Dehydration Of Specimens Research Articles

Scanning Electron Microscopy.

Scanning electron microscopy (SEM) remains distinct in its ability to allow topographical visualization of structures. Key elements to consider for successful examination of biological specimens include appropriate preparative and imaging techniques. Chemical processing induces structural artifacts during specimen preparation, and several factors need to be considered when selecting fixation protocols to reduce these effects while retaining structures of interest. Particular care for proper dehydration of specimens is essential to minimize shrinkage and is necessary for placement under the high-vacuum environment required for routine operation of standard SEMs. Choice of substrate for mounting and coating specimens can reduce artifacts known as charging, and a basic understanding of microscope settings can optimize parameters to achieve desired results. This article describes fundamental techniques and tips for routine specimen preparation for a variety of biological specimens, preservation of labile or fragile structures, immune-labeling strategies, and microscope imaging parameters for optimal examination by SEM. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Chemical preparative techniques for preservation of biological specimens for examination by SEM Alternate Protocol 1: Practical considerations for the preparation of soft tissues Alternate Protocol 2: Removal of debris from the exoskeleton of invertebrates Alternate Protocol 3: Fixation of colonies grown on agar plates Alternate Protocol 4: Stabilization of polysaccharide structures with alcian blue and lysine Alternate Protocol 5: Preparation of non-adherent particulates in solution for SEM Support Protocol 1: Application of thin layer of adhesive on substrate to improve adherence Support Protocol 2: Poly-L-lysine coating specimen substrates for improved adherence Support Protocol 3: Microwave processing of biological specimens for examination by SEM Basic Protocol 2: Critical point drying of specimens Alternate Protocol 6: Chemical alternative to critical point drying Basic Protocol 3: Sputter coating Alternate Protocol 7: Improved bulk conductivity through "OTOTO" Basic Protocol 4: Immune-labeling strategies Alternate Protocol 8: Immune-labeling internal antigens with small gold probes Alternate protocol 9: Quantum dot or fluoronanogold preparations for correlative techniques Basic Protocol 5: Exposure of internal structures by mechanical fracturing Basic Protocol 6: Exposure of internal structures of tissues by fracturing with liquid nitrogen Basic Protocol 7: Anaglyph production from stereo pairs to produce 3D images.

Cold temperature/Biodur® /S10/von Hagens'-Silicone plastination technique.

Plastination is a late 20th century preservation methodology which replaces tissue fluid within a specimen with a curable polymer, such as silicone. Plastination yields superb, beautiful, well-preserved specimens each with their own unique qualities. Silicone polymer is used around the world to preserve macroscopic cadavers or portions/organs thereof. Plastination was conceived by Dr. Gunther von Hagens, Universität Heidelberg, Heidelberg, Germany prior to 1977. Silicone polymer was the primary polymer which emerged initially for plastination. The Biodur® line of silicone polymer and additives was chosen and manufactured because it has consistently produced the best plastinates since the inception of plastination. Since the discovery of silicone, generic and similar silicone polymers are known and used around the World by many industries and used in numerous products. The plastination process has four steps: Specimen preparation, Specimen dehydration and degreasing, Vacuum-forced impregnation of specimens and Specimen hardening.

Effects of achilles tendon immersion in saline and perfluorochemicals on T2 and T2*

To determine if immersion of Achilles tendon segments into various solutions improved qualitative delineation of tendon and affected quantitative MR values of T2 and T2*. Six Achilles tendons were dissected, sectioned (proximal, midportion, and distal tensile pieces) and imaged at 3T both at baseline in air and after immersion into saline, Fomblin, and perfluorooctyl bromide (PFOB), respectively, for 24 h. Blinded readers qualitatively assessed the delineation of tendon boundaries and quantitatively Carr-Purcell-Meiboom-Gill (CPMG) T2 and ultrashort echo time (UTE) T2* was calculated. Comparison between images obtained in air and in solution was made. On qualitative evaluation, all images obtained in air had larger air-tissue susceptibility effects. Mean T2 values of saline, Fomblin, and PFOB groups were 16.1 ± 3.7, 16.6 ± 2.9, and 18.8 ± 2.6 ms at baseline in air, and 14.8 ± 4.6, 15.9 ± 3.0, and 17.7 ± 3.0 ms after immersion in the fluid, respectively. Mean T2* values of saline, Fomblin, and PFOB groups were 2.0 ± 0.8, 1.6 ± 0.5, and 1.5 ± 0.5 ms at baseline in air, and 2.1 ± 0.5, 1.6 ± 0.5, and 1.4 ± 0.5 ms after immersion in the fluid, respectively. There was no significant effect of immersion or fluid type on measured T2 or T2* (P > 0.1). These results validate the continued use of these solutions to prevent tendon specimen dehydration and to minimize susceptibility effects.

