Effect of sodium azide on the ultrastructural preservation of tissues.

An electron microscopic study was carried out to examine the quality of ultrastructural preservation of parenchymatous and mesenchymatous tissues and isolated cells fixed in glutaraldehyde with sodium azide (NaN3) as an additive. The dense tissues fixed with conventional glutaraldehyde containing calcium chloride demonstrated only a narrow zone of good tissue preservation on the surface of the specimens. Addition of azide at a concentration of 0.1% greatly improved the cellular preservation in the deeper region of tissues, in particular with respect to the mitochondrial morphology. There was no adverse effect on other cell organelles. The improvement in mitochondrial preservation and the enhancement of penetration of the fixative is presumably due to selective and instantaneous inhibition of mitochondrial metabolic activity by the azide, thus retarding anoxic degenerative effects on cellular structures until permanent fixation is completed by the comparatively slow-acting aldehyde. However, the addition of azide offers no significant improvement in the ultrastructural preservation of isolated lymphocytes and liver cells, or fibroblasts maintained in culture.     

The fixation,dehydration, drying and coating of cultured cells for SEM

Cultivated cells form a valuable model system for studies on the effects of various preparative protocols for scanning electron microscopy (SEM). The various effects of each preparative step can be followed in detail in the light microscope and no diffusion gradients complicate the fixation and other procedures as in the case of solid tissues. Studies on cultivated cells indicate that the glutaraldehyde component of a glutaraldehyde-based fixative does not contribute to the effective osmotic pressure of the fixative and thus the osmolarity of the buffer, and other components, must be equalized to that of the medium in which the cells grow. Even small deviations from this ideal effective osmotic pressure will result in osmotically induced artefacts. Disturbances of pH and temperature of the cultures prior to and during fixation will result in changes in the appearance of many cellular structures such as microspikes and ruffles. We find that osmium fixation is advisable in most instances for best possible membrane preservation and that even long periods of glutaraldehyde fixation do not compensate for osmium fixation. Dehydration always results in shrinkage. Freeze drying (FD) and critical point drying (CPD) also give rise to shrinkage, the former to a lesser degree than the latter. A gold-palladium alloy gives a less granular coating that does gold alone. When cultured cells are studied, a metal thickness of between 5 and 15 nm is usually sufficient to give rise to an adequate secondary electron production and to avoid charging even at accelerating voltages of 30–40 kV. Without treatment with OsO₄ a thicker metal coating is required.     

Use of microwave fixation in the preparation of cell cultures for observation with the scanning electron microscope

This paper describes a method for primary fixation of cultured cells for scanning (SEM) and transmission (TEM) electron microscopy using microwaves alone. This method of fixation takes 8 seconds and is therefore quicker and less expensive than conventional fixation techniques. The preservation of cell morphology is excellent and cultures of mammalian immune system cells and peripheral nervous tissue have been examined using this fixation.     

A new technique for perfusion fixation and contrast enhancement of foetal lamb myocardium for electron microscopy

A technique of perfusion fixation following elective diastolic cardiac arrest was developed to obtain excellent preservation of foetal lamb hearts for morphological studies with electron microscopy. A technique was developed to markedly enhance the contrast of embedded material by treating thin sections with tannic acid prior to lead staining. This technique has been particularly useful for quantitative morphometric analysis.     

Comparison of conventional and microwave-assisted processing of mouse retinas for transmission electron microscopy

Conventional fixation and processing of mammalian retinal tissues for transmission electron microscopic (TEM) examination is slow and produces ultrastructural artefacts in the photoreceptor cell layer. Among these artefacts are gaps between photoreceptor outer segment disc membranes and between photoreceptor cells in the region of the retina where the cell nuclei are located. A study was undertaken to determine whether a much more rapid microwave‐assisted fixation and processing protocol would have an effect on the quality of ultrastructural preservation of the retina, particularly on the photoreceptor cell artefacts. The overall ultrastructural preservation of the retina was similar for the conventional and microwave‐assisted techniques. However, the magnitudes of the photoreceptor artefacts were significantly reduced when microwave irradiation was used during primary fixation and processing. It is clear that, at least for the retina, employing microwave irradiation during specimen preparation for TEM results in superior ultrastructural preservation with a substantial reduction in the time required for sample preparation.     

