Relationship between structure and function in renal proximal tubule

Epithelia which support large transepithelial fluid movements are generally found to have histologic specializations which increase the surface areas of the cell membranes across which flows occur. A relationship between structure and function seems obvious in those cases. On the other hand, the area increasing specializations may also result in complicated shapes for the cells and their adjacent intercellular channels. In this paper we review the means for examining cell shape by quantitative stereologic techniques and the results obtained for the epithelium of the proximal renal tubule. We conclude that cell shape not only is a critical ingredient in any structure-function correlation for that tissue but also a “fingerprint” and a powerful tool with which one can predict and study epithelial absorptive flows and their driving forces.     

Identification of bacteria by studying one section under light microscopy,scanning, and transmission electron microscopy

A method for bacterial identification has been developed by means of studying the same histological sections through several types of microscopy. With this method, one section was processed and analyzed respectively for light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Sections of gingival biopsies were Gram stained and bacteria tentatively identified by LM. Photographs of the sections were taken and presketched transparent acetate sheets (PTAS) were made from the photos. The same section was later prepared for SEM, areas previously thought to contain bacteria were localized by placing the PTAS onto the SEM monitoring screen. The SEM specimens were subsequently processed for TEM, bacteria were located, and micrographs obtained. The results showed that out of ten diseased gingival biopsies observed under the LM, bacteria were found to be present in all the specimens and were identified as both Gram positive and Gram negative. By transferring the section from LM to SEM, the bacteria could be relocated and their morphotype (cocci, rods, etc.) clearly identified in most of the cases. Since cocci may resemble other biological granular structures under SEM, they require further analysis under TEM for additional positive identification. This study demonstrated that the method described here is a useful tool for assessing the presence and identifying bacteria within the gingival tissues.     

Sectionable microcarrier beads: An improved method for the preparation of cell monolayers for electron microscopy

Cross-linked dextran beads provide an excellent surface for tissue-cultured cell monolayers, and can be processed for transmission (TEM) and scanning (SEM) electron microscopy, as well as light microscopy (LM). Cells are grown to confluency on the surface of the microcarriers, where at any point aliquots can be removed and experimentally treated as desired (e.g. immunocytochemistry) providing a representative sample. Sample preparation for TEM follows standard procedures for any cell monolayer, but infiltration times must be at least doubled to allow penetration of the beads. The polymerized blocks can then be sectioned for TEM or LM with no additional steps required. SEM sample preparation involves attaching the fixed bead/cell suspension to a glass coverslip with poly-1-lysine, dehydration, critical point drying, and coating for conductivity. The fixed and dried sample can also be attached directly to the SEM stub as free beads and subsequently gold coated. These beads provide (1) an increased surface area of cells visible per area of thin section, (2) eliminates the careful orientation required for flat substrate methods of embedding, (3) decreases the amount of sample manipulation in the forms of re-embedding and gluing, and (4) decreases the amount of drying artifact seen as cracking in SEM monolayer preparations.     

Study of the tribologically transformed structure created during fretting tests

In fretting, without a lubricant, structures fail through wear involving debris. Various metallographic assessments show that this debris creation derives from a special structure, hereafter called the “tribologically transformed structure” (TTS). The latter is nucleated at the very beginning of a contact's life, and eventually masks the initial material's tribological behaviour. This TTS is very hard and brittle, and is quickly destroyed, which explains why TTS is rather difficult to observe. This study focuses on initiation mechanisms which, up to now, are still highly controversial. Large amounts of TTS are observed after fretting tests, which is the reason why they were chosen as the means of investigation. In this paper, the various possible creation mechanisms for TTS will be analysed. From this overview, two nucleation models are chosen: one is based on “mechanical alloying” considerations, and the other is linked to high-strain-recrystallisation phenomena. Transmission electron microscopy (TEM) investigations are used to check these assumptions.     

