A cryoclamp for the rapid cryofixation of the isolated blood-perfused rabbit cardiac papillary muscle preparation at predefined times during the contraction cycle

There is increasing evidence that the distribution of monovalent cations in cardiac cells may be non-uniform, particularly in the region immediately beneath the sarcolemma, and we have proposed that a build-up of sodium in this region could be an important factor in the development of ischaemia-reperfusion injury. Electron probe X-ray microanalysis is ideal for the study of such changes in distribution but the application of the technique to this problem imposes severe requirements on the specimen and on the method for cryofixation. The specimen must be perfused through its vasculature so that it can be made truly ischaemic and be successfully reperfused. It is necessary to be able to cryofix the specimen without disturbance of its blood supply, electrical stimulation or temperature. It is also important to know the time in the contraction cycle when cryofixation occurs. Here we describe the design of an automated cryofixation device which can be used to cryofix a blood perfused papillary muscle preparation at predetermined time points in the contraction cycle. Preliminary data obtained from the analysis of rabbit papillary muscles subjected to varying periods of ischaemia are included as an example of the use of the cryoclamp.     

Fast cryofixation technique for X-ray microanalysis

The proposed cryofixation technique uses a tubule-shaped needle chilled in liquid propane for simultaneous excision and freezing of a tissue specimen. Due to this simultaneity, ionic shifts created by traumatic influences are avoided even in the outermost cells of the specimen. Moreover, it is shown here that stopping the blood flow for more than about 10 s results in notable ionic shifts between cells and extracellular space in rat heart and liver. Such preparative ischaemic injury is minimized by the Fast Cryofixation Technique because it can be easily performed on organs within the circulatory system, whilst the heart of the animal is still beating. Intracellular concentrations of the monovalent ions in rat heart and liver, obtained by this method, tally well with recent results from different independent techniques reported in the literature. As demonstrated by cross-sectioning and freeze-fracturing, the structural preservation of the freezing technique is sufficient for X-ray microanalytical work.     

The cryopuncher: A pneumatic cryofixation device for X-ray microanalysis of tissue specimens

A cryopunching device is described which allows cryofixation of tissue specimens by quick contact with a precooled copper surface during excision. The advantage of the cryopuncher for analytical electron microscopy of cells and tissues in defined functional states is illustrated by electron probe X-ray microanalysis of freeze-dried cryosections from rat liver and dogfish kidney. In comparison with results obtained from specimens plunged into liquid propane, cryopunching in situ results in similar preservation of morphology and remarkably improved intracellular K/Na ratio.     

Structural changes in samples cryofixed by contact with a cold metal block

A common method of cryofixation is to bring a specimen rapidly in contact with a cold metal block. It is usually thought that during this process the surface of the specimen suffers little distortion since it freezes rapidly. Whether this is likely depends on the rate at which samples freeze compared with the speed at which the sample hits the cold block. There is some discrepancy between the published experimentally and theoretically determined freezing rates. As a contribution to this debate the distortion in cryofixed, freeze-substituted, striated muscle fibres has been investigated. In transverse sections, compression can be detected by deviations of the filament lattice from the hexagonal and used to estimate the time of freezing. Some specimens were frozen using a Gatan Cryosnapper, which freezes by catching the specimen between two nitrogen-cooled copper jaws. In addition, the speed with which the jaws close has also been determined. The results suggest that freezing of the well-preserved areas occurs in substantially less than 1 ms. This conclusion is supported by results obtained using metal-mirror apparatus in which the cushioned specimen was dropped onto a nitrogen- or helium-cooled copper block. All the specimens frozen against a cold block have a flat edge whereas muscle fibres are round. At the very edge there is evidence of structural damage as well as the more general lattice distortion.     

