Modified paraffin wax for improvement of histological analysis efficiency

Paraffin wax is usually used as an embedding medium for histological analysis of natural tissue. However, it is not easy to obtain enough numbers of satisfactory sectioned slices because of the difference in mechanical properties between the paraffin and embedded tissue. We describe a modified paraffin wax that can improve the histological analysis efficiency of natural tissue, composed of paraffin and ethylene vinyl acetate (EVA) resin (0, 3, 5, and 10 wt %). Softening temperature of the paraffin/EVA media was similar to that of paraffin (50–60°C). The paraffin/EVA media dissolved completely in xylene after 30 min at 50°C. Physical properties such as the amount of load under the same compressive displacement, elastic recovery, and crystal intensity increased with increased EVA content. EVA medium (5 wt %) was regarded as an optimal composition, based on the sectioning efficiency measured by the numbers of unimpaired sectioned slices, amount of load under the same compressive displacement, and elastic recovery test. Based on the staining test of sectioned slices embedded in a 5 wt % EVA medium by hematoxylin and eosin (H&E), Masson trichrome (MT), and other staining tests, it was concluded that the modified paraffin wax can improve the histological analysis efficiency with various natural tissues. Microsc. Res. Tech. 73:761–765, 2010. © 2010 Wiley‐Liss, Inc.     

Incident light microscopy of surfaces of plastic embedded hard tissues

To eliminate sectioning artefacts, we devised a method for the preparation of surfaces of plastic embedded hard tissues and their examination by incident light fluorescence microscopy. Flat surfaces produced by cutting on a microtome or using a low-speed saw were stained with dyes in aqueous solutions. Best results were obtained in tissues embedded in glycol-methacrylate. This polymer allowed good penetration of the dyes and its optical properties render cutting defects invisible with the present method. The relationship between hard and soft tissues was well preserved and the integrity of the mineralized component maintained. Cellular details and the distinction between osteoid and bone could be clearly demonstrated. The procedure is simple, rapid and produces good results even in instances when histological sectioning is unsatisfactory.     

Light microscopic identification and semiquantification of polyethylene particles in methylmethacrylate and paraffin-embedded experimental bone implant specimens

The aim of this study was to evaluate the identification of polyethylene (PE) particles in relatively thick methylmethacrylate (MMA) sections widely used in bone implant research. The sensitivity and specificity were compared between decalcified paraffin-embedded oil red O (ORO) stained and MMA-embedded sections using polarized light. Furthermore, we introduced a grading system to semiquantify the level of PE particles in peri-implant tissue. Paraffin-embedded and MMA-embedded sections were compared concerning intra-observer agreement of the grading system. Moreover, the semiquantitative assessment of particle level was compared between the two section types. We found a sensitivity and specificity of polarized light of 100% for both paraffin ORO-stained and MMA sections. The intra-observer agreement on both types was comparable and acceptable. The ratings of differently processed blocks (MMA- and paraffin-embedded) originating from the same bone implant specimen showed good correlation. Our study showed that relatively thick MMA sections were just as suitable as ORO-stained paraffin sections concerning peri-implant PE particle migration analysis. MMA sections do not allow analysis at cellular level, but unbiased estimation of bone ingrowth into the implant surface based on stereological principles is possible.     

Staining plastic sections

Many of the difficulties of staining plastic embedded tissues for light and electron microscopy derive from physical exclusion of hydrophilic staining reagents by hydrophobic embedding media. Structures which stain most intensely with hydrophilic reagents usually contain less hydrophobic plastic than do non-staining structures. Such incomplete infiltration is apparently caused by exclusion of viscous, hydrophobic monomers by physically dense and/or well hydrated tissue elements. In keeping with this, generalized staining of tissues embedded in hydrophobic media does occur when hydrophobic reagents are used. Staining of plastic-free structures with single hydrophilic reagents or with sequences of such reagents, is, however, largely rate-controlled. The surprising similarity of hydrophilic and hydrophobic plastic embedding media is discussed. Limits of this simple model are explored, with a consideration of the roles of fixative and of monomer-tissue reactions.     

