variable amount of specimen shrinkage. We studied the causes of this phenomenon is isolated mouse hepatocyte nuclei and in human erythrocytes and found that the critical point drying process itself caused most of the shrinkage that we observed (a 25-30% reduction in diameter in both specimens). Glutaraldehyde fixation and ethanol dehydration caused only minimal size reduction, prior to critical point drying. Substitution of an inert (ethylene glycol-ethylene glycol monethyl ether) dehydration technique did not alter the final result. Previous studies in our laboratory using high resolution SEM and correlative transmission microscopy of isolated nuclei have demonstrated that the shrinkage represents a miniaturization of the organelles in which all structural components retain their usual relationships.     

Measurements of critical point shrinkage of glutaraldehyde fixed mouse liver

The dimensional changes of small cubes of glutaraldehyde fixed mouse liver tissue were measured using a light microscope image projected into the Quantimet 720 Image Analysing computer system. The dimensional changes occurring in the critical drying bomb could be followed at all stages when violent turbulence was not occurring. The results show that liver tissue blocks shrink in four stages whilst in the critical point drying bomb: (1) during substitution of the intermediate solvent with the transitional fluid; (2) when the transitional fluid is warmed above the critical temperature; (3) when the transitional fluid, now a gas, is allowed to escape from the CPD bomb – the rate of shrinkage increasing as atmospheric pressure is approached; (4) at atmospheric pressure when all the gas has been allowed to escape from the bomb. Taken together with the authors' previous findings, it would seem that substantial shrinkage of animal soft tissue specimens must occur whilst they are undergoing “critical point drying”. This fact should be taken into account when interpreting SEM images of CPD tissues.     

Measurement of dimensional stability of heat treated southern red oak (Quercus falcata Michx.)

The objective of this study was to investigate the effect of heat treatment on physical and mechanical properties including oven-dry density, weight loss, swelling, shrinkage, and hardness of southern red oak (Quercus falcata Michx.). The samples were treated at a temperature level of 190 °C for 3 h and 8 h. After heat treatment of the specimens, their dimensional stability in the form of swelling and shrinkage were determined by soaking them in water for 2 and 24 h. Hardness of samples as function of heat treatment was also measured using Janka hardness (ASTM D 1037–12). Tangential, radial and longitudinal swelling values of the samples exposed to 8 h heat treatment and soaked in water for 2 h were 0.245%, 0.236%, 0.098%, respectively. Corresponding values for the control samples were 0.504%, 0.455%, 0.135%. Overall hardness of the specimens was adversely influenced due to heat treatment. Based on the findings in this study shrinkage and swelling of the samples improved as a result of heat exposure. It appears that heat treatment would be a viable method to enhance dimensional stability of red oak for more effective utilization where enhanced hygroscopicity of such species is desired.     

Pronounced loss of cell nuclei and anisotropic deformation of thick sections

The local deformation and variations in section thickness are studied in 100-μm thick vibratome sections of well-fixed human brain tissue. During processing, including drying on glass slides, the section thickness is reduced to less than half, but close to the edges there is less shrinkage of the section thickness. Close to both surfaces there is a pronounced reduction in the number of neuronal nucleoli. At the scale of the original section, the upper 15 μm and the lower 10 μm are depleted. The loss is most pronounced at the upper surface, which is unprotected during processing. In the central 70% of the section height, where one would ordinarily use an optical disector for sampling, there is no indication of non-uniform shrinkage. The simplest explanation for the observed loss of nucleoli is that all cells opened by the knife may lose their nuclei across an unprotected section surface. The observations do not generalize to other tissues and other preparation techniques, but illustrate the magnitude of some of the problems for uniform sampling and unbiased estimation in very thick sections. The uniform optical disector sampling of nucleoli in thick sections, as opposed to that of cell nuclei, raises a special problem, which is discussed briefly.     

Tissue shrinkage and unbiased stereological estimation of particle number and size*

This paper is a review of the stereological problems related to the unbiased estimation of particle number and size when tissue deformation is present. The deformation may occur during the histological processing of the tissue. It is especially noted that the widely used optical disector may be biased by dimensional changes in the z‐axis, i.e. the direction perpendicular to the section plane. This is often the case when frozen sections or vibratome sections are used for the stereological measurements. The present paper introduces new estimators to be used in optical fractionator and optical disector designs; the first is, as usual, the simplest and most robust. Finally, it is stated that when tissue deformation only occurs in the z‐direction, unbiased estimation of particle size with several estimators is possible.