A rapid and simple technique for correlating light microscopy,transmission and scanning electron microscopy of fixed tissues in Epon blocks

A simplified and standardized technique for close correlation between light microscopy (LM), transmission electron microscopy (TEM) and scanning electron microscopy (SEM) is described. Perfusion and immersion fixed tissue specimens were embedded in Epon 812 and cut for conventional LM and TEM. The Epon blocks with remaining tissue were thereafter treated with epoxy solvent (ethanol-NaOH solution) for partial epoxy resin removal only (dissolving rate approx 33μm/h). The blocks with partially blotted tissue specimens were then critically point dried and gold coated for SEM. This method, in an easy way, allows repeated observations with LM, TEM and SEM with preserved fine structure and exact correlation. Since the technique is so simple and there is no need for special equipment the method can easily be adopted in all laboratories with basic SEM standards.     

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.     

Comparative light microscopy,scanning electron microscopy and transmission electron microscopy of selected organic walled microfossils

Two simple techniques are described and illustrated. The first is for the study of one specimen by both light microscopy (LM) and scanning electron microscopy (SEM). The second is for the study of one selected specimen by LM, SEM and in ultrathin section by transmission electron microscopy (TEM). Although these techniques were developed for the comparative study of Precambrian organic walled microfossils (OWMs), they could be used for a wide range of other specimens.     

Staining tissue with methacrylate casts for light microscopy

Scanning electron microscopy (SEM) of methyl methacrylate casts and light microscopy (LM) of tissue are well-established methods for studying the microcirculation. The two are complimentary, but methacrylate is transparent and thus its presence is often not appreciated by LM. Histologic stains applied to methyl methacrylate in tissue sections would better identify by LM and allow the relationships with the SEM view of cast vasculature. We sought to test different stains on cast tissue to find one that would accent the cast. Surgically removed and autopsied human lungs were cast with methacry late and processed by routine light microscopic methods. They were stained with the hematoxylin and eosin, Masson trichome, elastic-van Gieson, Grocottmethenamine silver, Brown-Brennan, and Ziehl-Neelsen methods. The Ziehl-Neelsen procedure stained the methacry late best, giving it a red color. This procedure also worked well without heating. We conclude that (1) cast methacry late lung can be processed for routine LM with excellent results; (2) methacry late stains well with the Ziehl-Neelsen technique; (3) the acid-fast stained cast lung shows capillaries and cells in both normal and diseased lung better than the routine hematoxylin and eosin stain; (4) this technique can be used to assess filling and correlate findings on the same tissue with the two different microscopic methods.     

Quantitative DNA measurements in an instrument combining scanning electron microscopy and light microscopy

An instrument for combined scanning electron microscopy (SEM) and light microscopy (LM) to which a photometer unit is attached is described. A special stage in the vacuum chamber of a scanning electron microscope incorporates light microscope optics (objective and condenser) designed for transmission and epi-illumination fluorescence LM. An optical bridge connects these optics to a light microscope, without objective and condenser. The possibility of performing quantitative DNA measurements in this combined microscope (the LM/SEM) was tested using preparations of either chicken erythrocytes, human lymphocytes, or mouse liver cells. The cells were fixed, brought on a cover-glass, quantitatively stained for DNA, dehydrated, and critical point dried (CPD). After mounting the cells were coated with gold. The specimens were brought into the vacuum chamber of the combined microscope and individual cells were studied with SEM and LM. Simultaneously DNA measurements were performed by means of the photometer unit attached to the microscope. It is shown in this study that DNA measurements of cells in the combined microscope give similar results when compared to DNA measurements of embedded cells performed with a conventional fluorescence microscope. Furthermore, it is shown that although the gold layer covering the LM/SEM specimens weakens the fluorescence signal, it does not interfere with the DNA measurements.     

