Epoxy Embedding Research Articles

Critical issues, processes and solutions in ISFET packaging

PurposeTo provide an insight of the intricacies of ion‐sensitive field‐effect transistor (ISFET) encapsulation and describe the presently available packaging solutions, indicating how the packaging requirements can be complied for various applications.Design/methodology/approachISFET packaging is a complete subject in itself. The paper includes examples of the different packaging strategies that have been offered by literature and company findings over the past few decades.FindingsISFET packaging has progressed from the initial epoxy embedding of the wire bonds and contact pads to the more sophisticated techniques capable of automation wherein moulds are made for epoxy coating or the chip is tightened between contacting parts using elastomer gaskets.Research limitations/implicationsThe emerging packaging technologies have succeeded in making chip packaging more a science than an art, and the new methods are capable of large‐scale manufacturing with greater precision.Practical implicationsPackaging solutions for demanding applications of ISFETs have been provided by the upcoming technologies.Originality/valueThe information provided in this paper is of immense value to researchers working on ISFET encapsulation.

Peripheral nerve morphometry: Comparison between manual and semi-automated methods in the analysis of a small nerve

Manual nerve morphometry has been usually described as tedious, time consuming, difficult to perform correctly and subject to many sources of errors. The above considerations might suggest that fully automated image analysis systems could be ideally programmed to analyze myelinated fibers. However, operator intervention is necessary to manually eliminate dark tissue elements such as pericytes and Schwann cell nuclei. The aims of the present study were to compare the manual and semi-automated techniques in the evaluation of a small nerve, comparing the most commonly used morphometric parameters for nerve descriptions. The aortic depressor nerves (ADN) of male Wistar rats ( N = 12) were prepared with conventional techniques for epoxy resin embedding. Manual morphometry was performed on photomicrographs using a digitizing tablet. Semi-automated morphometry was performed with the aid of computer software, on the same negative images used on the photographic procedure, which were scanned and digitized to a microcomputer. Our results show no differences between data obtained with both methods, for any of the evaluated parameters (area, perimeter, diameters, myelin sheath thickness, g ratio, distribution histograms). In conclusion, manual morphometry reproduced data obtained with semi-automated technique in a small nerve, with the advantages of being less-expensive and an affordable method.

P53 Electron Microscopical Localisation of CD61 (Beta3 Integrin) on Placental Syncytiotrophoblast Microvilli

Introduction Neonatal alloimmune thrombocytopenia (NAIT) due to maternal antibodies to the human platelet antigen (HPA)‐1a can occur in first pregnancies. The HPA‐1 polymorphism is located on platelet glycoprotein (GP)IIIa (CD61, beta3 integrin). It is hypothesised that besides fetal platelets, an additional source of fetal CD61 could cause alloimmunisation early in pregnancy. CD61 was earlier identified on placental villous trophoblasts by immunocytochemistry.Methods Whole‐mount immuno‐electron microscopy was used to study the ultrastructural localisation of CD61 on placental tissue. Fragments of first trimester (n = 5, 8–12 weeks gestational age) and term (n = 1) placental chorionic villi were incubated with anti‐CD61 or IgG1 isotype control, washed, incubated with 18 nm colloidal gold‐conjugated anti‐mouse IgG, washed, fixed in phosphate‐buffered glutaraldehyde and processed through to embedding in epoxy resin. 100 nm sections were prepared from the EM blocks; these were stained with uranyl acetate and lead citrate, and examined with a Phillips CM10 electron microscope. Positive control was term tissue stained with anti‐placental alkaline phosphatase (PLAP).Results Microvilli were observed on the apical membrane of some but not all of the villous syncytiotrophoblast (STB) surfaces. Formation of this brush border occurs during the first trimester to increase the surface area of these cells for optimal transfer of nutrients and gases from the adjacent maternal blood to the fetus. All specimens had some microvilli. After staining with anti‐CD61, gold particles were present on and between some microvilli of all specimens, either singly or in small groups. Stronger staining with more frequent occurrence of groups of gold particles was evident on microvilli of the term tissue incubated with anti‐PLAP. No gold particles were seen on the isotype control specimens.Conclusion From early on in pregnancy, CD61 is present on the surface of fetal STB in contact with maternal blood. This protein carrying the HPA‐1 polymorphism might be the cause of the initial immunising event that leads to NAIT.