Graphene as a protein crystal mounting material to reduce background scatter.

The overall signal-to-noise ratio per unit dose for X-ray diffraction data from protein crystals can be improved by reducing the mass and density of all material surrounding the crystals. This article demonstrates a path towards the practical ultimate in background reduction by use of atomically thin graphene sheets as a crystal mounting platform for protein crystals. The results show the potential for graphene in protein crystallography and other cases where X-ray scatter from the mounting material must be reduced and specimen dehydration prevented, such as in coherent X-ray diffraction imaging of microscopic objects.

Combination of microscopic techniques reveals a comprehensive visual impression of biofilm structure and composition

Bacterial biofilms are imaged by various kinds of microscopy including confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). One limitation of CLSM is its restricted magnification, which is resolved by the use of SEM that provides high-magnification spatial images of how the single bacteria are located and interact within the biofilm. However, conventional SEM is limited by the requirement of dehydration of the samples during preparation. As biofilms consist mainly of water, the specimen dehydration might alter its morphology. High magnification yet authentic images are important to understand the physiology of biofilms. We compared conventional SEM, Focused Ion Beam (FIB)-SEM and CLSM with SEM techniques [cryo-SEM and environmental-SEM (ESEM)] that do not require dehydration. In the case of cryo-SEM, the biofilm is not dehydrated but kept frozen to obtain high-magnification images closer to the native state of the sample. Using the ESEM technique, no preparation is needed. Applying these methods to biofilms of Pseudomonas aeruginosa showed us that the dehydration of biofilms substantially influences its appearance and that a more authentic biofilm image emerges when combining all methods.

Scanning electron microscopy.

Scanning electron microscopy (SEM) remains distinct in its ability to allow topographical visualization of structures. Key elements to consider for successful examination of biological specimens include appropriate preparative and imaging techniques. Chemical processing induces structural artifacts during specimen preparation, and several factors need to be considered when selecting fixation protocols to reduce these effects while retaining structures of interest. Particular care for proper dehydration of specimens is essential to minimize shrinkage and is necessary for placement under the high-vacuum environment required for routine operation of standard SEMs. Choice of substrate for mounting and coating specimens can reduce artifacts known as charging, and a basic understanding of microscope settings can optimize parameters to achieve desired results. This unit describes fundamental techniques and tips for routine specimen preparation for a variety of biological specimens, preservation of labile or fragile structures, immune-labeling strategies, and microscope imaging parameters for optimal examination by SEM.

Rehydration of Forensically Important Larval Diptera Specimens

Established procedures for collecting and preserving evidence are essential for all forensic disciplines to be accepted in court and by the forensic community at large. Entomological evidence, such as Diptera larvae, are primarily preserved in ethanol, which can evaporate over time, resulting in the dehydration of specimens. In this study, methods used for rehydrating specimens were compared. The changes in larval specimens with respect to larval length and weight for three forensically important blow fly (Diptera: Calliphoridae) species in North America were quantified. Phormia regina (Meigen), Cochliomyia macellaria (F.), and Chrysomya rufifacies (Macquart) third-instar larvae were collected from various decomposing animals and preserved with three preservation methods (80% ethanol, 70% isopropyl alcohol, and hot-water kill then 80% ethanol). Preservative solutions were allowed to evaporate. Rehydration was attempted with either of the following: 80% ethanol, commercial trisodium phosphate substitute solution, or 0.5% trisodium phosphate solution. All three methods partially restored weight and length of specimens recorded before preservation. Analysis of variance results indicated that effects of preservation, rehydration treatment, and collection animal were different in each species. The interaction between preservative method and rehydration treatment had a significant effect on both P. regina and C. macellaria larval length and weight. In addition, there was a significant interaction effect of collection animal on larval C. macellaria measurements. No significant effect was observed in C. rufifacies larval length or weight among the preservatives or treatments. These methods could be used to establish a standard operating procedure for dealing with dehydrated larval specimens in forensic investigations.