Combined quick freezing,freeze-drying,and embedding tissue at low temperature and in low viscosity resins

The techniques of quick freezing and freeze-drying provide an alternative to the more classical methodologies of chemical fixation and dehydration with organic solvents. It is possible to embed freeze-dried tissue in low viscosity resins, either at room temperature or at subzero temperatures in Spurr's resin or Lowicryl K4M, respectively. The choice of embedding medium affords additional flexibility in postdrying and embedding conditions, since Spurr's resin allows vapor fixation with osmium tetroxide and thermal polymerization. Osmium tetroxide is not recommended for Lowicryl resins, but these media permit polymerization at subzero temperatures with ultraviolet light. Both resins have unique advantages that may be utilized, depending upon the purpose of the embedding. In this paper, we discuss the details of preparing smooth muscle, from rabbit renal artery, by quick freezing and freeze-drying, as well as methods for the embedding of the freeze-dried tissue in both Spurr's resin and Lowicryl K4M. Although we have previously reported the ultrastructure of smooth muscle embedded in Spurr's low viscosity resin, the combination of freeze-drying and infiltration in Lowicryl K4M represents a new approach that allows the elimination of chemical fixation, dehydration with organic solvents, and heat polymerization of the embedding medium.     

Study of potassium deficiency in cardiac muscle: quantitative X-ray microanalysis and cryotechniques

An imbalance of potassium in cardiac muscle causes an alteration of heart function. The distribution and concentration of potassium in rat papillary heart muscle was studied using cryofixation and X-ray microanalysis. Freeze-dried cryosections and sections of freeze-dried, embedded tissue were analysed. Bulk frozen specimens were freeze-dried either in a vacuum or by a new technique using liquid propane as a cryodehydration medium. These two methods of freeze-drying were tested for elemental retention in other specimens, with comparable results. A potassium concentration of 120 mmol/l was measured in normal myocytes of cardiac papillary muscle compared to 80 mmol/l in myocytes of animals stressed by a temperature of 45°C for 1 h. The presumed physiological significance of the findings is discussed.     

Imaging and elasticity measurements of the sarcolemma of fully differentiated skeletal muscle fibres

This study aimed to describe the three-dimensional structure and the elastic properties of the sarcolemma of adult, fully differentiated, skeletal muscle fibres combining Atomic Force Microscopy (AFM) and optical microscopy. Single fibres were enzymatically dissociated from Flexor Digitorum Brevis of adult mice and were maintained in culture up to 3 weeks. On the sixth day after dissociation, the upper surface of intact fibres, either alive in solution or fixed and kept in solution or fixed and exposed in air, was analysed with AFM. The most prominent features in AFM images were periodic transversal foldings with an interval that corresponded to the sarcomere length. More detailed analysis of the topography profile showed that the depth in the folding decreased with increasing sarcomere length and that the crests of the foldings corresponded to the Z-lines. Minor periodic structures could be detected in the valleys between the major foldings. AFM images also showed deep depressions on the sarcolemma likely corresponding to openings of T tubules and caveolae. Two-dimensional elasticity maps were obtained using AFM as an indenter and showed that the crests of the transversal foldings correspond to higher stiffness regions. This study provides the first complete three-dimensional topography and mechanical characterization of intact, living skeletal muscle fibres and might form the basis for further investigations aimed to compare healthy and dystrophic muscles.     

Some limitations of the use of tannic acid as a marker of damaged skeletal muscle fibres

Mouse biceps brachii muscles containing single transverse incisions were treated with low molecular weight tannic acid. In the absence of fibre degeneration, intracellular spread of tannic acid was confined to a short distance along the fibres from the damaged portions of the cell membranes. Beyond this, damaged and undamaged fibres could not be distinguished from one another. Tannic acid is not a reliable marker of localized trauma to skeletal muscle fibres.     