Feasibility of high pressure freezing with freeze substitution after long‐term storage in chemical fixatives

Fixation of biological samples is an important process especially related to histological and ultrastructural studies. Chemical fixation was the primary method of fixing tissue for transmission electron microscopy for many years, as it provides adequate preservation of the morphology of cells and organelles. High pressure freezing (HPF) and freeze substitution (FS) is a newer alternative method that rapidly freezes non‐cryoprotected samples that are then slowly heated in the FS medium, allowing penetration of the tissue to insure adequate fixation. This study addresses several issues related to tissue preservation for electron microscopy. Using mice liver tissue as model the difference between samples fixed chemically or with HPF immediately after excision, or stored before chemical or HPF fixation were tested with specific focus on the nuclear membrane. Findings are that immediate HPF is the method of choice compared to chemical fixation. Of the chemical fixatives, immediate fixation with 2.5% glutaraldehyde (GA)/formaldehyde (FA) is the best in preserving membrane morphology, 2.5% GA can be used as alternative for stored and then chemically processed samples, with 10% formalin being suitable as a storage medium only if followed by HPF fixation. Overall, storage leads to lower ultrastructural preservation, but HPF with FS can minimize these artifacts relative to other processing protocols. Microsc. Res. Tech. 76:942–946, 2013. © 2013 Wiley Periodicals, Inc.     

Functional morphology of the secretory pathway organelles in yeast

The glycoprotein secretory pathway of yeast serves mainly for cell surface growth and cell division. It involves a centrifugal transport of transit macromolecules among organelles, whose membranes contain resident proteins needed for driving the transport. These resident membrane proteins return by retrograde vesicular transport. Apart from this, the pathway involves endocytosis. The model yeast Saccharomyces cerevisiae and vertebrate cells were found to contain very similar gene products regulating the molecular mechanism of glycoprotein transport, and the cellular mechanism of their secretion pathways was therefore also presumed to be identical. Biochemists have postulated that, in S. cerevisiae, the translocation of peptides through the endoplasmic reticulum membranes into the lumen of ER cisternae and the core glycosylation is followed by a vector-mediated transport into the functional cascade of the Golgi system cisternae and between them. This is the site of maturation and sorting of glycoproteins, before the ultimate transport by other vectors involving either secretion from the cells (exocytosis across the plasmalemma into the cell wall) or transport into the lysosome-like vacuole via a prevacuolar compartment, which serves at the same time as a primary endosome. The established cellular model of secretion deals with budding yeast; interphase yeast cells, in which the secretion is limited and which predominate in exponential cultures, have not been taken into consideration. The quality of organelle imaging in S. cerevisiae ultra-thin sections depends on the fixation technique used and on specimen contrasting by metals. The results achieved by combinations of different techniques differ mostly in the imaging of bilayers of membrane interfaces and the transparence of the matrix phase. Fixation procedures are decisive for the results of topochemical localisations of cellular antigenic components or enzyme activities, which form the basis of the following survey of functional morphology of organelles involved in the yeast secretory pathway. The existing results of these studies do not confirm all aspects of the vertebrate model of the Golgi apparatus proposed by molecular geneticists to hold for S. cerevisiae, and alternative models of the cellular mechanism of secretion in this yeast are, therefore, also discussed.     

Histologic investigation of glycol methacrylate embedded chick embryonic tissue.

