Rapid method for resectioning of light microscopy sections for electron microscopy

A rapid method is described for obtaining ultrathin sections from light microscopy sections. Five-micrometre epoxy sections, heat-flattened to slides, were affixed to the tips of plastic blocks by light-curable dental bond, and cured while still on the microscope stage by illumination with blue light for 2 min. Sections were detached from the slides by rapid cooling and then resectioned for electron microscopy.     

Light- and electronmicroscopic study of infiltration of resin into initial caries lesions--a new methodological approach

It was the purpose of this pilot study to investigate resin infiltration into various types of initial subsurface caries lesions using a combined microscopic technique with polarized light microscopy and fluorescence microscopy and subsequent scanning microscopy with EDX-element analysis. Six extracted premolars with initial caries lesions were used. Five were infiltrated with resin after imbibition of the subsurface carious pore volume of enamel with sodium fluorescein solution. After light curing the unbound dye was removed by washing out in water. Serial sections were cut through the lesions and investigated with polarized light microscopy, fluorescence microscopy and simultaneously with both microscopic techniques. The same sections were then studied with scanning electron microscopy and EDX-element analysis to prove the infiltration of the resin into the lesions. The results showed, that the combination technique adds further morphologic information to infiltration behaviour of the resin. The individual volume of early acute lesions versus chronic lesions involving dentin, and the fluorescein bound by resin was well documented in serial sections. The EDX calcium and phosphorus signals correlate negatively with the lesion extension, and the carbon signal correlates positively, thus labelling the resin infiltration. It could be demonstrated that resin infiltration is dependent from the pore volume of the lesion. It can be concluded that the combined polarized light microscopy with fluorescence microscopy is an advantageous tool for studying infiltration of resin into hard tissues.     

Three-Dimensional imaging of fertilization and early development

The field of biological microscopy has recently enjoyed major technical advances, exemplified by the development of field-emission low-voltage scanning electron microscopes and laser scanning confocal light microscopes. In addition, computer processing of microscopical data is revolutionizing the way morphological information is imaged. In this paper, we illustrate methods by which this new technology can be used to examine events in fertilization and early development in three dimensions. Different types of specimen preparation protocols, using both echinoderm and mammalian gametes and embryos, are evaluated for their ability to preserve accurately the threedimensional organization of these specimens for imaging by both low-voltage scanning electron microscopy and laser scanning confocal light microscopy.     

Iso-butanol saturated water: a simple procedure for increasing staining intensity of resin sections for light and electron microscopy

The addition of the alcohol iso‐butanol (2‐methylpropan‐1‐ol) to water was found to improve the post‐staining procedures for semi‐thin and ultrathin resin sections, for both light and electron microscopy. Stain penetration was enhanced with samples embedded in both acrylic and epoxy resins and provided structural information not previously seen. These improvements were found with general (non‐specific) stains and a fluorescence stain for light microscopy, as well as for a range of heavy metal stains for electron microscopy. The use of this water/solvent medium also gave improved results when used in a variety of immunogold labelling procedures, resulting in a greater specific label density without affecting background gold levels. The use of this solvent/water medium may have wider applications for other types of staining.     

Gridded Aclar: preparation methods and use for correlative light and electron microscopy of cell monolayers,by TEM and FIB–SEM

Aclar, a copolymer film with properties very similar to those of tissue culture plastic, is a versatile substrate to grow cells for light (including fluorescence) and electron microscopic applications in combination with both chemical fixation and cryoimmobilization. In this paper, we describe complete procedures to perform correlative light and electron microscopy using Aclar as substrate for the culture of cell monolayers to be finally embedded in plastic. First, we developed straightforward, efficient and flexible ways to mark the surface of the Aclar to create substrates to locate cells first at the light microscopy and then the electron microscopy level. All the methods enable the user to self‐design gridded Aclar pieces, according to the purpose of the experiments, and create a large number of substrates in a short time. Second, we confirmed that marked Aclar supports the normal growth and morphology of cells. Third, we validated the correlative light and electron microscopy procedure using Aclar. This validation was done for the high‐resolution analysis of endothelial cells using transmission electron microscopy and focused ion beam–scanning electron microscopy in combination with the use of fluorescence, phase contrast and/or bright field microscopy to map areas of interest at low resolution. The methods that we present are diverse, easy to implement and highly reproducible, and emphasize the versatility of Aclar as a cell growth substrate for diverse microscopic applications.     

