Quantitative X-ray microanalysis of biological specimens

Qualitative X-ray microanalysis of biological specimens requires an approach that is somewhat different from that used in the materials sciences. The first step is deconvolution and background subtraction on the obtained spectrum. The further treatment depends on the type of specimen: thin, thick, or semithick. For thin sections, the continuum method of quantitation is most often used, but it should be combined with an accurate correction for extraneous background. However, alternative methods to determine local mass should also be considered. In the analysis of biological bulk specimens, the ZAF-correction method appears to be less useful, primarily because of the uneven surface of biological specimens. The peak-to-local background model may be a more adequate method for thick specimens that are not mounted on a thick substrate. Quantitative X-ray microanalysis of biological specimens generally requires the use of standards that preferably should resemble the specimen in chemical and physical properties. Special problems in biological microanalysis include low count rates, specimen instability and mass loss, extraneous contributions to the spectrum, and preparative artifacts affecting quantitation. A relatively recent development in X-ray microanalysis of biological specimens is the quantitative determination of local water content.     

Vitreous cryo-sectioning of cells facilitated by a micromanipulator

Sectioning vitrified cells and tissues for cryo‐electron microscopy is more challenging than room‐temperature sectioning of plastic‐embedded samples. As the sample must be kept very cold (相似文献   

Serial reconstruction of inner hair cell afferent innervation using semithick sections

The afferent innervation pattern of inner hair cells in the apex of the guinea pig cochlea was studied using serial reconstruction of semithick (0.25–μm) sections and high-voltage electron microscopy (HVEM). This thickness produced a good compromise between the ability to resolve details of the synaptic contacts between the hair cells and sensory neurons and the number of sections required to reconstruct the nerve terminals within the receptor organ. The use of a goniometer allowed the sections to be tilted to angles optimum for viewing either the synaptic membrane specializations or the presynaptic bodies. Reasonably good images of 0.25-μm sections could be obtained using a conventional 120-keV microscope, but the images produced by the HVEM were clearly superior. The sensory nerve terminals and hair cells were reconstructed using a microcomputer-based computer-aided-design system. Nerve terminals with complex shapes could be successfully rendered as surface models viewed as stereo pairs. The advantages and limitations of the techniques used are discussed.     

Light microscope based analysis of three-dimensional structure: Applications to the study of Drosophila salivary gland nuclei. I. Data collection and analysis

Many biological structures of interest are large enough that they may be viewed by light microscope methods, yet they are sufficiently complicated that interpretation of what is seen is quite difficult. The salivary gland nuclei from Dipterans are an example of this. Previous attempts at determining the path of the giant chromosomes in these nuclei have depended on the laborious construction of models by hand. A unified Computer Aided Modelling and Analysis system (CAMA) has been implemented, allowing data collection and analysis of structures visible by light microscopy. This system is extendible to the analysis of electron micrographs of serial sections or of other data consisting of images present in a stack.     

Low temperature techniques for X-ray microanalysis in pathology: Alternatives to cryoultramicrotomy

Many diseases are associated with a change in the distribution of diffusible ions at the cell or tissue level. These diseases can profitably be studied by X-ray microanalysis. This technique for the study of ion distribution requires the use of cryoprepared specimens. Analysis at low or medium resolution can be carried out on thick or semi-thick cryosections, or on frozenhydrated or freeze-dried embedded bulk samples. Such analyses are particularly useful in the initial stages of an investigation or when data from a large number of samples have to be acquired. Also X-ray microanalysis of cultured or single cells prepared by freeze-drying can be used to rapidly collect information on a large number of cells. Analysis at high resolution has to be carried out on thin sections: Cryosections or sections of freeze-substituted or freeze-dried embedded tissue. For the latter type of specimens, the use of low-temperature embedding methods may have important advantages.     