Micro-computed tomography-based phenotypic approaches in embryology: procedural artifacts on assessments of embryonic craniofacial growth and development

BackgroundGrowing demand for three dimensional (3D) digital images of embryos for purposes of phenotypic assessment drives implementation of new histological and imaging techniques. Among these micro-computed tomography (μCT) has recently been utilized as an effective and practical method for generating images at resolutions permitting 3D quantitative analysis of gross morphological attributes of developing tissues and organs in embryonic mice. However, histological processing in preparation for μCT scanning induces changes in organ size and shape. Establishing normative expectations for experimentally induced changes in size and shape will be an important feature of 3D μCT-based phenotypic assessments, especially if quantifying differences in the values of those parameters between comparison sets of developing embryos is a primary aim. Toward that end, we assessed the nature and degree of morphological artifacts attending μCT scanning following use of common fixatives, using a two dimensional (2D) landmark geometric morphometric approach to track the accumulation of distortions affecting the embryonic head from the native, uterine state through to fixation and subsequent scanning.ResultsBouin's fixation reduced average centroid sizes of embryonic mouse crania by approximately 30% and substantially altered the morphometric shape, as measured by the shift in Procrustes distance, from the unfixed state, after the data were normalized for naturally occurring shape variation. Subsequent μCT scanning produced negligible changes in size but did appear to reduce or even reverse fixation-induced random shape changes. Mixtures of paraformaldehyde + glutaraldehyde reduced average centroid sizes by 2-3%. Changes in craniofacial shape progressively increased post-fixation.ConclusionsThe degree to which artifacts are introduced in the generation of random craniofacial shape variation relates to the degree of specimen dehydration during the initial fixation. Fixation methods that better maintain original craniofacial dimensions at reduced levels of dehydration and tissue shrinkage lead to the progressive accumulation of random shape variation during handling and data acquisition. In general, to the degree that embryonic organ size and shape factor into μCT-based phenotypic assessments, procedurally induced artifacts associated with fixation and scanning will influence results. Experimental designs will need to address these significant effects, either by employing alternative methods that minimize artifacts in the region of focus or in the interpretation of statistical patterns.

Histological Processing of pH-Sensitive Hydrogels Used in Corneal Implant Applications

Hydrogels are hydrophilic, crosslinked polymers that are being used with increasing frequency in a variety of biomedical applications. The ability to obtain high-quality histological sections, without artifacts, after in vivo implantation is critical to their evaluation. Unfortunately, because of their high water content and environmental sensitivities, many hydrogels are unable to be successfully processed for histology using typical tissue processing methods. In this work, we present a method for successfully processing pHsensitive hydrogels for histological analysis. A modified glycol methacrylate (GMA) processing and embedding protocol is described. One cornea each from 10 New Zealand Red rabbits was implanted with a poly(ethylene glycol) and poly(acrylic acid) interpenetrating double network hydrogel disc for 2 weeks and processed for histology. Maintaining a neutral pH during fixation with glutaraldehyde is a critical initial step for processing. In addition, typical tissue processing methods require the use of alcohol and/or xylene for specimen dehydration, causing severe distortion of hydrogel morphology. The substitution of these chemicals with deionized water during the processing steps also proved to be vital for maintaining hydrogel structure and morphology. Slides stained with cresyl violet showed excellent tissue and hydrogel morphology with minimal histological artifacts or distortion (The J Histotechnol 30:157, 2007)Submitted August 27, 2006; accepted with revisions March 12, 2007.