A simple method for correlative light,scanning electron microscopic and X-ray microanalytical examination of the same section

Thin paraffin sections, mounted on scanning specimen holders previously coated with polyester film tape (Minnesota Mining and MFG Co., Scotch film tape No. 850 gold), were processed for light microscopy (LM) in the conventional way, then covered with celloxin shellac and examined in the LM by using the upper illuminating source. After removal of the shellac from the surface of the sample by immersion in acetone, the sections were air-dried, coated with a copper layer in a vacuum evaporator and examined in a scanning electron microscope (SEM). The method allows: (i) high-quality LM possibilities for establishment of the diagnosis in pathological cases; (ii) SEM examination of the same area as observed in LM; and (iii) EPMA measurements of insoluble precipitates embedded in the tissue. The usefulness of the proposed method is obvious in cases where the composition of a precipitate on LM scale is to be compared with the LM appearance of the surrounding tissue.     

Developing 3D SEM in a broad biological context

When electron microscopy (EM) was introduced in the 1930s it gave scientists their first look into the nanoworld of cells. Over the last 80 years EM has vastly increased our understanding of the complex cellular structures that underlie the diverse functions that cells need to maintain life. One drawback that has been difficult to overcome was the inherent lack of volume information, mainly due to the limit on the thickness of sections that could be viewed in a transmission electron microscope (TEM). For many years scientists struggled to achieve three‐dimensional (3D) EM using serial section reconstructions, TEM tomography, and scanning EM (SEM) techniques such as freeze‐fracture. Although each technique yielded some special information, they required a significant amount of time and specialist expertise to obtain even a very small 3D EM dataset. Almost 20 years ago scientists began to exploit SEMs to image blocks of embedded tissues and perform serial sectioning of these tissues inside the SEM chamber. Using first focused ion beams (FIB) and subsequently robotic ultramicrotomes (serial block‐face, SBF‐SEM) microscopists were able to collect large volumes of 3D EM information at resolutions that could address many important biological questions, and do so in an efficient manner. We present here some examples of 3D EM taken from the many diverse specimens that have been imaged in our core facility. We propose that the next major step forward will be to efficiently correlate functional information obtained using light microscopy (LM) with 3D EM datasets to more completely investigate the important links between cell structures and their functions.     

Correlative light and backscattered electron microscopy of bone--Part I: Specimen preparation methods

A method for preparing nondecalcified bone and tooth specimens for imaging by both light microscopy (LM) and backscattered electron microscopy in the scanning electron microscope (BSE-SEM) is presented. Bone blocks are embedded in a polymethylmethacrylate (PMMA) mixture and mounted on glass slides using components of a light-cured dental adhesive system. This method of slide preparation allows correlative studies to be carried out between different microscopy modes, using the same histologic section. It also represents a large time savings relative to other mounting methods whose media require long cure times.     

Identification of monocot flora using pollen features through scanning electron microscopy

Pollen used to track structural and functional evolution in plants as well as to investigate the problems relative to plant classification. Pollen characters including ornamentation, shape, apertural pattern, pollen symmetry, colpus length, width, and margins used to detect the similarities and dissimilarities between genera and also species of the same genus. In this study pollen features of 20 monocot species belonging to 15 genera of the Amaryllidaceae, Asparagaceae, Iridaceae, Ixioliriaceae, Liliaceae, and Xanthorrhoeaceae were studied using scanning electron microscopy (SEM) and light microscopy (LM). In this study two species that is Zephyranthes citrina and Tulbaghia violacea were reported for the first time from Pakistan. Pollen grains were visualized with LM. Non‐acetolyzed and acetolyzed pollen were examined using SEM. A taxonomic key was developed to highlight the variation in pollen features in order to make their systematic application for correct species identification.     