Material contrast in SEM: Fermi energy and work function effects

‘Is it possible to assign various grey levels of a scanning electron microscope (SEM) image to different components of a given sample? Among other instrumental effects, the answer is not only a function of the respective secondary electron emission (SEE) yields of the components, δ, but also of the angular fraction of the secondary electrons (SE)s being collected, k_α and of a possible voltage contact effect between sample and detector, k_?. Expressed as a function of E_F, Fermi energy, and ?, work function of the components of interest, equations of spectral, (∂δ/∂E_k), and angular, (∂δ/∂α) distributions of the emitted SEs permit to evaluate k_α and k_? for Au and Si. It has been established that collected SE spectra, ∂δ_α/∂E_k, are distorted with respect to the emitted and fraction k_α is material dependent for a solid angle of detection Ω° less than 2π (or maximum semi-apex angle α_max<90°) In particular, for coaxial detections around the normal incident beam the detected fraction of SEs from Au, k_α(Au), is slightly larger than that for Si, k_α(Si). For simple geometries in the vacuum gap, similar investigations show that parameter k_φ is also larger for gold than for n-doped Si as well as for p-doped Si with respect to n-doped Si. Then Au is always quite brighter than n-doped Si in the SEM images while a doping contrast, C, due to a work function effect may reach ∼15% for a Si p/n junction with N_p∼10¹⁶ and N_n∼10¹⁵ cm⁻³. The present analysis may be extended to some metals such as Ag, Cu, Pb, Pd, Pt, and Zn that are expected to appear brighter than Si(n) and Ge in the SEM images.     

Local crystal structure analysis with several picometer precision using scanning transmission electron microscopy

We report a local crystal structure analysis with a high precision of several picometers on the basis of scanning transmission electron microscopy (STEM). Advanced annular dark-field (ADF) imaging has been demonstrated using software-based experimental and data-processing techniques, such as the improvement of signal-to-noise ratio, the reduction of image distortion, the quantification of experimental parameters (e.g., thickness and defocus) and the resolution enhancement by maximum-entropy deconvolution. The accuracy in the atom position measurement depends on the validity of the incoherent imaging approximation, in which an ADF image is described as the convolution between the incident probe profile and scattering objects. Although the qualitative interpretation of ADF image contrast is possible for a wide range of specimen thicknesses, the direct observation of a crystal structure with deep-sub-angstrom accuracy requires a thin specimen (e.g., 10 nm), as well as observation of the structure image by conventional high-resolution transmission electron microscopy.     

Comparison between the conventional method and the simple desiccation method in porcine sperm processing for scanning electron microscopy

Sperm quality has been studied using different microscopic methods. Light microscopy gives similar percentages of sperm typologies (mature, immature and aberrant) as scanning electron microscope (SEM) techniques using both normal (critical-point drying) and simple desiccation techniques. Artefactual forms, which are easily recognizable, are not taken into account. The artefactual forms observed by the simple desiccation SEM method were spermatozoa with tails folded in a simple loop, and spermatozoa with short tails coiled at their extreme. The simple desiccation SEM method is a useful and rapid technique for the testing of porcine sperm quality.     

High-Resolution scanning electron micrographs of freeze-cracked cells in testes from normal and irradiated rats at different stages of gonadal development

Newly developed techniques in high-resolution scanning electron microscopy (SEM) and for tissue-processing procedures have been applied to an investigation of structures of various cells in rat testes at different stages of gonadal maturation. A series of high-resolution SEM micrographs are presented which survey the surfaces of different types of testis cells during normal development, and which also illustrate ultrastructural features of some of their intracellular organelles. In addition, a series of high-resolution SEM micrographs are presented which compare the structural features of Sertoli cells in normal testes with those in germ-cell-depleted testes obtained from rats killed at varying times after having been irradiated in utero. We describe our observations on the structural properties of surfaces and intracellular organelles in Sertoli cells, Leydig cells, peritubular myoid cells, and some classes of germinal cells. We also consider the possible role of Sertoli cell apical cytoplasmic processes in lumen formation. Similarities are pointed out between the structure of germ-cell-depleted testes, resulting from irradiation in utero, and the structure of germ-cell-depleted testes in seasonal breeders during periods of involution. Finally, we discuss advantages and disadvantages of methods employed to reveal the fine structure of intracellular organelles in cells of the testis.     