Structure of GlnK1 with bound effectors indicates regulatory mechanism for ammonia uptake

A binary complex of the ammonia channel Amt1 from Methanococcus jannaschii and its cognate P(II) signalling protein GlnK1 has been produced and characterized. Complex formation is prevented specifically by the effector molecules Mg-ATP and 2-ketoglutarate. Single-particle electron microscopy of the complex shows that GlnK1 binds on the cytoplasmic side of Amt1. Three high-resolution X-ray structures of GlnK1 indicate that the functionally important T-loop has an extended, flexible conformation in the absence of Mg-ATP, but assumes a compact, tightly folded conformation upon Mg-ATP binding, which in turn creates a 2-ketoglutarate-binding site. We propose a regulatory mechanism by which nitrogen uptake is controlled by the binding of both effector molecules to GlnK1. At normal effector levels, a 2-ketoglutarate molecule binding at the apex of the compact T-loop would prevent complex formation, ensuring uninhibited ammonia uptake. At low levels of Mg-ATP, the extended loops would seal the ammonia channels in the complex. Binding of both effector molecules to P(II) signalling proteins may thus represent an effective feedback mechanism for regulating ammonium uptake through the membrane.

High-Resolution Mapping of the Desmosomal Plaque and Adhesive Interface

A precise structural definition of the desmosome has so far remained elusive, likely in part because of its insolubility in aqueous buffers (Matoltsy, 1975). Despite this limiting factor, several key papers have attempted to address this question. What is becoming clear is that the desmosome is both a complex and dynamic structure whose different states may not be readily appreciated by its textbook presentation in single static images. Several approaches have been used to generate a structural map of the desmosome (Figure 1). In vitro binding assays, immunoprecipitation, immunofluorescent colocalization and yeast two-hybrid analyses have been useful in defining protein–protein interactions among desmosome components. Immunogold labeling with N-terminaland C-terminal-specific antibodies to known desmosome proteins was used by North et al. (1999) to generate a molecular map of desmosomes in ultrathin cryosections of bovine epidermis. Using this approach, they were able to generate quantitative measures of the distance of each label from the plasma membrane and thus define the orientation, intermolecular distance and ordering of desmosome protein components. Thus, desmoglein and desmocollin desmosomal cadherins are single-pass transmembrane proteins whose aminoterminal extracellular domains form the adhesive interface. The cytoplasmic tail of desmoglein binds to plakoglobin, which in turn binds to desmoplakin. Desmoplakin binds to keratin intermediate filaments and plakophilins bind to desmoplakin, plakoglobin, desmogleins and desmocollins. Complexes of this unit repeat structure localize into regions of the plasma membrane to form the desmosome, yielding a polyvalent adhesive interface. New specimen preparation techniques and imaging technologies are being applied to the study of desmosome structure. Vitrification, a process in which biological tissues are converted to solids free of crystalline water, facilitates preservation of native structures. Cryo-electron microscopy of vitreous sections is a technique that avoids specimen dehydration and heavy metal deposition of traditional electron microscopy, and thus offers the promise of significantly higher resolution images of the desmosome. Applying this technique to mouse skin, Al-Amoudi et al. (2004) generated an image of the desmosome in which the extracellular inter-desmosomal interface was characterized by highly ordered straight rod-like structures with a 5 nm periodicity. In electron microscopic tomography, multiple two-dimensional images are obtained by tilting a sample at angles to the electron beam; computer software is then used to reconstruct a