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 determination of thickness and surface relief in reembedded sections of an epoxy- and a melamine-resin containing ferritin as size standard

Chemical and physical data of two electron microscopic embedding media (the non-polar epoxy resin Epon 812 and the polar melamine resin Nanoplast FB 101) suggest that less kinetic energy must be applied for cutting a section from a Nanoplast block than from an Epon block of the same hardness and that, consequently, the cutting qualities of Nanoplast are better. To test this hypothesis, normal and extremely thin sections of Epon- and Nanoplast-embedded horse spleen ferritin micropellets were reembedded and resectioned for a determination of thickness and surface roughness. The ease with which extremely thin sections can be cut from the Nanoplast resin (8 nm versus 15 nm in Epon) and the smooth surface of these sections support the hypothesis that the cutting quality of an embedding material is determined primarily by its energy balance, i.e. by the kinetic energy which must be introduced for sectioning and the bonding energy which is released exothermically from a polymer while being sectioned.     

Thin sections for hard tissue histology: a new procedure

We describe a simple method by which thin sections (∼100 µm) from modern and archaeological teeth and bones can be obtained. A detailed embedding–cutting–mounting procedure is proposed, suggesting the use of a dental adhesive system, composite resins and conventional embedding resins, with the aims of improving the quality of the sections and substantially reducing the steps and time needed to prepare specimens for histological analysis. The introduction of this dental materials-based system allows an accurate positioning of the sample embedded inside the resin, prevents cracks and distortions of the section during the cutting phase and generally improves mounting sections on slides.     

Postembedment staining of complex carbohydrates: Influence of fixation and embedding procedures

In recent years technological advancements have led to improvements in ultrastructural cytochemical methods for localizing and characterizing complex carbohydrates. In particular the introduction of lectins with specific affinities for various sugars and sugar sequences as histochemical probes has increased knowledge concerning the cellular and subcellular distribution of glycoconjugates. Development of nonepoxy-based embedding materials has provided increased sensitivity compared to the earlier less specific methods and the current lectin methods for localizing sugar moieties. Postembedment staining based on the reactivity of functional groups present in sugars, such as hydroxyl groups, vicinal diol groups, carboxyl groups, and sulfate esters, requires specific conditions for tissue fixation and embedding. The same requirements pertain to staining based on lectin binding. The influence of fixation and embedment using older and newly developed embedding mixtures on the ultrastructural demonstration of complex carbohydrates is considered in this discussion. Fixation with osmium tetroxide and embedment in epoxy resins provides the least sensitive combination for the detection of the reactive groups of complex carbohydrates. The best ultrastructural demonstration of glycoconjugates is achieved when nonosmicated tissues are embedded in nonepoxy resins.     

An image-based methodology to establish correlations between porosity and cutting force in micromilling of porous titanium foams

Porous titanium foam is now a standard material for various dental and orthopedic applications due to its light weight, high strength, and full biocompatibility properties. In practical biomedical applications, outer surface geometry and porosity topology significantly influence the adherence between implant and neighboring bone. New microfabrication technologies, such as micromilling and laser micromachining opened new technological possibilities for shape generation of this class of products. Besides typical geometric alterations, these manufacturing techniques enable a better control of the surface roughness that in turn affects to a large extent the friction between implant and surrounding bone tissue. This paper proposes an image analysis approach for optical investigation of the porosity that is tailored to the specifics of micromilling process, with emphasis on cutting force monitoring. According to this method, the area of porous material removed during micromilling operation is estimated from optical images of the micromachined surface, and then the percentage of solid material cut is calculated for each tool revolution. The employment of the aforementioned methodology in micromilling of the porous titanium foams revealed reasonable statistical correlations between porosity and cutting forces, especially when they were characterized by low-frequency variations. The developed procedure unlocks new opportunities in optimization of the implant surface micro-geometry, to be characterized by an increased roughness with minimal porosity closures in an attempt to maximize implant fixation through an appropriate level of bone ingrowth.     

The use of an ultra-low viscosity medium (VCD/HXSA) in the rapid embedding of plant cells for electron microscopy

The VCD/HXSA ultra-low viscosity medium is characterized by a viscosity of only 20 cps at 298 K. This is extremely useful for rapid embedding schedules and facilitates cutting large sections even from difficult materials. The suitability of this medium for ultrastructural studies is tested with plant specimens ranging from soft, highly vacuolated parenchymatous tissue to hard thick-walled cells impregnated with a variety of substances or covered with mucilage. The results, when compared with those from similar materials infiltrated with Spurr's and Epon embedding media, show that the general preservation of the cellular structure is comparable for all the three media tested. In addition, embeddment in VCA/HXSA medium results in better preservation of some vacuolar features and in the reduction of plasmolysis.     