Simplified nerve cell counting in the rat brainstem with the physical disector using a drawing-microscope

A simple modification of the physical disector is presented, which is used to count the number of neurons in the hypoglossal nucleus of the rat in a series of paraffin sections. One disector consists of two adjacent sections (6 μm thick) that have been Nissl-stained with cresyl fast violet. In the first step of the procedure each of the two sections is investigated separately with a drawing-microscope. The boundary of the hypoglossal nucleus and the position of neurons devoid of, or containing a part of, the cell nucleus in the plane of the section are marked on transparent paper. In the second step, these two drawings are placed one upon another, aligned and the number of cell profiles that show a cell nucleus in one but not in both drawings counted. This modification of the disector method for cell counting needs no specialized equipment, simply a light microscope with drawing apparatus, and can be combined with histochemical studies of other sections from the same tissue block.     

Rat kidney glomerular basement membrane visualized in situ by embedment-free sectioning and subsequent platinum-carbon replication

Following the removal of polyethylene glycol (PEG) from thin sections, and viewing through the endothelial fenestrae and/or the interpedicel spaces, the rat renal glomerular basement membrane in situ was revealed to consist of meshworks and to be electron-transparent when examined at right angles to the plane of the membrane. By subsequent platinum replication of the embedment-free sections, the lamina densa of the basement membrane appeared as a veil composed of rather closely packed particles. The architecture of the slit diaphragm and the surface morphology of the endothelial cell membrane were also clearly revealed. The present results indicate that the PEG method, with or without replication, can provide valuable information on basement membrane morphology.     

Estimating mean particle volume and number from random sections by sampling profile boundaries

We describe some new shape-independent stereological estimates of particle mean volume and surface area. Finding volumes or surface areas of cell nuclei, from electron micrographs of random thin sections, is a central problem of biological stereology. The well-known point-sampled intercept (PSI) method samples profile interiors to find the volume-weighted mean volume. This can be used in place of the true mean volume, but to do so introduces bias when volumes vary a great deal, as they do in fixed specimens. Jensen and Gundersen quite recently extended the PSI estimator to provide particle surface area, with no bias in the case of uniform surface areas. Here we extend the PSI volume estimator in a different way, sampling profile boundaries rather than their interiors. We obtain a boundary-sampled intercept (BSI) volume estimator, simpler than the PSI surface area estimator, but also unbiased for uniform surface areas. Both of these estimators are attractive, for example, in measuring and counting cell nuclei, where membrane surface area varies less than volume. Furthermore, they have no shape bias whatsoever. This paper also examines the general relationship between boundary- and area-sampled estimates, and we clarify the formal connection between our volume estimator and the PSI surface area estimator. We also calculate and compare their theoretical efficiencies.     

Gap-junction quantification in biological tissues: freeze-fracture replicas versus thin sections

The relative efficiency of freeze-fracture replicas versus thin sections for the visualization and quantification of gap junctions in biological tissues has been evaluated. Both methods may underestimate gap-junction number—thin sections for reasons of tissue resolution and freeze-fracture replicas due to the mechanics of the fracturing process. Freeze-fracture misses gap junctions in regions of plasma membrane which are highly contoured, such as the overlapping basal cell processes of Drosophila imaginal wing discs and the interdigitating lateral membrane plications of intercalated discs in cardiac tissue. If the missed gap junctions are relatively large, as they are in both of these examples, freeze-fracture significantly underestimates the total gap-junctional area. Thin sections may miss small gap junctions, but in tissues which contain a range of gap-junction sizes the lost junctions constitute a relatively small fraction of the total junctional area. In neoplastic imaginal wing discs, thin sections were as efficient as freeze-fracture replicas in identifying even the smallest gap junctions. Although freeze-fracture may be the better technique for the qualitative and quantitative documentation of small gap junctions in tissues with relatively flat to gently contoured plasma membranes, and thin sections may be the superior method for gap-junction quantification in tissues containing a range of gap-junctional sizes and highly contoured cellular processes, the data suggest that a combination of the two approaches should be utilized whenever possible.     