Experimental deformation of flint in axial compression

Abstract The experimental deformation of flint, a water-rich (1–2 wt%) fine-grained (1 μm) microcrystalline quartz, has been studied using a gas-confining medium apparatus at 300 MPa confining pressure in the temperature range 500–1000 °C. In constant strain-rate axial compression tests a mechanically unstable behaviour with peaked and undulating stress-strain curves, especially at the faster strain rates, is manifest. The rheology of these tests can be approximately described by the traditional power-law equation with a stress exponent ( n ) of between 3 and 5, and an apparent activation energy ( Q ) of 108 kJ mol −1 . However, the strain-independent (‘steady-state’) equation is only a partial description of the data. The mechanical properties were studied at lower stresses using stress relaxation testing, this method has the advantage of recording specimen response over a very small strain interval ( c. 1%), i.e. nearly constant structure. These tests revealed that in a high stress regime above 100 MPa the rheology was only weakly dependent on strain and very similar to the constant strain-rate behaviour. Below 100 MPa the rheology can be described by a power law with Q = 64 kJ mol −1 and n = 1. The low-stress behaviour is extremely sensitive to specimen strain, the strain rate decreasing with increasing strain for a given stress. The optical microstructure reveals that the mechanical instabilities are related to localized displacement zones, sometimes en echelon, consisting of relatively large grains (10 μm). The important reduction of the integral infrared absorption of deformed specimens, a 98% reduction of the initial water content, suggests that a pore fluid was developed by specimen dehydration in the sealed (undrained) assembly. The presence of pores or bubbles decorating grain boundaries and microfractures normal to the piston-specimen interface both testify to the presence of the pore fluid during deformation. The pore fluid pressure may have approached the confining pressure resulting in a near-zero effective pressure. Transmission electron microscopy revealed the presence of many Brazil micro-twins parallel to grain boundaries. The twins are thought to be growth defects indicating rapid grain growth during deformation. Dislocations in the basal plane were observed with a 1/3 〈a〉-type Burgers vector. Dislocations were also observed in the grain boundaries, possibly caused by grain-boundary sliding in the low stress regime. The complete lattice preferred orientation (LPO) of homogeneously deformed specimens has been determined by X-ray texture goniometry. The inverse pole figures have a strong concentration of compression axes parallel, and a weaker concentration normal, to the c -axis. The local c -axis pole figures in heterogeneously deformed specimens were characterized by the photometric technique. The c -axis pole figures in the bulk are identical to that determined by X-rays in homogeneously deformed specimens. In the displacement zones the fabrics are asymmetric with respect to the zone boundaries. The asymmetry is not consistent with the shear sense imposed by the deformation geometry and the usually accepted dislocation slip models. Fabric development by a grain growth mechanism in which the mechanically nucleated micro-twins (Brazil law) favour growth of grains with low-energy twin grain-boundary orientation relationships with their neighbours is proposed. The mechanism is consistent with the microstructure (twins parallel to grain boundaries, low density of dislocations on glide planes) and the c -axis pole figures. In natural deformations under similar conditions (e.g. subsolidus granites in the β-quartz field) we suggest caution in using fabric asymmetry as it may not be a dependable indicator of shear sense, as grain growth and dislocation slip will give rise to different interpretations.

The influence of short-term water storage on the flexural properties of unidirectional glass fiber-reinforced composites

Objective: The aim of this study was to determine flexural properties of unidirectional E-glass fiber-reinforced composite (FRC) with polymer matrices of different water sorption properties. Methods: Rhombic polymer and FRC test specimens made of three commercially available diacrylate resin (Sinfony Activator, Triad Gel, 3M Scotchbond Adhesive) and different volume fractions of fibers were tested with three-point bending test according to ISO 10477 after storing in water for 30 days. Water sorption of specimens was also measured. Results: Flexural strength of specimens with 45 vol% fraction E-glass fibers varied from 759 to 916 MPa in dry conditions. Water-stored specimens showed flexural strengths of 420–607 MPa. ANOVA analysis revealed that the fiber-volume fraction and the water sorption of the polymer matrix had a significant effect ( p<0.001) on the flexural properties. Dehydration of specimens recovered the mechanical properties. Decrease of flexural properties after water immersion was considered to be mainly caused by the plasticizing effect of water and the decrease depended on water sorption. Significance: Use of polymers with low-water sorption seems to be beneficial in order to optimize the flexural properties of FRC.