A Simplified Method for Formulation of Epoxy Resin Embedding Media

Abstract In a recent paper on the revised formulation of Spurr low viscosity embedding medium with ERL 4221 the importance of maintaining an appropriate anhydride:epoxide (A:E) ratio was discussed. By understanding a few simple concepts about epoxy resin formulations and setting up a formulation table it is possible to create new resin mixtures with good sectioning properties and other desirable properties such as decreased viscosity and increased beam stability. Before starting a formulation you need to know the molecular weight of the anhydride and the WPE (weight per epoxide equivalent) of the epoxy resin component. The molecular weights and WPEs are usually printed on the bottle or can be obtained from the vendor. An A:E ratio of 0.7:1.0 -1.0:1.0 is used for most biological specimens. Increasing the A:E ratio results in a harder block; decreasing the A:E ratio results in a softer block. Table 1 shows a basic formulation spreadsheet where the molecular weights of the anhydrides and the WPEs of the epoxy resin components can be entered. The A:E ratio is entered under the anhydride for the molar ratio and the molar ratios of the epoxy components are entered under the epoxy components. The calculations are done as shown in each column and row.

Description of a new fossil Pseudogarypus (Pseudoscorpiones: Pseudogarypidae) with the use of X-ray micro-CT to penetrate opaque amber

Pseudogarypus pangaea Henderickx n. sp., a new fossil pseudoscorpion from Baltic amber, is described. Epoxy embedding and X-ray micro-CT are used to visualize the morphology of the single available specimen.

Use of Anti-fluorophore Antibody to Achieve High-sensitivity Immunolocalizations of Transporters and Ion Channels

We have discovered that the immunoreactivity of the fluorophore Alexa Fluor 488 survives glutaraldehyde and osmium tetroxide fixation and epoxy resin embedding and etching. We have developed new localization methods that for the first time take advantage of this property. The antigen is localized in cryosections using suitable primary antibody and an Alexa Fluor 488-conjugated secondary antibody. Cryosection fluorescence can be photographed for later correlation with electron microscopy (EM) findings. The sections are then further fixed with glutaraldehyde and OsO4, if desired and flat-embedded in epoxy resin. Semi-thin sections are etched completely with sodium ethoxide, whereas thin sections are partially etched. Alexa Fluor 488 is then localized with rabbit anti-Alexa Fluor 488 and goat anti-rabbit conjugated to Alexa Fluor 488 [light microscopy (LM)] or to colloidal gold (EM). A second antigen may also be localized using Alexa Fluor 568. When used without postfixation, these methods produce high-resolution semi-thin, or even thin, sections that retain a high level of fluorescence for LM observations. These methods allow highly sensitive immunolocalizations in tissue while preserving cell fine structure through traditional fixation and epoxy embedding. In demonstration of the methods, we describe the localization of the thiazide-sensitive sodium/chloride cotransporter and the epithelial sodium channel in rat kidney.

Arachidonic acid cascade and epithelial barrier function during Caco-2 cell differentiation