Rapid combined light and electron microscopy on large frozen biological samples

The use of large unfixed frozen tissue samples (10 × 10 × 5 mm³) for combined light microscopy (LM) and electron microscopy (EM) is described. First, cryostat sections are applied for various LM histochemical approaches including in situ hybridization, immunohistochemistry and metabolic mapping (enzyme histochemistry). When EM inspection is needed, the tissue blocks that were used for cryostat sectioning and are stored at −80 °C, are then fixed at 4 °C with glutaraldehyde/paraformaldehyde and prepared for EM according to standard procedures. Ultrastructurally, most morphological aspects of normal and pathological tissue are retained whereas cryostat sectioning at −25 °C does not have serious damaging effects on the ultrastructure. This approach allows simple and rapid combined LM and EM of relatively large tissue specimens with acceptable ultrastructure. Its use is demonstrated with the elucidation of transdifferentiated mouse stromal elements in human pancreatic adenocarcinoma explants grown subcutaneously in nude mice. Combined LM and EM analysis revealed that these elements resemble cartilage showing enchondral mineralization and aberrant muscle fibres with characteristics of skeletal muscle cells.     

Characterization of an oil-degrading Microcoleus consortium by means of confocal scanning microscopy, scanning electron microscopy and transmission electron microscopy

A consortium of microorganisms with the capacity to degrade crude oil has been characterized by means of confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). The analysis using CLSM shows that Microcoleus chthonoplastes is the dominant organism in the consortium. This cyanobacterium forms long filaments that group together in bundles inside a mucopolysaccharide sheath. Scanning electron microscopy and transmission electron microscopy have allowed us to demonstrate that this cyanobacterium forms a consortium primarily with three morphotypes of the heterotrophic microorganisms found in the Microcoleus chthonoplastes sheath. The optimal growth of Microcoleus consortium was obtained in presence of light and crude oil, and under anaerobic conditions. When grown in agar plate, only one type of colony (green and filamentous) was observed.     

Rapid diagnosis of fungal infection of intravascular catheters in newborns by scanning electron microscopy.

Intravascular catheters carry a significant risk of becoming colonized with bacteria and fungi and are important risk factors of septicemia in premature neonates. The study was undertaken to evaluate whether scanning electron microscopy (SEM) examination of removed catheters can be useful in early diagnosis of plastic infection by Candida, providing information useful for initiation of an eventual therapy. The evolution of biofilms in 28 catheters (umbilical or central) implanted in 24 newborns for prematurity was studied by SEM and transmission electron microscopy (TEM). In 4 of 24 patients, SEM examination revealed the presence of Candida in form of yeast or hyphae. In one of these patients, TEM confirmed the presence of organisms. In each case, hemoculture and culture of the catheter itself confirmed the diagnosis. The study demonstrates that SEM can identify fungi in the biomaterials covering the catheter surface in a few hours, allowing an early diagnosis of plastic infection.     

Evaluation of demineralized dentin contraction by stereo measurements using environmental and conventional scanning electron microscopy

Numerous investigations of etched human dentin are performed using scanning electron microscopy (SEM). Usually specimens are fractured and cross sections of etched layers with underlying unaffected dentin are observed. Results from this study showed that the edge of the etched layer contracted and became curved after fracture of wet specimens and that tensile stresses were developed in this layer by acid etching. The degree of contraction was determined utilizing profiles of the specimen edges obtained with the help of stereo measurements. Fixation in glutaraldehyde decreased the contraction in wet specimens prepared for environmental scanning electron microscopy (ESEM). Fixation also decreased shrinkage of the demineralized layer due to gradual desiccation in the ESEM during observation. For conventional SEM, the contraction was minimized if specimens of etched and fixed dentin were fractured in the dry condition after dehydration.     

The fine structure of fenestrated adrenocortical capillaries revealed by in-lens field-emission scanning electron microscopy and scanning transmission electron microscopy