Preparing and analyzing fractured archaeological fibers

A technique was developed to prepare archaeological fiber cross sections for electron microscopic examination and x-ray analysis. Use of this new method allows chemical and morphological information to be obtained from the interior of a single fiber or yarn. Fibers are fractured while frozen and then freeze dried. Following mounting and carbon coating, fibers are examined by scanning and backscatter electron microscopy and then analyzed by using energydispersive spectrometry. Elemental distribution is mapped by using image-processing software. In this report, the described technique is employed in the examination of ancient fibers from three different long-term storage environments (moist buried, dry buried, museum stored). Data obtained by examining the interior of fibers such as these provide insight into the conditions of a fiber's growth, the treatments applied during the fiber's processing and use, and the conditions in which the fiber was stored.     

Quantitative analysis of synapse structure with a semiautomatic interactive computer system: A study on identified pyramidal tract neurons in the sensory-motor cortex of cat

Pyramidal tract (Pt) neurons in the sensory-motor cortex of cat were labeled by injection of HRP into the spinal cord. Ultrastructural and quantitative analysis of the synaptic covering of their soma and apical dendrite (up to 100 μm from soma) was undertaken. We intercalated a visual-manual treatment between composite electron micrographs and a fully automated computer system and developed specific programs for evaluation of the morphometric data. Programs are included. A total of 412 synaptic boutons were examined that were found in contact with large Pt neurons. The mean linear percentage of the surface area covered by boutons was 26.2 ± 8.4% and the mean contacting length and cross-sectional area of the bouton profiles were 1.28 ± 0.58 μm and 0.89 ± 0.59 μm², respectively. All types of boutons with active zones accounted for 41.2% of the total. The distribution of two types of bouton (S- and F-type boutons, showing asymmetric and symmetric contacts, respectively) was examined quantitatively. The mean proportion of F-type boutons was 89.1% with a soma and S-type boutons contacting apical dendrites was 10.9%. In addition, GABAergic boutons were identified with the soma by immunocytochemistry with antibodies against glutamic acid decarboxylase. They formed symmetric synaptic contacts with the Pt cells that were identical to those formed by F-type boutons. The quantitative analysis revealed that synaptic clefts are narrower and synaptic vesicles are smaller in symmetric F-type boutons than in S-type boutons forming asymmetric contacts. These data establish that at least three parameters (postsynaptic density, synaptic cleft, and size of vesicles) can be utilized singly or in combination to identify GABAergic inhibitory synapses in neocortex.     

Construction of 35-millimeter camera adaptors for scanning electron microscopes

Described here is the construction of two 35-mm camera adaptors for attachment to the recording monitors of scanning electron microscopes (SEM). The designs are simple and readily adaptable to almost any SEM. The choice of this camera format for recording SEM images is one of convenience as well as economy and does not sacrifice micrograph quality.     

Application of two-bath development processing to transmission electron films and plates

In particular areas of materials research transmission electron microscopy has traditionally been plagued by the seemingly inherent problems of excessive photographic contrast and high densities. This need not always be the case. This article will suggest that the use of a development process in which the developing agent and its accelerator are split into two baths can accomplish the desirable effects of compressing the contrast range of the negative and limiting excessive density without loss of recorded detail. The developer used in the study is a commercially available formula called Diafine. Two-bath developers can be formulated by individuals. At the time of writing, Diafine was the only off-the-shelf split developer known to the author.     