Comparison of reflection contrast microscopy and electron microscopy on the histopathological diagnosis of various kidney diseases

Our aim in this study was to compare reflection contrast microscopy (RCM) with transmission electron microscopy (TEM) to understand whether RCM could be used in the histopathological diagnosis of various kidney diseases as a less expensive and an easier alternative to TEM. The diagnoses of kidney pathologic lesions included Alport syndrome, thin membrane disease, Ig A nephropathy. RCM is a form of light microscope that works in the reflected mode, suitable to observe ultrathin (50-100 nm) plastic sections that is also used in TEM. Our findings showed that RCM showed similar results compared with TEM on these lesions described earlier.     

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.     

Aclar discs: a versatile substrate for routine high-pressure freezing of mammalian cell monolayers

High‐pressure freezing avoids the artefacts induced by conventional chemical fixation, and, in combination with freeze‐substitution and plastic embedding, is a reliable method for the ultrastructural analysis of mammalian cell monolayers. In order to high‐pressure freeze mammalian cell monolayers, cells have to be seeded on a suitable substrate. Unfortunately, electron microscopy analysis is often hampered by poor cell growth, changes in cell morphology induced by the cell substrate or cell loss during processing. We report a method to culture, high‐pressure freeze, freeze‐substitute and plastic embed mammalian cell monolayers. The method is based on the use of Aclar, a copolymer film with properties very similar to those of tissue culture plastic. We show that Aclar discs support the normal growth and morphology of a wide variety of mammalian cell types, and form an ideal starting point for high‐pressure freezing, freeze‐substitution and plastic embedding. We present a complete protocol, which, because of its simplicity and reproducibility, provides a method suitable for the routine analysis of mammalian cell monolayers by electron microscopy and tomography.     

Ultrastructural characteristics of ovine bone marrow‐derived mesenchymal stromal cells cultured with a silicon stabilized tricalcium phosphate bioceramic

Bioceramics are being used in experimental bone engineering application in association with bone marrow derived mesenchymal stem cells (BM‐MSCs) as a new therapeutic tool, but their effects on the ultrastructure of BM‐MSCs are yet unknown. In this study we report the morphological features of ovine (o)BM‐MSCs cultured with Skelite, a resorbable bioceramic based on silicon stabilized tricalcium phosphate (SiTCP), able to promote the repair of induced bone defect in sheep model. oBM‐MSCs were isolated from the iliac crest, cultured until they reached near‐confluence and incubated with SiTCP. After 48 hr the monolayers were highly damaged and only few cells adhered to the plastic. Thus, SiTCP was removed, and after washing the cells were cultured until they became confluent. Then, they were trypsinizated and processed for transmission electron microscopy (TEM) and RT‐PCR analysis. RT‐PCR displayed that oBM‐MSCs express typical surface marker for MSCs. TEM revealed the presence of electron‐lucent cells and electron‐dense cells, both expressing the CD90 surface antigen. The prominent feature of electron‐lucent cells was the concentration of cytoplasmic organelles around the nucleus as well as large surface blebs containing glycogen or profiles of endoplasmic reticulum. The dark cells had a multilocular appearance by the presence of peripheral vacuoles. Some dark cells contained endocytic vesicles, lysosomes, and glycogen aggregates. oBM‐MSCs showed different types of specialized interconnections. The comparison with ultrastructural features of untreated oBM‐MSCs suggests the light and dark cells are two distinct cell types which were differently affected by SiTCP bioceramic. Skelite cultured ovine BM‐MSCs display electron‐dense and electron‐lucent cells which are differently affected by this bioceramic. This suggests that they could play a different role in bioceramic based therapy.     

Histomicroscopy and normal anatomy of the adult killifish Aphanius hormuzensis (Teleostei; Aphaniidae) from the Persian Gulf coastal environment