The small intestinal epithelium is a highly dynamic system continuously renewed by a process involving cell proliferation and differentiation. The intestinal epithelium constitutes a permeability barrier regulating the vectorial transport of ions, water, and solutes. Morphological changes during cell differentiation, as well as changes in the activity of brush-border enzymes and the expression of transport proteins, are well established. However, little is known about the arachidonic acid (AA) cascade underlying epithelial cell differentiation or its role in the development of epithelial barrier function. The main purpose of this study was to examine the activity of the high-molecular-weight phospholipases A(2) (PLA(2)) and cyclooxygenase (COX) pathway during differentiation, with particular emphasis on paracellular permeability. PLA(2) activity, AA release, COX-2 expression, prostaglandin E(2) (PGE(2)) production, and paracellular permeability were studied in preconfluent, confluent, and differentiated Caco-2 cell cultures. Our results show that Caco-2 differentiation induces a decrease in both calcium-independent PLA(2) activity and COX-2 expression and, consequently, a decrease in AA release and PGE(2) synthesis in parallel with a reduction in paracellular permeability. Moreover, the addition of PGE(2) to differentiated cells, at concentrations similar to those detected in nondifferentiated cultures, induces the disruption of epithelial barrier function. These results suggest that AA release by calcium-independent PLA(2), COX-2 expression, and subsequent PGE(2) release are important for the maintenance of paracellular permeability in differentiated Caco-2 cells.

There is no relation between the fascicular and the endoneural blood vessel areas on the cervical vagus nerve of normotensive rats

The aim of this study was to determine the existence of a relation between the fascicular area and total vessel area on the cervical vagus nerve of Wistar-Kyoto female rats. Animals aged 30, 180 or 360 days had their right vagus nerves prepared for epoxy resin embedding and light microscopy study. Semithin serial sections were studied every 100μm of the longitudinal extension of the nerve. The fascicular area and the total endoneural vessel area were obtained for each nerve segment (proximal, middle and distal) and compared between segments and ages. The relation between fascicular area and endoneural vessel area was accessed by linear regression analysis and correlation coefficient calculations. No significant relationship between the two variables was detected in all nerves studied. This suggests that the entrance of blood vessels to the endoneural space is not accompanied by an increase in the fascicular area and this may have implications on the fiber density calculations since, in the presence of large vessels, this density can be underestimated if the vessel area is not discounted. The endoneural vessel area increased from proximal to distal in all groups but got smaller with ageing, suggesting that older animals may be more susceptible to nerve ischemia.

Loss of PECAM-1 Function Impairs Alveolarization

The final stage of lung development in humans and rodents occurs principally after birth and involves the partitioning of the large primary saccules into smaller air spaces by the inward protrusion of septae derived from the walls of the saccules. Several observations in animal models implicate angiogenesis as critical to this process of alveolarization, but all anti-angiogenic treatments examined to date have resulted in endothelial cell (EC) death. We therefore targeted the function of platelet endothelial cell adhesion molecule, (PECAM-1), an EC surface molecule that promotes EC migration and has been implicated in in vivo angiogenesis. Administration of an anti-PECAM-1 antibody that inhibits EC migration, but not proliferation or survival in vitro, disrupted normal alveolar septation in neonatal rat pups without reducing EC content. Three-dimensional reconstruction of lungs showed that pups treated with a blocking PECAM-1 antibody had remodeling of more proximal branches resulting in large tubular airways. Subsequent studies in PECAM-1-null mice confirmed that the absence of PECAM-1 impaired murine alveolarization, without affecting EC content, proliferation, or survival. Further, cell migration was reduced in lung endothelial cells isolated from these mice. These data suggest that the loss of PECAM-1 function compromises postnatal lung development and provide evidence that inhibition of EC function, in contrast to a loss of viable EC, inhibits alveolarization.