Cell biologists probing the physiologic movement of macromolecules and solutes across the fenestrated microvascular endothelial cell have used electron microscopy to locate the postulated pore within the fenestrae. Prior to the advent of in-lens field-emission high-resolution scanning electron microscopy (HRSEM) and ultrathin m et al coating technology, quick-freeze, platinum-carbon replica and grazing thin-section transmission electron microscopy (TEM) methods provided two-dimensional or indirect imaging methods. Wedge-shaped octagonal channels composed of fibrils interwoven in a central mesh were depicted as the filtering structures of fenestral diaphragms in images of platinum replicas enhanced by photographic augmentation. However, image accuracy was limited to replication of the cell surface. Subsequent to this, HRSEM technology was developed and provided a high-fidelity, three-dimensional topographic image of the fenestral surface directly from a fixed and dried bulk adrenal specimen coated with a 1 nm chromium film. First described from TEM replicas, the “flower-like” structure comprising the fenestral pores was readily visualized by HRSEM. High-resolution images contained particulate ectodomains on the lumenal surface of the endothelial cell membrane. Particles arranged in a rough octagonal shape formed the fenestral rim. Digital acquisition of analog photographic recordings revealed a filamentous meshwork in the diaphragm, thus confirming and extending observations from replica and grazing section TEM preparations. Endothelial cell pockets, first described in murine renal peritubular capillaries, were observed in rhesus and rabbit adrenocortical capillaries. This report features recent observations of fenestral diaphragms and endothelial pockets fitted with multiple diaphragms utilizing a Schottky field-emission electron microscope. In-lens staging of bulk and thin section specimens allowed tandem imaging in HRSEM and scanning TEM modes at 25 kV.     

Correlated light optical and scanning electron microscopy of Gram smears of bacteria and paraffin sections of cardiac muscle

Light-microscope slides (3 in. × 1 in.) bearing Gram smears of Erysipelothrix rhusiopathiæ, or Staphylococcus aureus, after preliminary examination under the light-optical microscope (LM), were cut down in size, glued onto specimen stubs, coated with gold and examined in the scanning electron microscope (SEM). These preparations served as a control for investigations into bacteria-cell junctions in tissue. Cover-slips from stained sections of staphylococcal or swine erysipelas endocarditis mounted on 3 in. × 1 in. microscope slides (which had been intensively studied previously with conventional light microscopy) were floated off by immersing the slides in xylol. After dehydration of the tissues on the slides, the preparations were treated similarly to the Gram smears, and were examined with the SEM. Lesions of endocarditis were thus examined, and the information gained from these preliminary examinations shed new light on the pathogenesis of the disease. This information had not previously been available by any other technique. Because of this, and in view of the simplicity of preparing sections for scanning electron microscopy, it is suggested that the SEM might be a useful tool to be applied to routine histological sections.     

Implication of scanning electron microscopy as a tool for identification of novel,nonedible oil seeds for biodiesel production

Biodiesel is a promising, bio-based, renewable, nontoxic, environment friendly, and alternative fuel for petroleum derived fuels which helps to reduce dependency on conventional fossil fuels. In this study, six novel, nonedible seed oil producing feedstock were explored for their potential for sustainable production of biodiesel. It is very important to correctly identify oil yielding plant species. Scanning electron microscopy (SEM) was used as reliable tool for authentic identification of oil yielding seeds. Macromorphological characters of seeds were studied with light microscopy (LM). Outcomes of LM of seeds exposed distinctive variation in seed size from 16.3 to 3.2 mm in length and 12.4 to 0.9 mm in width, shape varied from oval to triangular, and color from black to light brown. Oil content of nonedible seed ranged from 25 to 30% (w/w). Free fatty acid content of seed oil varied from 0.32 to 2.5 mg KOH/g. Moreover, ultra structural study of seeds via SEM showed variation in surface sculpturing, cell arrangement, cell shape, periclinal wall shape, margins, protuberances, and anticlinal wall shape. Surface sculpturing varied from rugged, reticulate, varrucose, papillate, and striate. Periclinal wall arrangements confirmed variation from rough, wavy, raised, depressed, smooth, and elevated whereas, anticlinal walls pattern showed variation from profuse undulating, smooth, raised, grooved, deep, curved, and depressed. It was concluded that SEM could be a latent and advanced technique in unveiling hidden micromorphological characters of nonedible oil yielding seeds which delivers valuable information to researchers and indigenous people for precise and authentic identification and recognition.     