The histomicroscopy and normal anatomy of the major body organ systems were investigated in the adult killifish, Aphanius hormuzensis using histological examination, X‐ray imaging, double staining, light microscopy and scanning electron microscopy (SEM). Based on the histomicroscopic observations, the kidney, liver and swim bladder in the studied species were comparable to other fish models. The anterior portion of the kidney is bulbous, while the posterior portion is narrow and elongated; the liver has a single lobe and the swim bladder is a single‐chambered organ with no connection to the digestive tract (physoclistous). X‐ray imaging and double staining examination showed 12 abdominal and 15 caudal vertebrae and a single hypural plate in the caudal skeleton. According to light microscopy, the scales were rounded to pentagonal in shape with three types of radii (primary, secondary and tertiary), and the urohyal bone was elongated. SEM microscopy showed a single row of tricuspid teeth on the upper and lower jaw, respectively, each tooth has two lateral cusps that are shorter than the middle one. The number of teeth was 17–18 in the upper jaw and 19–20 in the lower jaw. The saccular otoliths were rounded‐trapezoid in shape with a moderately incised and V‐shaped excisura. The members of killifishes are an important group for biologists because of their evolutionary properties, regeneration capacity and usefulness as biological control and also for the ecotoxicological assessment of environmental pollution. The outcomes of this study may provide a useful basis for future research on the genus Aphanius.     

Correlative microscopy: a potent tool for the study of rare or unique cellular and tissue events

Biological studies have relied on two complementary microscope technologies – light (fluorescence) microscopy and electron microscopy. Light microscopy is used to study phenomena at a global scale to look for unique or rare events, and it also provides an opportunity for live imaging, whereas the forte of electron microscopy is the high resolution. Traditionally light and electron microscopy observations are carried out in different populations of cells/tissues and a 'correlative' inference is drawn. The advent of true correlative light-electron microscopy has allowed high-resolution imaging by electron microscopy of the same structure observed by light microscopy, and in advanced cases by video microscopy. Thus a rare event captured by low-resolution imaging of a population or transient events captured by live imaging can now also be studied at high resolution by electron microscopy. Here, the potential and difficulties of this approach, along with the most impressive breakthroughs obtained by these methods, are discussed.     

Detection of food fraud of meat products from the different brands by application of histological methods

In Sohag City, 400 samples were collected from different food markets of different meat products from two companies with high and low prices (e.g., minced meat, kofta sausage, beef burger, and luncheon meat) for determining food fraud. Light, fluorescence, and scanning electron microscopy (SEM) were used to examine the samples. “Special histochemical stains” permit the microscopic examination of different cell types, structures, and/or microorganisms. Histological examination revealed variant tissue types, besides skeletal muscles. Nuchal ligaments, bones, hyaline cartilages, white fibrocartilages, large and medium arteries, cardiac muscles, tendons, and collagenous connective tissues comprised the capsule of a parenchymatous organ. Additionally, a crystal of food additives was recognized using light microscopy and SEM. SEM allows the visualization of bacterial contamination. Using different microscopic anatomy techniques is an efficient methodology for qualitative evaluations of various meat products. No difference in quality was observed between low- and high-priced meat products.     

Integration of a high‐NA light microscope in a scanning electron microscope

We present an integrated light‐electron microscope in which an inverted high‐NA objective lens is positioned inside a scanning electron microscope (SEM). The SEM objective lens and the light objective lens have a common axis and focal plane, allowing high‐resolution optical microscopy and scanning electron microscopy on the same area of a sample simultaneously. Components for light illumination and detection can be mounted outside the vacuum, enabling flexibility in the construction of the light microscope. The light objective lens can be positioned underneath the SEM objective lens during operation for sub‐10 μm alignment of the fields of view of the light and electron microscopes. We demonstrate in situ epifluorescence microscopy in the SEM with a numerical aperture of 1.4 using vacuum‐compatible immersion oil. For a 40‐nm‐diameter fluorescent polymer nanoparticle, an intensity profile with a FWHM of 380 nm is measured whereas the SEM performance is uncompromised. The integrated instrument may offer new possibilities for correlative light and electron microscopy in the life sciences as well as in physics and chemistry.     

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.     

The Ralph knife in practice

Long edge glass knives, ‘Ralph knives’, were produced in an LKB Histo KnifeMaker. The edge angles were measured by light microscopy; depending on the breaking conditions the angles varied between 12° and 58°, as measured close to the edge in the mid-portion of the knives. Hackle marks were more common in the left portion of the edge than in the middle and right portions. Some of the knives were used for cutting sections from urinary bladder tumours embedded in paraffin or in glycol methacrylate. Following microtomy the sections were allowed to stretch on a water surface; this procedure resulted in an increased width of the plastic sections, whereas the paraffin sections were not affected. The compression of the whole sections averaged 15% in the paraffin sections and 11% in the plastic sections; in both cases the compression factors were positively correlated to the angle of the knife edge. Smaller compression factors were found for the cell nuclei in the embedded tissue.     