High resolution observations of friction-induced oxide and its interaction with the worn surface

A detailed transmission electron microscopy study of oxide and oxygen-containing phase formation during the sliding wear of metals, composites and coatings is provided. A wide range of different materials types are reported in order to compare and contrast their oxidational wear behaviour: a low carbon stainless steel, a H21 tool steel containing 7%TiC particles, a 17%Cr white iron, an Al–Si/30%SiC composite, an Al–alloy (6092)–15%Ni₃Al composite and finally a 3rd generation TiAlN/CrN ‘superhard’ multilayer coating. For the ferrous alloys, nanoscale oxides and oxygen-containing phases were formed that exhibited excellent adhesion to the substrate. In all cases, an increase in oxide coverage of the surface was associated with a decrease in Lancaster wear coefficient. The oxide at the surface of the 316L and H21+7%TiC was found to deform with the substrate, forming a mechanically mixed layer that enhanced surface wear resistance. Evidence of oxidational wear is presented for the wear of the Al–Si–30%SiC composite, but this did not give a beneficial effect in wear, a result of the brittle nature of the oxide that resulted in detachment of fine (150nm) thick fragments. The worn surface of the Al–alloy (6092)–15%Ni₃Al and TiAlN/CrN coating was characterized by reaction with the counterface and subsequent oxidation, the product of which enhanced wear resistance. The observations are related to the classical theory of oxidational wear.     

Comparison of osmium/sonication and edta/sonication microdissection techniques in exposing the adepithelial basal lamina surface of developing rat colon

We have used two epithelial-stripping techniques in our studies of the basal lamina in the developing rat colon. The first involves prolonged osmication followed by sonication; the second uses chelation of calcium by EDTA followed by sonication. Both techniques remove the epithelium from the basal lamina; however, the EDTA/sonication technique appears to produce a cleaner adepithelial surface of the basal lamina. In addition, the fine structure of the basal lamina appears to be better preserved in specimens prepared by the EDTA/sonication technique. In contrast, the basal lamina of specimens prepared by the osmium/sonication technique has a shattered appearance that we believe is due to an increase in the fragility of the delicate fetal basal lamina.     

Backscattered electron imaging of immunogold-labeled and silver-enhanced microtubules in cultured mammalian cells

This technique permits the visualization of microtubules in situ by employing silver-enhanced immunogold labeling and backscattered electron imagery. For best results, monolayer cultures of PtK₂ cells are lysed with Triton X-100 in a microtubule stabilizing buffer, fixed with 1% glutaraldehyde, reduced with NaBH₄, incubated with monoclonal antitubulin and 5-nm gold-labeled anti-IgG, silver enhanced, freeze dried, lightly coated with aluminum, and examined in an SEM equipped with a backscattered electron detector. A high contrast view of the entire microtubule complex of each cell is obtained. Microtubules in freeze-dried preparations have relatively smooth surfaces, whereas those in critical point dried preparations are more irregular or beaded. At high magnifications, an unstained inner core of each microtubule can be resolved. Backscattered electron imaging appears to be a promising technique for localizing cytoskeletal proteins and other intracellular antigens that can be labeled with immunogold and enhanced with silver.     

Specimen handling and data interpretation in x-ray microanalysis of frozen hydrated etched gastrointestinal tract

Low temperature scanning electron microscopy of mammalian tissue allows microanalysis data and morphological results to be considered together, without the risk of elemental diffusion introduced by liquid fixatives and processing agents. The technique, however, is time consuming and there are many areas where errors are possible and standardization of technique is advisable. The present paper describes some of the problems inherent in handling frozen tissue and comparison is made between microanalytical measurements from luminal contents and those from villous epithelium.     

Dark field electron holography for strain measurement

Dark field electron holography is a new TEM-based technique for measuring strain with nanometer scale resolution. Here we present the procedure to align a transmission electron microscope and obtain dark field holograms as well as the theoretical background necessary to reconstruct strain maps from holograms. A series of experimental parameters such as biprism voltage, sample thickness, exposure time, tilt angle and choice of diffracted beam are then investigated on a silicon-germanium layer epitaxially embedded in a silicon matrix in order to obtain optimal dark field holograms over a large field of view with good spatial resolution and strain sensitivity.