Stevenel's Blue, an excellent stain for optical microscopical study of plastic embedded tissues

Stevenel's Blue is a reliable, rapid, and clean, one-step polychromatic stain for 1 micron thick epoxy sections. The staining solution, originally used by L. Stevenel (1918) to stain human parasites, is made by adding diluted potassium permanganate (2%) to an aqueous solution of the methylene blue (1.3%) and redissolving the precipitate thoroughly, by boiling in water bath and filtering. Staining is carried out in a Coplin jar at 60 degrees C for approximately 10 minutes for tissues embedded in Epon 812 or Poly 812, or 20 minutes for tissues embedded in Spurr's medium. The sections are rinsed, air dried, and mouted in Permount. The staining solution is very stable, and does not tend to form precipitates on the tissue. The stain brings excellent histological differentiation to nuclear, cytoplasmic, and extracellular components. Incorporation of the stain by elements within each tissue varies from intense to light with a subtle gradation of intermediate shades of purple and blue tones. For most cell structures the density of the stain parallels the electron density of that structure as seen under the electron microscope. For example, nucleoli and heterochromatin stain in dark purple while euterochromatin appear in a light blue shade. In all cases, the embedding media remains unstained. The bond between Stevenel's Blue and the tissues is stable, remaining unaltered by the mounting medium. It is also resistant to time-fading.     

Feasibility of detecting trabecular bone around percutaneous titanium implants in rabbits by in vivo microfocus computed tomography

Objectives: The goal of this study was to examine the feasibility of in vivo imaging of trabecular bone around titanium implants by means of microfocus computed tomography (micro‐CT) and the use of rabbits for this purpose. Materials and Methods: Ten male rabbits type Hollander, received a titanium implant (1.7 mm diameter and 10 mm length) in the trabecular bone of the left tibia. Seven weeks later a micro‐CT scan was taken. Four rabbits were used to monitor potential harmful effects from X‐ray absorption until 4 weeks after scanning. A second group of six rabbits was used for testing the hypothesis that a good correlation exists between in vivo micro‐CT images and histological images of trabecular bone around titanium implants. The six rabbits were scanned and sacrificed immediately. The tibias were extracted and submitted to standard histological procedures. This resulted in a total of 12 histological sections and their corresponding 12 micro‐CT images. Bone area measurements were performed at the left and right side of the implant in three regions: 0–500, 500–1000 and 1000–1500 μm distance from the implant interface. Intra‐class correlations (ICC) were calculated between both techniques. Results: The four rabbits did not show any sign of radiodermatitis 4 weeks after scanning. In the micro‐CT images of the group of six rabbits, trabeculae are visible, but not well defined, due to the presence of noise in the image. The ICC for the right implant side were 0.44 for zone 0–500 μm, 0.48 for zone 500–1000 μm and 0.40 for zone 1000–1500 μm. The ICC for the left implant side could not be calculated. Conclusion: A low agreement was found between the bone measurements from histology and in vivo micro‐CT images. The use of the in vivo micro‐CT for trabecular bone imaging around metallic implants should be restricted to track tendencies in follow‐up studies.     

Assessment of In vivo behavior of polymer tube nerve grafts simultaneously with the peripheral nerve regeneration process using scanning electron microscopy technique

In this study, scanning electron microscopy (SEM) has been applied for instantaneous assessment of processes occurring at the site of regenerating nerve. The technique proved to be especially useful when an artificial implant should have been observed but have not yet been extensively investigated before for assessment of nerve tissue. For in vivo studies, evaluation of implant's morphology and its neuroregenerative properties is of great importance when new prototype is developed. However, the usually applied histological techniques require separate and differently prepared samples, and therefore, the results are never a 100% comparable. In our research, we found SEM as a technique providing detailed data both on an implant behavior and the nerve regeneration process inside the implant. Observations were carried out during 12‐week period on rat sciatic nerve injury model reconstructed with nerve autografts and different tube nerve grafts. Samples were analyzed with haematoxylin‐eosin (HE), immunocytochemical staining for neurofillament and S‐100 protein, SEM, TEM, and the results were compared. SEM studies enabled to obtain characteristic pictures of the regeneration process similarly to TEM and histological studies. Schwann cell transformation and communication as well as axonal outgrowth were identified, newly created and matured axons could be recognized. Concurrent analysis of biomaterial changes in the implant (degradation, collapsing of the tube wall, migration of alginate gel) was possible. This study provides the groundwork for further use of the described technique in the nerve regeneration studies. SCANNING 35: 232‐245, 2013. © 2012 Wiley Periodicals, Inc.     