Freeze-fracture electron microscopic and low temperature X-ray scattering studies of the effect of cryofixation upon serum low density lipoprotein structure

We report here a correlated X-ray diffraction and freeze-fracture electron microscope study of the effects of several cryofixation procedures upon human serum low density lipoprotein (LDL₂) structure. Only when the LDL₂ solutions contained 75%, by weight, glycerol were the room temperature and post cryofixation low temperature LDL₂ X-ray scattering curves indistinguishable from one another. Other cryofixation procedures, slow or rapid, with or without glycerol, resulted in differences between the room temperature and low temperature LDL₂ X-ray scattering curves, in part due to the effect of quenching upon the solvent. Freezeetching electron microscopy of the slowly cryofixed LDL₂ showed marked aggregation of the particles and an unusual morphological appearance. In contrast, after rapid cryofixation or cryofixation in the presence of glycerol, freeze-etch electron microscopy revealed well-isolated particles which had a knobby morphology. The results demonstrate that under certain conditions (in the presence of 75% glycerol) cryofixation results in minimal, if any, structural alteration of, at least, the LDL₂ lipid moiety. Further, this study underlines the more general conclusion that any high resolution structural study employing a cryofixation step must be interpreted with caution and the effect of cryofixation upon the sample structure need be evaluated by independent means.     

Morphological and histochemical study of the masseter muscle after occlusal alteration

The aim of this study was to evaluate alterations in the masseter muscle of 30 male guinea-pigs submitted to occlusal alteration. The animals were divided into 2 equal groups, the control group (C) only submitted to surgical stress, and the occlusal altered group (T) submitted to teeth extraction. Each group was subdivided into 3 groups, with 5 animals, for the following studies: macroscopy and vessels distribution, light microscopy and histochemical analysis, with animals’ perfusion 2 months after surgery. Data were submitted to statistical analysis. Macroscopically, an anteroposterior orientation of the muscular fibers was found and statistical difference between C and T groups in width (p<0.05). Microscopically, C and T groups showed polygonal muscular fibers with variable diameters, and on the left side of the T group these differences were more pronounced. Histochemically, in both groups, the prevalence of intermediate reactivity fibers and several high reactivity fibers spread out in the deeper area was observed, with no significant differences between superficial fibers on both sides of C and T groups. It was possible to conclude that the masseter muscle in guinea-pigs was sensible to functional chewing alteration.     

Rapid freezing,cryosectioning, and x-ray microanalysis on cardiac muscle preparations in defined functional states

Electrically stimulated heart muscle preparations can be quickly frozen in undercooled propane at defined times of the mechanically controlled contraction cycle. The apparatus for triggered freezing of the muscle strips in undercooled propane is described in detail. Freeze substitution of some strips after freezing shows the degree of ice crystal formation without the potential interference of artifacts introduced later by cryosectioning and freeze drying. Ultrathin longitudinal and transversal cryosections are cut with a LKB cryoultramicrotome at temperatures of −130 to −140°C, freeze-dried at 10⁻⁶ Torr vacuum and carbon-coated before analysis. The freeze-dried cryosections are analyzed in a Siemens Elmiskop 102 electron microscope equipped with a Kevex energy dispersive system, and the elemental concentrations (in mMol/kg d.w.) of Na, Mg, P, S, Cl, K, and Ca are determined in subcellular compartments of muscle frozen in different functional states. The methodology of quantitation, i.e, determination of elemental net peak and continuum, correction of continuum, preparation of standards, and deconvolution of overlapping peaks are described. The minimum detectable elemental concentration using the reported methods is in the range of a few mMol/kg d.w. This also applies to Ca, which can be accumulated in heart muscle in readily detectable amounts in intracellularly located stores as well as structures connected with the cell membrane. The present report shows that cryotechniques and x-ray microanalysis can be successfully applied to heart physiology.     

Candida albicans biofilms: comparative analysis of room‐temperature and cryofixation for scanning electron microscopy