ESEM Study of Dental Adhesive Polymerization

Dental adhesives are bonded to a wet surface of live tissue, dentin. There are two important stages of surface preparation for adhesive application: 1) Etching the tooth structure creating a layer of collagen network free from mineral, and 2) Applying a primer, in this case a water solution of hydrophilic monomer of 2-hydroxyethylmethacrylate (ScotchBond Multipurpose®, 3M). During the priming stage, the resin monomer should penetrate the fibril network and coat collagen fibrils. Subsequent adhesive resin penetration into the collagen network (hybrid layer) is not uniform, influenced by the presence of residual water and degree of resin polymerization. Dependence of bond strength of dental adhesives on time or intensity of light activation is well known, but no direct microscopic observations of the dynamics of polymerization were performed. This was due to the fact that during specimen dehydration for conventional SEM the polymerization process is still proceeding and residual monomer could be washed out.

An ultrastructural study of the application of dentine adhesives to acid-conditioned sclerotic dentine

Objective: This in vitro study examined the ultrastructure of resin-infiltrated sclerotic dentine following the application of a self-etching primer, with or without the adjunctive use of phosphoric acid pre-conditioning. Materials and Methods: Non-carious, natural cervical sclerotic lesions were hand-cleaned with a slurry of pumice and chlorhexidine and bonded without further cavity preparation. One group was bonded using Clearfil Liner Bond 2V (Kuraray Co. Ltd, Osaka, Japan) alone. Specimens from the other group were pre-conditioned with K-etchant (40% phosphoric acid gel, Kuraray) prior to the application of the same self-etching primer. Artificially prepared wedge-shaped lesions were also made in sound bicuspids, bonded using the two methods, and used as controls for the two groups. For SEM examination, each specimen was cryofractured into two halves through a pre-formed slit made on the lingual surface, after the respective conditioning treatment. Different locations within the lesions were examined after rinsing of the phosphoric acid/self-etching primer and specimen dehydration. For TEM investigation, specimens were bonded with the adhesive. Both demineralised and undemineralised ultrathin sections were prepared from the occlusal, gingival and deepest part of the wedge-shaped bonded lesions following specimen fixation, dehydration and resin embedding. Results: A hypermineralised surface layer was present on the surface of etched sclerotic dentine. This layer was thicker in the deepest part of the natural lesions, where bacterial colonisation of the lesion surface was also apparent. Both treatment protocols were unable to effectively dissolve sclerotic casts that occluded the dentinal tubules. Depending upon the thickness of the surface layers at different locations in the natural lesion, self-etching primer treatment alone resulted in reduction of the thickness of the authentic hybrid layer (i.e. hybridised intertubular dentine). This was also true of phosphoric acid pre-conditioning along the deepest part of the natural lesions. Within this region, intertubular dentine completely devoid of an authentic hybrid layer could be seen in both treatment groups. Resin tags were also sparsely observed in such regions. Conclusions: Adhesive strategies that rely mostly on micromechanical retention alone may be compromised by the sporadic absence of the hybrid layer and resin tags in sclerotic dentine. Based on the ultrastructural features presented, it is further speculated that adaptive strategies such as removal of the surface layers and extended etching time may not be completely effective in improving bonding efficacy in highly sclerotic dentine. Interdisciplinary research should be continued to develop alternative procedures for bonding resins equally well to sound and sclerotic dentine.

440X線顕微鏡で細胞内に何が見えるか?

X-ray microscopy is promised to be a useful tool for studying living biological specimen without dehydration and fixation. A laboratory-sized x-ray microscopy system was realized as a flash contact microscopy using a laser-produced plasma as a flash x-ray source. X-ray images of living biological specimens obtained by the system were provided. Some of these images indicate fragile structures in living states, which are damaged by dehydration of specimens.

The Effect of Repeated Freeze-thaw Cycles on the Biomechanical Properties of Canine Cortical Bone