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.     

Effects of different epoxies on avoiding wrinkles in thin sections of botanical specimens

Fold or wrinkles appear frequently in thin sections cut from botanical material embedded in epoxy resin such as Epon 812 (Polysciences) or EM-bed-812 (Electron Microscopy Sciences). Small wrinkles may occur more or less perpendicular to the cutting direction and mostly across the cell wall, causing problems in the examination of the ultrastructure. We studied the occurrence of wrinkles and folds by methodological sectioning, with regard to block hardness, use of Formvar-support film, and chloroform vapor. Methodological sectioning or changing the block hardness did not reduce the occurrence of wrinkles in sections. Chloroform vapor improved the result as did also the use of uncoated grid. Wrinkles and folds could be avoided completely by embedding the specimens in LX-112 (Ladd) or ERL-4206 (Polysciences) low-viscosity epoxy resins, in which case Formvar-coated grids could be used, and chloroform vapor was not necessary.     

MicroCT examination of human bone specimens: effects of polymethylmethacrylate embedding on structural parameters

X‐ray microtomography permits the nondestructive investigation of trabecular and cortical bone specimens without special preparation of the sample. To do a quantitative characterization, the cross‐section images have to be binarized, separating bone from nonbone. For this purpose, a widely used method is uniform thresholding. However, for commonly available microtomography scanners which use a polychromatic X‐ray source, it is unclear what effect the surrounding medium (e.g. air, saline solution, polymethylmethacrylate) has on the threshold value used for the binarization. In the literature an easy procedure to find the optimal uniform threshold value for a given acquisition condition is reported. By applying this procedure, the present work investigated whether a microtomography scan of trabecular bone samples in air or embedded in polymethylmethacrylate gave the same results in terms of structural parameters. The gold standard, that is, histological sections, was used as a reference. Two fixed threshold values were found, one for the microtomography scans performed in air and one for the scans with the same samples embedded in polymethylmethacrylate. These were applied on the correspondent microtomography images for the estimation of structural parameters, such as bone volume fraction, direct trabecular thickness, direct trabecular separation and structure model index. Paired comparisons were made in bone volume fraction between histological sections and microtomography cross‐sections for the same bone samples scanned first in air and then embedded in polymethylmethacrylate, by which no significant differences were found. Paired comparisons were also made in bone volume fraction, direct trabecular thickness, direct trabecular separation and structure model index for the same samples over volumes of interest of 4 × 4 × 4 mm³ between microtomography scans in air and scans with the samples embedded in polymethylmethacrylate. Neither these comparisons showed significant differences. This leads to the conclusion that structural parameters estimated by microtomography for human trabecular bone samples scanned either in air or embedded in polymethylmethacrylate are not affected by the surrounding medium (i.e. presence or absence of polymethylmethacrylate), provided that the corresponding optimal threshold value is applied for each acquisition condition.     

线切蓝宝石晶片面形质量评价方法的试验研究

多线切割广泛应用于硬脆材料的切片加工,线切后晶片的面形质量会对后续研磨抛光加工工艺产生较大影响。提出了一种针对线切片面形质量的评价指标：线切片最小加工余量(MMA);设计出MMA的测量原理,并给出具体实施方法;分析了不同工艺参数下线网不同位置处MMA的变化规律。试验结果表明,提出的评价指标能够更好地反映线切加工工艺参数对晶片面形质量的影响规律。     

Friable rock embedding in methyl methacrylate for thin-sectioning in geological preparations

In most cases, technical problems prevent the production of good geological thin-slabs from friable rocks such as muds, sands or decomposed granites. Usual impregnation methods using Araldite or epoxies lead to incompletely impregnated blocks. Furthermore the excessive heat occurring during polymerization alters the micromorphology of heat sensitive minerals. Methyl methacrylate (MMA) is a widely used plastic for bone embedding. We have developed a new procedure using vacuum infiltration with MMA in a cold environment. Wafers from friable rocks are easily obtained and can be processed in the same way as those of firm and coherent rocks, without grain loss nor mineral heat alterations.     

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.