Biofilms are frequently related to invasive fungal infections and are reported to be more resistant to antifungal drugs than planktonic cells. The structural complexity of the biofilm as well as the presence of a polymeric extracellular matrix (ECM) is thought to be associated with this resistant behavior. Scanning electron microscopy (SEM) after room temperature glutaraldehyde‐based fixation, have been used to study fungal biofilm structure and drug susceptibility but they usually fail to preserve the ECM and, therefore, are not an optimised methodology to understand the complexity of the fungal biofilm. Thus, in this work, we propose a comparative analysis of room‐temperature and cryofixation/freeze substitution of Candida albicans biofilms for SEM observation. Our experiments showed that room‐temperature fixative protocols using glutaraldehyde and osmium tetroxide prior to alcohol dehydration led to a complete extraction of the polymeric ECM of biofilms. ECM from fixative and alcohol solutions were recovered after all processing steps and these structures were characterised by biochemistry assays, transmission electron microscopy and mass spectrometry. Cryofixation techniques followed by freeze‐substitution lead to a great preservation of both ECM structure and C. albicans biofilm cells, allowing the visualisation of a more reliable biofilm structure. These findings reinforce that cryofixation should be the indicated method for SEM sample preparation to study fungal biofilms as it allows the visualisation of the EMC and the exploration of the biofilm structure to its fullest, as its structural/functional role in interaction with host cells, other pathogens and for drug resistance assays.     

Computation of a tetrahedral mesh for striated muscle deformation simulation

Competing concepts exist regarding surgery for instance of the cleft lip and palate to date. Morphology‐based simulations at histological scale may one day be used to help the surgeon predict the possible outcome of a variety of approaches. It however can be a challenge to generate volume meshes that are applicable to the mathematical modelling of three‐dimensional spatial modifications. Computation of surface meshes may be considered less delicate. The aim of this study is to design and evaluate a novel algorithm that supports finite element methods. Images of histological serial sections of a striated muscle were segmented. Results of the three‐dimensional reconstruction of multiple layers of the polygonal segmentation data characterized the hull of the muscle. The corresponding surface mesh was then converted into a tetrahedral mesh to generate volume. This was achieved by mapping multiple template types onto neighbouring intersection polygons. Muscle contraction was subsequently simulated by mesh deformation. The technique successfully generated volumes and was able to provide data on contraction directions. The mesh supported a novel approach to simulate representations of contraction. However, several drawbacks were evident. Mathematical modelling of scenarios with more than one striated muscle will require considerable modification of the currently presented approach. Future studies need to then evaluate the applicability of volume meshes to represent arrays of three‐dimensional biological objects. Surface mesh based mathematical modelling of cleft lip and palate surgery and its results are therefore not yet in reach.     

Contribution of cryofixation and freeze-substitution to analytical microscopy: a study of Tritrichomonas foetus hydrogenosomes

The hydrogenosome, an organelle that produces molecular hydrogen and ATP from the oxidation of pyruvate or malate under anaerobic conditions, presents some characteristics common to mitochondria. It is found in several trichomonad species, protists living in oxygen-poor environments, as well as certain free-living ciliates, rumen ciliates, and some fungi. We performed a comparative microanalytical study (energy dispersive X-ray analysis and electron spectroscopic imaging) of different fixation methods for electron microscopy analyzing hydrogenosomes of the bovine parasite Tritrichomonas foetus. The study included the elemental composition and the mapping of calcium, phosphorus, and oxygen. A preparation of T. foetus cells, based on cryoimmobilization by high-pressure freezing and freeze-substitution, was compared to a second preparation based on chemical fixation followed by dehydration and routine processing. The ultrastructural preservation achieved by the cryotechnique was far superior to the chemical fixation, since it allowed the successful cryoimmobilization of intracellular ion contents. The detection of several cations (Al, Mg, Co, Ca, Fe) by X-ray microanalysis inside the peripheral vesicle of the hydrogenosome was only possible in cryofixed cells. The presence of aluminum and cobalt ion in the hydrogenosomal vesicle was established for the first time. Electron-spectroscopic images of calcium showed that this element, in addition to the vesicle compartment, is present in the hydrogenosome's membrane in varying concentrations, which might reflect changes in the physiology of this organelle.     