Determination of the size distribution of lecithin liposomes: a comparative study using freeze fracture, cryoelectron microscopy and dynamic light scattering

The size distribution of liposomes is often determined using freeze fracture, cryoelectron microscopy or dynamic light scattering. However, the resulting size distributions do not directly coincide owing to the different weighting of the techniques. We present several methods which correct for these effects and allow a comparison of liposome size distributions as obtained by freeze fracture, cryoelectron microscopy or dynamic light scattering. These methods are based on theoretical models for the weighting of the size distribution of liposomes, which result from the preparation procedure for freeze fracture electron microscopy and from the measurement by dynamic light scattering. The proposed transformation methods are then experimentally tested with a sample of lecithin liposomes, whose size distribution was determined by dynamic light scattering, freeze fracture and cryoelectron microscopy. Furthermore, the weaknesses of the experimental techniques and hence of the resulting size distributions are discussed.     

Combination of normal light and fluorescence microscopy for authentication of five Lonicera species flower buds

The flower buds of five Lonicera species, Lonicera japonica Thunb., L. macranthoides Hand.-Mazz., L. hypoglauca Miq., L. confusa DC. and L. fulvotomentosa Hsu et S.C. Cheng are confusable and usually utilized under the same name "Jinyinhua" in different areas for morphological similarity. Studies found that these five species possess extreme differences in chemical compounds, correspondingly showing different pharmacological activities and clinical applications. To ensure efficacy and safety of these herbal medicines and prevent unknown adverse effect, in this work, a simple, rapid and effective method combining normal light and fluorescence microscopy was developed for authentication. Surface slides and transverse sections of these buds were investigated to reveal their differences. As a routine technique, normal light microscopy which gives detailed microscopic features such as glandular hairs and nonglandular hairs, can easily distinguish four species except L. confusa. Fluorescence technique, which could present different distribution of fluorescence materials, is further employed to identify three species including L. confusa successfully. It is the first report to identify these five Lonicera species by combining normal light and fluorescence microscopy. This work indicated combining normal light and fluorescence microscopy could be a powerful method in authentication of confused species.     

A grinding method for producing sections of large areas of plastic embedded tissues

We have developed a method utilizing relatively thick ground sections of plastic embedded tissue which affords the resolution obtained with 0·5 μm cut sections. The sections, which are permanently affixed to plastic microscope slides, are much larger in area than ultramicrotome sections. Additional advantages are: sections can be destained and restained and selected areas can be examined with various forms of electron microscopy. Autoradiographic studies are also possible. Although the method has a broader application, it is particularly useful in examining the interface between hard and soft tissues.     

Nanostructural analysis by atomic force microscopy followed by light microscopy on the same archival slide

Integrated information on ultrastructural surface texture and chemistry increasingly plays a role in the biomedical sciences. Light microscopy provides access to biochemical data by the application of dyes. Ultrastructural representation of the surface structure of tissues, cells, or macromolecules can be obtained by scanning electron microscopy (SEM). However, SEM often requires gold or coal coating of biological samples, which makes a combined examination by light microscopy and SEM difficult. Conventional histochemical staining methods are not easily applicable to biological material subsequent to such treatment. Atomic force microscopy (AFM) gives access to surface textures down to ultrastructural dimensions without previous coating of the sample. A combination of AFM with conventional histochemical staining protocols for light microscopy on a single slide is therefore presented. Unstained cores were examined using AFM (tapping mode) and subsequently stained histochemically. The images obtained by AFM were compared with the results of histochemistry. AFM technology did not interfere with any of the histochemical staining protocols. Ultrastructurally analyzed regions could be identified in light microscopy and histochemical properties of ultrastructurally determined regions could be seen. AFM-generated ultrastructural information with subsequent staining gives way to novel findings in the biomedical sciences. Microsc. Res. Tech., 2009. © 2009 Wiley-Liss, Inc.     

Resolution in nonlinear laser scanning microscopy

The lateral and depth resolution of nonlinear microscopy was studied systematically. Nonlinear microscopy can be classified into several categories depending on the coherence properties of the process that generates the imaging signal from the illuminating light, on whether a single- or a two-beam geometry is used, and whether the optical setup is Type I or Type II. An evaluation of the imaging equations shows that (i) lateral and depth resolution improve with increasing nonlinearity, (ii) the differences between coherent and incoherent imaging diminish, and (iii) nonlinear imaging allows depth discrimination in Type I microscopy.