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

A review of rapid microwave fixation technology: Its expanding niche in morphologic studies

Microwave (MW) fixation methods are important because excellent preservation of both cell structure and antigenicity can be attained several orders of magnitude faster than by routine chemical fixation methods. However, because of the limitations of commercial MW ovens, fixation results are often irreproducible. We present a standardization protocol for MW fixation in household MW ovens that emphasizes magnetron warm-up; the use of a water load during sample irradiation, of an agar/saline/Giemsa model to evaluate uniformity of irradiation within the MW cavity, and of specimen containers with one dimension less than 1.5 cm; and fast specimen handling to prevent conductive heating artifacts after irradiation. We describe a prototypic MW device that improves the precision of sample irradiation and fixes blocks of tissue and cells in suspension in milliseconds. The solutions used to immerse the specimen during irradiation influence the specimen morphology. Aldehyde- or osmium-containing solutions used simultaneously with MW irradiation resulted in the best morphologic preservation of specimens up to 1 cm³. Using MW fixation methods and a postembedding, ultrastructural immunogold-labeling approach, we have localized granule chymase and histamine in rat mast cells and amylase in rat parotid acinar cells.     

Influence of aldehyde fixation on the morphology of endosomes and lysosomes: quantitative analysis and electron tomography

Cryoimmobilization is regarded as the most reliable method to preserve cellular ultrastructure for electron microscopic analysis, because it is both fast (milliseconds) and avoids the use of harmful chemicals on living cells. For immunolabelling studies samples have to be dehydrated by freeze‐substitution and embedded in a resin. Strangely, although most of the lipids are maintained, intracellular membranes such as endoplasmic reticulum, Golgi and mitochondrial membranes are often poorly contrasted and hardly visible. By contrast, Tokuyasu cryosectioning, based on chemical fixation with aldehydes is the best established and generally most efficient method for localization of proteins by immunogold labelling. Despite the invasive character of the aldehyde fixation, the Tokuyasu method yields a reasonably good ultrastructural preservation in combination with excellent membrane contrast. In some cases, however, dramatic differences in cellular ultrastructure, especially of membranous structures, could be revealed by comparison of the chemical with the cryofixation method. To make use of the advantages of the two different approaches a more general and quantitative knowledge of the influence of aldehyde fixation on ultrastructure is needed. Therefore, we have measured the size and shape of endosomes and lysosomes in high‐pressure frozen and aldehyde‐fixed cells and found that aldehyde fixation causes a significant deformation and reduction of endosomal volume without affecting the membrane length. There was no considerable influence on the lysosomes. Ultrastructural changes caused by aldehyde fixation are most dramatic for endosomes with tubular extensions, as could be visualized with electron tomography. The implications for the interpretation of immunogold localization studies on chemically fixed cells are discussed.     

铈在铝铜合金凝固过程中的作用

本文在定向凝固的条件下考察了稀土元素Ce对Al-Cu合金平面生长、胞状生长及枝晶生长的界面稳定性,一次及二次枝晶间距的影响;借此探讨了一般微量合金元素在凝固过程中的作用。研究表明,Ce降低Al-Cu合金的界面稳定性,平面生长时,临界G/V值与Ce含量关系的回归处理结果是：G/V＝0.38[4.5＋(2～3)C (Ce)]（Al－4.5%Cu）。Ce含量较小时减小一次枝晶间距,但含量较大时反而增加一次枝晶间距。随着Ce含量的增加,二次枝晶间距减小。分析表明,只有当微量合金元素的平衡分配系数k很小及C 0/k较大时,才会对合金的凝固过程产生明显影响。     

Atomic force microscopy applied to study macromolecular content of embedded biological material

We demonstrate that atomic force microscopy represents a powerful tool for the estimation of structural preservation of biological samples embedded in epoxy resin, in terms of their macromolecular distribution and architecture. The comparison of atomic force microscopy (AFM) and transmission electron microscopy (TEM) images of a biosample (Caenorhabditis elegans) prepared following to different types of freeze-substitution protocols (conventional OsO4 fixation, epoxy fixation) led to the conclusion that high TEM stainability of the sample results from a low macromolecular density of the cellular matrix. We propose a novel procedure aimed to obtain AFM and TEM images of the same particular organelle, which strongly facilitates AFM image interpretation and reveals new ultrastructural aspects (mainly protein arrangement) of a biosample in addition to TEM data.     

Preparation of resin embedded unicellular organisms without the use of fixatives and dehydration media.