一种车床快速定位实用夹具的设计与制造

分析了现有的传统夹具在装夹具有较高形位公差要求的轴类零件进行加工时存在的弊端和不足,并提出了相应的解决方案。开发了一种＂两顶一面＂快速定位夹具,并介绍了其结构组成、工作原理、特点及加工制造方法。     

Freeze-substitution of rabbit tibial articular cartilage reveals that radial zone collagen fibres are tubules

Investigations of the micromorphology of rabbit tibial articular cartilage using scanning and transmission electron microscopy revealed that the collagenous elements in the tissue form fluid-containing tubular structures. The commonly described radial or deep zone longitudinal fibres were found to be tubular structures with internal diameters of 1–2 μm. The walls of the tubules were composed of tightly packed fibrils of collagen. The tangential zone, close to the tibial plateau, was composed mainly of a spongy arrangement of collagen fibrils, containing bunches of tangentially lying small (< 1 μm) diameter tubules. The application of conventional chemical fixation techniques resulted in the fine detail of this tissue being obscured. When the tissue was frozen, followed by cryo-scanning electron microscopy or freeze-drying, prior to observation in the scanning electron microscope the tubule structures were not obviously present. It was only by applying freeze-substitution techniques, followed by critical point drying or resin embedding, that the structure was revealed clearly. Segregation of water into ice crystals did occur during the freezing process, but the formation of those crystals played no part in creating the tubular morphology observed. A similar structure was still revealed following pre-treatment with glycerol, methanol or Triton X-100, provided that concentration of these additives was not too high. The walls of the tubules in the radial region were composed of straight, longitudinally arranged as well as helically arranged, 30 nm diameter fibrils. The lumen of the tubules appears to be lined by a circumferentially arranged array of approximately 10 nm diameter fibres, spaced at regular intervals of 50–70 nm.     

带CAN总线的智能温度变送器的研制

利用带ＣＡＮ控制器的高性能８位单片机８０Ｃ５９２,研制了能同时接受８路不同测温元件输入的带现场总线接口的新型智能温度变送器,并对该变送器的软硬件组成和使用方法作简要介绍。     

Vascular smooth muscle orientation in straight portions of human cerebral arteries

A technique of embedding, sectioning and analysis has been developed for studying the orientation and proportional composition of smooth muscle in the straight portions of human major cerebral arteries. Various distortions, which occurred during processing and sectioning, were measured quantitatively. Nerve fibres were implanted as a reference frame within the paraffin block containing an arterial bifurcation. The nuclei of smooth muscle have been treated as three-dimensional vectors of cellular orientation. The projected length in the plane of the section and the section thickness were used to define section pitch. To relate these vectors to the overall geometry of cerebral arteries they were transformed such that the resultant pitch would be the same as that observed if the sections were cut normal to the arterial longitudinal axis. Matrix transformations of nuclear vectors were of the expansion and Eulerian forms. The average pitch for the five sections from three straight portions was ?0.22° ± 2.36 (SD of five means) with a range between ?2.8° and 3.2°. Because this pitch is small it is possible to use 6.9 μm thick longitudinal and mid-plane sections of straight arteries to obtain estimates of proportional composition. Stereological point counting was used to determine that smooth muscle comprised 72.0% ± 4.76 (SD for ten segments) of the tunica media in cerebral arteries.     

Combined biochemistry and histocytochemistry as a tool to investigate Ecto-ATPase in the cardiac muscle

Ecto-ATPase (ecto-adenosine triphosphatase), a key enzyme of cardiac metabolism, is responsible for modulation of the concentration of extracellular nucleotides in the heart. We present methodology consisting of the combined use of biochemical and histocytochemical techniques to study its properties. Using samples from essentially the same preparation, we applied biochemistry and histocytochemistry to determine biochemical characteristics of ecto-ATPase and an in situ localization of its reactivity. Our results indicated that detected enzyme resists fixation, depends on divalent ions, and hydrolyzes ATP, but not AMP or ADP-beta-S. Reaction product of the enzyme activity was found confined to the extracellular surface of the plasma membrane of cardiac myocytes and endothelial cells due to the corresponding orientation of the enzyme active sites. Experiments using an inhibitor justified specificity of the reaction. When used together with molecular biological and immunocytochemical techniques, the present methodological approach should be capable of yielding important information about the actual ability of ecto-ATPase to operate.