Ultrastructural anatomy of the renal nerves in rats

The innervation within mammalian kidneys (intrinsic innervation) has been extensively described in the literature, particularly for rats. In contrast, few studies have provided a detailed description of the morphology of the extrinsic renal nerves leading to the kidneys. The extrinsic renal nerves are of interest because they are the site for recording of sympathetic nerve activity and electrical stimulation in functional studies. Thus, the aim of the present study was to describe the ultrastructural anatomy of the extrinsic renal nerves in rats. Male Wistar rats (N=6) were killed and the renal nerves were prepared for transmission electron microscopy by means of conventional techniques for epoxy resin embedding. Morphometry was carried out with the aid of computer software. The renal nerve average fascicular area was 5027±813 μm2. The average number of fibers in each nerve was 22±6 for the myelinated and 1236±115 for the unmyelinated. The average diameter of the myelinated fiber was 3.4±0.2 μm, distributed on a bimodal histogram, with peaks at 3.0 and 9.5 μm. The average unmyelinated fiber diameter was 0.6±0.1 μm, distributed on a unimodal histogram, with the peak at 0.6 μm. The relation between the myelin area and the axon diameter for the myelinated fibers was linear and positive (r2=0.85). This is the first study to describe ultrastructural morphometric details of the extrinsic renal nerves in rats, thus providing morphological basis for further experiments involving renal nerve pathologies, such as diabetes and hypertension. Support: FAPESP 04/01390-8, FAPESP 04/09139-2, CNPq 501230/2003-3 and FAEPA

The Niemann-Pick C1 protein in recycling endosomes of presynaptic nerve terminals

Niemann-Pick type C (NPC) disease is a fatal, neurodegenerative disorder caused in 95% of cases by loss of function of NPC1, a ubiquitous endosomal transmembrane protein. A biochemical hallmark of NPC deficiency is cholesterol accumulation in the endocytic pathway. Although cholesterol trafficking defects are observed in all cell types, neurons are the most vulnerable to NPC1 deficiency, suggesting a specialized function for NPC1 in neurons. We investigated the subcellular localization of NPC1 in neurons to gain insight into the mechanism of action of NPC1 in neuronal metabolism. We show that NPC1 is abundant in axons of sympathetic neurons and is present in recycling endosomes in presynaptic nerve terminals. NPC1 deficiency causes morphological and biochemical changes in the presynaptic nerve terminal. Synaptic vesicles from Npc1(-/-) mice have normal cholesterol content but altered protein composition. We propose that NPC1 plays a previously unrecognized role in the presynaptic nerve terminal and that NPC1 deficiency at this site might contribute to the progressive neurological impairment in NPC disease.

SEM Evaluation of Advanced Refractory Failures in Slagging Gasifiers

The SEM is an invaluable tool in the evaluation of advanced refractories and their failure. A reaction vessel?s refractory liner, at minimum, must protect the reaction vessel from elevated temperatures, corrosive slag and thermal cycling. To understand the failure mechanisms ARC staff had first to determine how an advanced chrome rich refractory was attacked by various components that make up a slag. Refractory cups were made from the refractory of interest and various compounds that can be found in a slag such as CaO, SiO2, Fe2O3, NaCl were placed into the test cups and fired for 24 hours at the required temperature with the desired atmosphere. The cups are prepared for examination by embedding in epoxy and cross sectioning. SEM examination revealed how various slag compositions attacked and penetrated the refractory. The slag could corrode, free refractory grains or react with the refractory and from a new compound. It was found that the only way to measure slag component penetration was with multiple elemental X-ray maps. SiO2 penetrated deeply and in many instances moved through the cup. The knowledge of slag refractory interactions gather during cup testing was applied to actual spent refractory from reaction vessels. Obtaining samples from themore » reaction vessel itself proved difficult due to time constraints imposed in relining. Samples were selected based on spent brick shape, color or location in the heap of spent refractory. Sample preparation affected the results dry, water or oil coolant during cutting may dissolve reaction products. The complex reactions between the slag and refractory made for very interesting and time consuming evaluation. Elemental X-ray maps at low and high magnification combined with point analysis aided in locating regions of interest. Crystals were found growing in voids and appear to be from vapor deposition. Other crystal structures are from the slag refractory interaction. Knowledge gathered from this and other supporting research resulted in a new patented refractory composition that resists slag penetration.« less

Effects of enamel matrix proteins on tissue formation along the roots of human teeth