A method is presented for processing single cells for conventional ultrathin sectioning without the use of fixatives and dehydration media. The cells were fixed by a physical method--spray freezing--which provides extremely high cooling rates, needs no pretreatment with cryoprotective agents and is therefore assumed to maintain the in vivo morphology of the cell. Hitherto cells prepared in this way have been investigated exclusively by freeze etching. To combine the advantages of this method with those of conventional ultrathin sectioning we have processed spray frozen cells with widely varying water contents (spermatozoa and lymphocytes) by freeze drying at 188 K and vacuum embedding. When compared to conventional chemical fixation the differences found in ultrastructural preservation of spermatozoa using this kind of preparation were confined to the arrangement of spermhead membranes and middlepiece structures. Lymphocyte structure was much closer to that known from chemical preparation, the only differences being a denser cytoplasm, denser mitochondrial matrices and thicker plasma membranes. These differences are probably due to the absence of eluating and dissolving effects present in conventional chemical preparations. The ultrastructural preservation of spray frozen cells is not different after freeze etching or after freeze-drying and vacuum embedding. This indicates clearly that drying and resin embedding does not produce artefacts and that structural preservation is therefore limited by the quality of cryofixation. Therefore this method is considered a contribution to the problem of preservation of the in vivo assembly of cellular substructure. Furthermore it seems to be a potential basis for preparation of soluble or diffusible substances or cellular compounds which would be influenced by fixatives and dehydrating agents.     

Transmission electron microscopy of yeast

The challenges of sample preparation can limit a researcher's selection of transmission electron microcopy (TEM) for analysis of yeast. However, with the exception of thin sectioning, preparation of well-fixed and infiltrated samples of yeast cells is achievable by any reasonably equipped laboratory. This review presents a general overview of TEM sample preparation methods and detailed protocols for chemical fixation of yeast for ultrastructural analysis and immunolabeling. For ultrastructural analysis, the most commonly used chemical fixation involves treatment with glutaraldehyde followed by either potassium permanganate or osmium. Prior to osmium postfixation, the cell wall must be enzymatically digested to allow optimal fixation and embedding. Freeze substitution methods continue to provide the highest quality of fixation, but equipment needed for these protocols is not generally available to many labs. The low viscosity of Spurr's resin makes it the resin of choice for ultrastructure studies. Immunoelectron microscopy has enjoyed great success in analysis of yeast molecular organization. For immunoelectron microscopy, glutaraldehyde/formaldehyde-fixed cells are embedded in LR White resin. The thin sections are then treated in much the same way as an immunoblot: following blocking, they are incubated in primary antiserum, washed, and then incubated in gold-labeled secondary antiserum.     

Differential sensitivity of neurosecretory and non-neurosecretory neurones of an aphid to sucrose during fixation for electron microscopy

The Group 1 neurosecretory cells and a proportion of the non-neurosecretory cells in the protocerebrum of the aphid Megoura viciae show gross differences in preservation when sucrose is used to increase the osmolarity of a glutaraldehyde primary fixative and an osmium tetroxide secondary fixative. The neurosecretory cells are well preserved whilst a proportion of the non-neurosecretory cells are shrunken and electron dense, the proportion of affected cells increases with the concentration of sucrose used. This difference is not seen when either the primary or the secondary fixatives are used alone, when a mixture of the primary and secondary fixatives is used or when the osmotic pressure is increased with inorganic salts.     