Preparation of cultured mammalian cells for transmission and scanning electron microscopy using aclar film

Common methods for the preparation of cultured cells for concurrent light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) are not completely satisfactory. This article describes how we grow mammalian cells on plastic disks made from Aclar film. Aclar is a transparent fluorinated-chlorinated thermoplastic that contains no volatile components and is, for all practical purposes, chemically inert. Cells adhere to it readily and remain attached after fixation, dehydration, and critical-point drying or embedding. The film also accepts heavy metal coating by ionic bombardment and is extremely stable in the vacuum of the SEM. LM observations are unhindered by Aclar, since the film is as transparent as glass. Fluorescence microscopy is possible with this film, since it exhibits no detectable autofluorescence. During SEM observation, the film has great dimensional stability, and the cells and heavy metal coating remain attached to the Aclar even under high-resolution operating conditions. TEM processing of specimens grown on Aclar is simplified by the fact that Aclar does not stick to the epoxy resins used in EM. Furthermore, Aclar is easily sectioned and does not damage knives used in ultramicrotomy. The use of Aclar film considerably simplifies the preparation of cultured cells for all types of microscopy. This method is particularly useful in correlating surface features between SEM and TEM observations.     

Investigation of cholesterol,bilirubin, and protein distribution in human gallstones by color cathodoluminescence scanning electron microscopy and transmission electron microscopy

The application of color cathodoluminescent scanning electron microscopy (CCL-SEM) for qualitative luminescence analysis of cholesterol, bilirubin, and protein in human gallstones was demonstrated. Images of these deposits (cholesterol, bilirubin, and protein) were formed in real colors (blue—cholesterol, red, orange—bilirubin, yellow, green—protein) in accordance with the cathodoluminescent spectrum for each control material. The other method described for transmission electron microscopy (TEM) of ultra-thin sections provides more detailed characterization of the ultrastructure of cholesterol-containing regions and their spatial interrelations with bilirubin-containing regions. Using CCL-SEM combined with TEM permits the receipt of more complete information about the chemical composition and ultrastructure of gallstones and may lead to more effective understanding of the pathogenesis of cholesterol cholelithiasis.     

Micro‐CT scouting for transmission electron microscopy of human tissue specimens

Transmission electron microscopy (TEM) provides sub‐nanometre‐scale details in volumetric samples. Samples such as pathology tissue specimens are often stained with a metal element to enhance contrast, which makes them opaque to optical microscopes. As a result, it can be a lengthy procedure to find the region of interest inside a sample through sectioning. We describe micro‐CT scouting for TEM that allows noninvasive identification of regions of interest within a block sample to guide the sectioning step. In a tissue pathology study, a bench‐top micro‐CT scanner with 10 μm resolution was used to determine the location of patches of the mucous membrane in osmium‐stained human nasal scraping samples. Once the regions of interest were located, the sample block was sectioned to expose that location, followed by ultra‐thin sectioning and TEM to inspect the internal structure of the cilia of the membrane epithelial cells with nanometre resolution. This method substantially reduced the time and labour of the search process from typically 20 sections for light microscopy to three sections with no added sample preparation.     

Correlative light and backscattered electron microscopy of bone--part II: automated image analysis

Detailed studies of biological phenomena often involve multiple microscopy and imaging modes and media. For bone biology, various forms of light and electron microscopy are used to study the microscopic structure of bone. Integrating information from the different sources is necessary to understand how different aspects of the bone structure interact. To accomplish this, methods were developed to prepare and image thin sections for correlative light microscopy (LM) and backscattered electron imaging in the scanning electron microscope (BSE-SEM). Images of the same fields of view may then be analyzed for degrees of relationships between specimen features not observed by LM or SEM alone. These methods are applied here to study possible associations between the degree of bone mineralization and pattern of collagen fiber orientation in the mid-shaft of the human femur. The "relational images" obtained allow us to examine the relationship between these two variables, both objectively and quantitatively.