Enamel matrix-derived proteins (EMD) are thought to trigger the formation of acellular extrinsic fibre cementum (AEFC), while other reports indicate that EMD may have osteogenic potential. The aim of the present study was to characterize the tissues developing on the root surface following application of EMD. Twelve human periodontitis-affected teeth, scheduled for extraction, were treated with EMD. Two to 6 weeks later, the teeth were extracted, demineralized and processed for embedding in acrylic and epoxy resins. New tissue formation was analysed by light and transmission electron microscopy. New tissue formation on the root was observed in the notch and on both scaled and unscaled root surfaces distant of the notch area in six defects. The newly formed tissues on the root were thick, collagenous, devoid of extrinsic fibres, and had an irregular surface contour. The presence of electron-dense, organic material in the collagenous matrix indicated at least partial mineralization. Embedded cells were numerous and the cells on the matrix surface were very large in size. Abundant rough endoplasmic reticulum and a prominent Golgi complex were evident. The presence of a split between the treated root surfaces and the newly formed tissue was a common observation, as was the presence of bacteria and host cells in the interfacial gap. Following treatment with EMD, a bone-like tissue resembling cellular intrinsic fibre cementum may develop on the root surfaces, instead of AEFC. Furthermore, EMD may both induce de novo formation of a mineralized connective tissue on scaled root surfaces and stimulate matrix deposition on old native cementum. Interfacial bonding appeared to be weak after 6 weeks of healing.

Morphological Measurement of Neurotoxic Injury in the Peripheral Nervous System: Preparation of Material for Light and Transmission Electron Microscopic Evaluation

An important method of assessing experimental neurotoxic injury is the pathologic examination of the nervous system. Methods for fixation, sampling, and preparation of peripheral nervous system tissues for critical pathological neurotoxicology studies are presented. Fixation of tissue is carried out using either perfusion-fixation of laboratory animals or immersion-fixation of dissected nerve segments. Dissection of the peripheral nervous system (from perfusion-fixed animals) is done to allow for multilevel sampling. Focus is on use of epoxy resin embedding tissue sections for optimal light microscopic resolution. Protocols for processing, sectioning, and staining for light and transmission electron microscopy are provided. A protocol for teasing and microscopic study of individual myelinated fibers is provided.

Lecithin-retinol acyltransferase is essential for accumulation of all-trans-retinyl esters in the eye and in the liver.

Lecithin-retinol acyltransferase (LRAT), an enzyme present mainly in the retinal pigmented epithelial cells and liver, converts all-trans-retinol into all-trans-retinyl esters. In the retinal pigmented epithelium, LRAT plays a key role in the retinoid cycle, a two-cell recycling system that replenishes the 11-cis-retinal chromophore of rhodopsin and cone pigments. We disrupted mouse Lrat gene expression by targeted recombination and generated a homozygous Lrat knock-out (Lrat-/-) mouse. Despite the expression of LRAT in multiple tissues, the Lrat-/- mouse develops normally. The histological analysis and electron microscopy of the retina for 6-8-week-old Lrat-/- mice revealed that the rod outer segments are approximately 35% shorter than those of Lrat+/+ mice, whereas other neuronal layers appear normal. Lrat-/- mice have trace levels of all-trans-retinyl esters in the liver, lung, eye, and blood, whereas the circulating all-trans-retinol is reduced only slightly. Scotopic and photopic electroretinograms as well as pupillary constriction analyses revealed that rod and cone visual functions are severely attenuated at an early age. We conclude that Lrat-/- mice may serve as an animal model with early onset severe retinal dystrophy and severe retinyl ester deprivation.