Preparation of fetal rat brains for light and electron microscopy

To study cellular shapes, growth patterns, and fine structure during early stages of CNS development in rat embryos, preparative procedures were evaluated and modified to meet two criteria: (1) Coronal semithin sections should reveal undeformed telencephalic hemispheres that were symmetrically expanded on both sides of midline structures and were surrounded by contiguous mesenchyme. (2) In electron micrographs, cells should have intact, undistorted surface membranes, evenly distributed nucleoplasm and well preserved cytoplasmic organelles. To meet these criteria, 378 fetuses with a gestational age of 11–20 days (E11–E20) were used to test and modify procedures for anesthesia, embryo removal and handling, dissection, fixation, dehydration, and embedding of the embryonic CNS. Most specimens were in an early stage of development (E11–E13), which, in case of the neopallial wall, is the preneural period. The tests produced methods that met the above criteria and identified the most common artifacts and their causes. Deformities of the cerebral hemispheres and separations between the brain and its coverings were usually caused by trauma during embryo removal and during handling before fixation. Changes in cellular volumes, especially swelling during fixation and dehydration, were the most important causes of histological artifacts. The procedures and methods that consistently produced the best light and electron microscopic preservation of the E11–E13 rat CNS are described. Fixation was best when the brains were treated with glutaraldehyde and s-collidine buffer, followed by osmium tetroxide in s-collidine buffer. A surprisingly beneficial effect of sodium chloride in the dehydrating alcohol was noted.     

A different fixation of the femoral component in total knee arthroplasty may lead to preservation of femoral bone stock

Good femoral bone stock is important for the stability of the femoral component in revision knee arthroplasty. However, the primary total knee replacement (TKR) may cause significant loss of bone stock in the distal anterior femur. Earlier stress-induced bone remodelling simulations have suggested that a completely debonded component may save bone stock in the distal anterior region. However, these simulations did not consider the fixation of a debonded implant and possible secondary effects of micromotions and osteolysis at the interface. The current study tries to combine the preservation of bone stock with adequate component fixation. Different bone remodelling simulations were performed around femoral knee components with different sizes of bonding area and different friction characteristics of the debonded area. The fixation of the femoral component with different bonding characteristics is quantified with calculated implant-bone interface stresses. The results show that a bonded femoral component with a debonded inner side of the anterior flange may significantly reduce bone resorption in the endangered distal anterior femur, without jeopardizing the fixation of the femoral implant. This effect may be obtained in vivo by using a femoral component with a highly polished inner side of the anterior flange.     

Transpiration-assisted perfusion fixation provides in situ preservation of developing ray parenchyma cells in Eucalyptus nitens

The technique of animal vascular perfusion fixation was adapted for in situ fixation of the fragile and difficult to access cells of the ray parenchyma system in stems of 10-year-old Eucalyptus nitens trees. In situ fixation enabled tissue to be safely dissected for histological processing without risk of damage to microstructure or initiation of wound response. Fixative was perfused through the active vascular system under hydrostatic and transpiration pressure directly to vessel-associated ray cells. Diffusion from vessels allowed fixative to access nonvessel-associated ray cells. Acrolein was included to aid diffusion fixation and safranin dye was included to define fixed regions within the xylem. Sections prepared for light and electron microscopy from samples cut from regions showing intense safranin staining showed good microstructural preservation and were free of artefacts caused by mechanical injury or wound response.     

Tracing DiO-labelled tumour cells in liver sections by confocal laser scanning microscopy

Investigating rare cellular events is facilitated by studying thick sections with confocal laser scanning microscopy (CLSM). Localization of cells in tissue sections can be done by immunolabelling or by fluorescent labelling of cells prior to intravenous administration. Immunolabelling is technically complicated because of the preservation of antigens during fixation and the problems associated with the penetration of the antibodies. In this study, an alternative and simple approach for the labelling of cells in vitro with the fluorescent probe DiO and its subsequent application in vivo will be outlined. The method was applied to trace DiO‐labelled colon carcinoma cells (CC531s) in 100 µm thick liver sections. In vitro and in vivo experiments revealed that DiO‐labelling of CC531s cells had no cytotoxic or antiproliferative effect and the cells preserved their susceptibility towards hepatic NK cells or Kupffer cells. In addition, DiO remained stable for at least 72 h in the living cell. DiO‐labelled CC531s cells could be traced all over the tissue depth and anti‐metastatic events such as phagocytosis of tumour cells by Kupffer cells could be easily observed. In situ staining with propidium iodide (nucleic acids) or rhodamine‐phalloidin (filamentous actin) resulted in additional tissue information. The data presented improved the understanding of the possible effects of the vital fluorescent probe DiO on cell function and provided a limit of confidence for CLSM imaging of DiO‐labelled cells in tissue sections.