Comparison of different methods for thin section EM analysis of Mycobacterium smegmatis

Bacteria are generally difficult specimens to prepare for conventional resin section electron microscopy and mycobacteria, with their thick and complex cell envelope layers being especially prone to artefacts. Here we made a systematic comparison of different methods for preparing Mycobacterium smegmatis for thin section electron microscopy analysis. These methods were: (1) conventional preparation by fixatives and epoxy resins at ambient temperature. (2) Tokuyasu cryo-section of chemically fixed bacteria. (3) rapid freezing followed by freeze substitution and embedding in epoxy resin at room temperature or (4) combined with Lowicryl HM20 embedding and ultraviolet (UV) polymerization at low temperature and (5) CEMOVIS, or cryo electron microscopy of vitreous sections. The best preservation of bacteria was obtained with the cryo electron microscopy of vitreous sections method, as expected, especially with respect to the preservation of the cell envelope and lipid bodies. By comparison with cryo electron microscopy of vitreous sections both the conventional and Tokuyasu methods produced different, undesirable artefacts. The two different types of freeze-substitution protocols showed variable preservation of the cell envelope but gave acceptable preservation of the cytoplasm, but not lipid bodies, and bacterial DNA. In conclusion although cryo electron microscopy of vitreous sections must be considered the 'gold standard' among sectioning methods for electron microscopy, because it avoids solvents and stains, the use of optimally prepared freeze substitution also offers some advantages for ultrastructural analysis of bacteria.     

Comparison of polyethylene glycol and diethylene glycol distearate embedding methods for the preservation of fungal cytoskeletons

Hyphae of the fungus Basidiobolus magnus were embedded in extractable polyethylene glycol (PEG) or diethylene glycol distearate (DGD) to prepare embedment-free sections in order to seek components of the cytoskeleton that may be obscured in epoxy-embedded sections. All methods showed that hyphae possess an intricate filamentous matrix, which is apparently unoriented. However, the appearance of the cytoskeleton depended on the embedding medium, the solvent used during embedding, and whether cells were fixed by conventional fixation or freeze-substitution. PEG proved to be the best embedding medium for fungal cells because DGD caused cell shrinkage and produced a more lamellar than filamentous matrix. Also, the cytoskeletal filaments were clearer in freeze-substituted cells than in conventionally-fixed cells. Since we failed to find microtubules in the embedment-free sections, we re-embedded cells in Epon to discern whether microtubules or other cytoplasmic components had changed. Neither PEG nor DGD adequately preserved microtubules as compared to regular Epon-embedded sections, and other cellular structures were significantly altered. Therefore, alternative methods need to be employed to further characterize fungal cytoskeletons.     

Electron microscopy of cellulose in entire tissue

Microfibrillar structures can be seen in ultrathin sections of cell walls which have been post-stained with uranyl acetate and lead citrate. This stain is removable by washing, suggesting that a physical mechanism is involved. It is suggested that these structures are the cellulose microfibrils of the wall; they appear to be 3.5 nm in diameter and not fasciated into larger units. Freeze-etching of untreated tissue supports this conclusion. These two techniques seem to have many advantages over methods previously used for the study of microfibril arrangement and structure.     

Method for preparing thin sections of untreated equine hoof horn for electron microscopic examination

The preparation of hard tissues such as the equine hoof horn for electron microscopic examination is very difficult. In particular the penetration of fixatives and chemicals used during fixation and embedding is a problem. The objective of this study was to find and implement an alternative method enabling the preparation of high-quality thin sections of hoof horn and other hard tissue, which maintains the hard tissue ultrastructure and can be used for immuno-labeling. Compared to commonly used fixation and embedding techniques, the preparation of thin sections from untreated material method saves time and material and provides equivalent ultrastructural information. Furthermore, thin sections from untreated material are significantly larger and more homogeneous, more resistant to the electron ray, as well as more suitable for sectioning. The electron microscopical pictures obtained allow a comparison to previous test results achieved with fixed and embedded material. Using the preparation of thin sections from untreated material method, fixation and embedding artifacts are avoided, providing a clearer interpretation of the electron microscopical findings. Considerable advantages are achieved by using immunohistochemical techniques with untreated horn specimens because fixation invariably decreases antigenicity.     