A comparison of resins for embedding teeth, with special emphasis on adaptation to enamel surface as evaluated by scanning electron microscopy

Objective: Eight commercially available resins, Epoxy Embedding Medium (“Epon”), Spurr, Epofix ®, Epo-Kwick ®, Epo-Thin ®, Technovit ® 9100 New, Unicryl™, and Vestopal W ®, all intended for embedding specimens for microscopy, were tested and compared for binding and adaptation to the enamel surface of human third molar crowns that had been embedded in them. Design: After sectioning, grinding and acid etching, the specimens were observed in the SEM. The presence or absence of a gap between enamel and resin was recorded together with the width of the gap, when present. Results: A pilot study, where gaps were observed before and after etching, indicated that the etching did not widen the gap appreciably. The resins could be divided into three groups according to gap width: “Epon” and Spurr performed best with a mean gap width of only 0.6 μm. Vestopal W ®, Epofix ®, Epo-Kwick ®, Epo-Thin ®, and Technovit ® 9100 New constituted a group of medium performance with mean gap widths of 2.3, 3.4, 3.8, 3.9 and 3.9 μm, respectively. Less satisfactory performance was observed for Unicryl™, with a mean gap width of 7.4 μm. Conclusions: It is concluded that “Epon” and Spurr should be chosen for high quality embedding of undecalcified teeth, while Epo-Kwick ® is an acceptable alternative if a faster procedure is needed.

Benzyldimethylamine (BDMA): Catalyst of Choice with Epoxy Embedding Media

Benzyldimethylamine (BDMA): Catalyst of Choice with Epoxy Embedding Media

Preparation of serial sections of arthropods using 2,2-dimethoxypropane dehydration and epoxy resin embedding under vacuum

Improved methods are described for anatomical investigation of small insects and other arthropods using serial semithin sections. The specimens were dehydrated with acidified 2,2-dimethoxypropane and embedded in ERL 4206 epoxy resin under vacuum. This procedure ensures good resin impregnation of thin, long body compartments and appendages. Furthermore, it produces excellent overall preservation of the specimen and its fragile anatomical structures. This procedure saves time and gives excellent results when sectioning difficult arthropod material. A continuous recording of serial semithin sections is possible when diamond knives are used.

Ossification in the human calcaneus: a model for spatial bone development and ossification

Perichondral bone, the circumferential grooves of Ranvier and cartilage canals are features of endochondral bone development. Cartilage canals containing connective tissue and blood vessels are found in the epiphysis of long bones and in cartilaginous anlagen of small and irregular bones. The pattern of cartilage canals seems to be integral to bone development and ossification. The canals may be concerned with the nourishment of large masses of cartilage, but neither their role in the formation of ossification centres nor their interaction with the circumferential grooves of Ranvier has been established. The relationships between cartilage canals, perichondral bone and the ossification centre were studied in the calcaneus of 9 to 38-wk-old human fetuses, by use of epoxy resin embedding, three-dimensional computer reconstructions and immunhistochemistry on paraffin sections. We found that cartilage canals are regularly arranged in shells surrounding the ossification centre. Whereas most of the shell canals might be involved in the nourishment of the cartilage, the inner shell is directly connected with the perichondral ossification groove of Ranvier and with large vessels from outside. In this way the inner shell canal imports extracellular matrix, cells and vessels into the cartilage. With the so-called communicating canals it is also connected to the endochondral ossification centre to which it delivers extracellular matrix, cells and vessels. The communicating canals can be considered as inverted 'internal' ossification grooves. They seem to be responsible for both build up intramembranous osteoid and for the direction of growth and thereby for orientation of the ossication centre.

A simple method for orienting silk and other flexible fibres in transmission electron microscopy specimens.

When microstructures are characterized by transmission electron microscopy (TEM), the interpretation of results is facilitated if the material can be sectioned in defined orientations. In the case of fibres, it is especially useful if transverse and longitudinal sections can be obtained reliably. Here we describe a procedure for orienting spider silk and other flexible fibres for TEM investigation. Prior to embedding in epoxy resin, the silk is wound around a notched support made from polyester film. No glue is required. After the silk and its supporting film have been embedded and the resin has been cured the film can be peeled away to reveal nearly perfectly orientated silk threads. Both transverse and longitudinal sections can then be cut with a microtome. The method can be extended to obtain sections at any intermediate orientation.