SummaryAs orthopaedic investigations have become more intricate, bone specimens have sometimes undergone multiple freeze-thaw cycles prior to biomechanical testing. The purpose of this study was to determine if repeated freezing and thawing affected the mechanical properties of canine cortical bone. Six pairs of third-metacarpal bones were tested in three-point bending and six pairs of femurs were tested in torsion. At the time of collection, one member of each pair was tested destructively. The other member was tested nondestructively at the time of collection and after each of five freeze-thaw cycles, followed by destructive testing after the fifth cycle. For destructive tests, the material properties (modulus, maximum stress, maximum strain and absorbed energy) of a specimen at the time of collection were compared to those of the corresponding contralateral specimen that had undergone five freeze-thaw cycles. For repeated nondestructive tests, the modulus of a specimen at the time of collection was compared to modulus of the same specimen at each of the five thaw intervals. During destructive testing, there was a significant (p = 0.02) decrease (20%) in maximum torsional strain. Other changes in bending and torsional destructive properties were not statistically significant. During repeated nondestructive testing, there were solitary significant (p &lt; 0.05) increases (8% and 9%, respectively) in both bending and torsional modulus. However, these isolated changes were not correlated to the number of freeze-thaw cycles. The pattern of alterations in destructive and non-destructive biomechanical properties was most consistent with varying specimen dehydration at each thaw interval. Despite using accepted methods to maintain specimen hydration, repeated freezing, thawing, handling and testing of cortical bone increased the risk of moisture loss. Unless stringent efforts are made to ensure proper hydration, the mechanical properties of canine cortical bone will be altered by repeated freezing and thawing, affecting the results of studies utilizing this technique.The effect of five freeze-thaw cycles on paired canine cortical bone specimens was evaluated using destructive and repeated non-destructive three-point bending and torsion tests. A significant decrease in destructive torsional strain and isolated significant increases in nondestructive bending and torsional modulus were most consistent with varying specimen dehydration at each thaw interval.

Chemical Dehydration of Specimens with 2,2-Dimethoxypropane (DMP) for Paraffin Processing of Animal Tissues: Practical and Economic Advantages over Dehydration in Ethanol

Chemical dehydration can be accomplished using 2,2-dimethoxy-propane (DMP). In the presence of an acid catalyst, this liquid reacts with water generating methanol and acetone as products. Although DMP is more expensive per milliliter than ethanol and other solvents used for dehydration, it is an economical alternative because a much smaller volume is needed. Slow penetration of DMP was previously thought to restrict its use to tiny specimens, but we now show that pieces of tissue as thick as 2 cm are dehydrated by overnight immersion in acidified DMP. We also show that dehydration in acidified DMP does not impair the staining of UNA or other basophilic components of animal tissues. The temperature and concentrations of methanol and H+ in the chemical dehydrating agent are too low to produce histochemicaUy detectable methylation or nucleic acid extraction.

Micro-PIXE analyses of thick frozen lenses with cataracts

The microprobe of 27 MeV α particles has been utilized for the study of a possible role of ions in causing hereditary cataracts of mice. The distributions of S to Ca in lenses were determined at a resolution around 50 μm. In order to obtain the elemental concentrations on wet weight basis, thick frozen hydrated specimens were analyzed. The sample preparation method and the experimental setup developed to minimize redistributions of diffusible elements as well as dehydration of specimens are described. The ratio of the X-ray peak intensity to the continuum background intensity was used for quantification. The accuracy of this P/B method used for PIXE analyses of thick soft tissues was examined both by computer calculations and by experiments with biological standards such as Spurr's epoxy resins and NBS SRMs. The elemental distributions in cataractous lenses were obtained for two different types of hereditary cataract: Nakano mice and its congenic strain. The features of distributions and quantitative concentrations of S, Cl, K and Ca were compared.

Artifacts caused by dehydration and epoxy embedding in TEM

Various means have been devised to preserve biological specimens for electron microscopy, the most common being chemical fixation followed by dehydration and resin impregnation. It is intuitive, and has been amply demonstrated, that these manipulations lead to aberrations of many tissue elements. This report deals with three parts of this problem: specimen dehydration, epoxy embedding resins, and electron beam-specimen interactions. However, because of limited space, only a few points can be summarized.Dehydration: Tissue damage, or at least some molecular transitions within the tissue, must occur during passage of a cell or tissue to a nonaqueous state. Most obvious, perhaps, is a loss of lipid, both that which is in the form of storage vesicles and that associated with tissue elements, particularly membranes. Loss of water during dehydration may also lead to tissue shrinkage of 5-70% (volume change) depending on the tissue and dehydrating agent.