A loop for the transfer of thin tissue sections

The construction and use of a simple wire loop for the transfer of thin tissue sections are described. The loop picks up, carries, and releases a drop of liquid containing the sections. The loop is opened and closed by manipulation of the forceps to which it is attached. This controls the attraction of the loop for the water drop and ensures reliable pickup and release of the sections.     

Cylindrical diameters method for calibrating section thickness in serial electron microscopy

An estimate of section thickness is required for measuring structures in serial section microscopy. Mean section thickness is estimated reliably by averaging the ratios of the diameters of cylindrical objects, such as mitochondria, to the number of sections they span. This cylindrical diameters method improves the accuracy of section thickness as inferred from the colour of sections floating in water. The cylindrical diameters method gives the same answer as that obtained by the minimal folds method. It is preferable because it can be done in a series that has no folds that can distort and obscure the objects that are being measured.     

Imaging thin and thick sections of biological tissue with the secondary electron detector in a field-emission scanning electron microscope

A field-emission scanning electron microscope (FESEM) equipped with the standard secondary electron (SE) detector was used to image thin (70–90 nm) and thick (1–3 μm) sections of biological materials that were chemically fixed, dehydrated, and embedded in resin. The preparation procedures, as well as subsequent staining of the sections, were identical to those commonly used to prepare thin sections of biological material for observation with the transmission electron microscope (TEM). The results suggested that the heavy metals, namely, osmium, uranium, and lead, that were used for postfixation and staining of the tissue provided an adequate SE signal that enabled imaging of the cells and organelles present in the sections. The FESEM was also used to image sections of tissues that were selectively stained using cytochemical and immunocytochemical techniques. Furthermore, thick sections could also be imaged in the SE mode. Stereo pairs of thick sections were easily recorded and provided images that approached those normally associated with high-voltage TEM.     

Pronounced loss of cell nuclei and anisotropic deformation of thick sections

The local deformation and variations in section thickness are studied in 100-μm thick vibratome sections of well-fixed human brain tissue. During processing, including drying on glass slides, the section thickness is reduced to less than half, but close to the edges there is less shrinkage of the section thickness. Close to both surfaces there is a pronounced reduction in the number of neuronal nucleoli. At the scale of the original section, the upper 15 μm and the lower 10 μm are depleted. The loss is most pronounced at the upper surface, which is unprotected during processing. In the central 70% of the section height, where one would ordinarily use an optical disector for sampling, there is no indication of non-uniform shrinkage. The simplest explanation for the observed loss of nucleoli is that all cells opened by the knife may lose their nuclei across an unprotected section surface. The observations do not generalize to other tissues and other preparation techniques, but illustrate the magnitude of some of the problems for uniform sampling and unbiased estimation in very thick sections. The uniform optical disector sampling of nucleoli in thick sections, as opposed to that of cell nuclei, raises a special problem, which is discussed briefly.     

A technique for studying one and the same section of a cell in sequence with the light and electron microscope

Methods for mounting and staining relatively thin sections on electron microscope grids, in order that one and the same section of a cell can be photographed in sequence with the light and electron microscope are described. Toluidine blue is used as a stain and hexachlorabuta-1,3 diene as a medium which enables the grid carrying the stained sections to be temporarily mounted under a coverslip and examined with an oil-immersion lens. Results obtained with pollen mother cells of Fritillaria lanceolata at zygotene are illustrated.     

A technique for revealing tissue embedded within resin blocks

A technique is described for revealing in fine detail large pieces of tissue still embedded within rough, scratched or partly-trimmed blocks of epoxy and other resin before sectioning. The pieces of tissue immersed in baths of oil or glycerol and illuminated by an appropriate optical system, may be photographed to provide a guide to subsequent sectioning, aiding the precise location of elements in the sections back to their position in the parent tissue, and for tracing the connections of the element between serial sections. The method has been used by the authors in sectioning for light microscopy, but could equally be used with sections for electron microscopy.