Techniques for combining light microscopy and scanning electron microscopy: a survey of the literature

A survey of methods combining light microscopy and scanning electron microscopy is presented. A simple correlation is made when two preparations from adjacent parts of one specimen are investigated in two different microscopes. A more sophisticated method is the consecutive investigation of one specimen with two microscopes. A major problem in this method is the relocation of the area of interest. Several authors have presented solutions for this problem. It is preferable when one preparation is investigated in only one instrument, combining the two microscopical (LM and SEM) techniques, thus making relocation redundant.     

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.     

A new method for investigating the undersurface of cell monolayers by scanning electron microscopy

Glow discharge is commonly used for cleaning the inside of coating units and for cleaning hard surfaces before carbon or metal evaporation procedures. In this study it has been used to remove the embedding medium to reveal, for scanning electron microscope (SEM) study, the undersurfaces of Balb/c 3T3 fibroblastic cells that have been cultured on Thermanox discs and embedded in LR White resin. Ten to twenty-minute ionization times were found to reveal the largest area of the undersurface without causing damage to the cells. Chemical etching of the resin was also shown to reveal the undersurface of the cells, but caused some damage, preventing successful re-embedding for transmission electron microscopy, and at higher magnifications revealed less detail. A circular impression within the main outline of the cells was observed in many cells, which is considered to reflect the presence of a nucleus. The undersurfaces of most cells, after applying both methods of etching, displayed a number of very short processes. Subsequent transmission electron microscopy of ultrathin sectioned, re-embedded, areas of the gold sputter-coated blocks revealed the depth of ionization that had occurred and confirmed that the specimens observed in SEM were the undersurfaces of cells. This method can be modified to study the attaching surface of any organism to a substratum.     

Imaging an optical disc by the combined use of scanning tunnelling microscopy and scanning electron microscopy

We present the data obtained by scanning tunnelling microscopy combined with scanning electron microscopy of the digitally encoded structure on a stamper used to fabricate optical discs. The combination allows us to focus the STM tip on a preselected spot with a precision of ?0·3 μm. The data show the superiority of STM for a more detailed characterization of shape, width, length, height and fine structure appearing on the sample. We also show the influence of tip shape on STM resolution. Simultaneous use of both microscopes is possible but high electron doses produce an insulating layer of contaminants thick enough to make STM operation impossible.     

Imaging of thermal and elastic surface properties by scanning electron acoustic microscopy

Scanning electron acoustic microscopy is a new technique for imaging the thermal and elastic properties of surfaces and detecting subsurface flaws. It can be carried out in a modified scanning electron microscope. The effects of electron beam energy and phase angle on scanning electron acoustic images of the thermal and elastic properties of surfaces were studied with an alumina fiber/aluminum matrix composite for fiber directions both transverse and coaxial to the surface. Images produced with 10- and 30-keV electrons at beam modulation frequencies of 80–1200 kHz appeared to be identical, with the exception of a lower signal-to-noise ratio for the lower electron energy. This observation suggests that the energy input from the beam can be considered to occur at the surface for electron energies below 30 keV and frequencies below 1200 kHz. Images recorded at 0° phase angle mapped regions of different thermal and elastic properties. Images recorded at 90° phase angle highlighted the boundaries between such regions. Scanning electron acoustic microscopy can image features of different thermal and elastic properties at greater depth than traditional imaging with backscattered electrons. The practical application of the technique to the study of surfaces is illustrated by the imaging of grain structure and subsurface particles for an extruder barrel.     

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.     

The preparation of leaf surfaces for scanning electron microscopy: a comparative study

Cryopreservation is the superior technique for viewing leaf surfaces in the SEM. Epidermal cells become distorted when freeze dried and disrupt the orientation of epicuticular wax structures. The latter are largely lost during critical point drying. Nevertheless, the appearance of surface structures after subjecting them to each drying method is valuable in interpreting the features observed by cryopreservation.     

Angioarchitecture of squamous cell carcinoma from hamster buccal pouch: a scanning electron microscopy study

Tumoral angiogenesis has been widely studied by histochemical analysis but little has been done regarding morphology of these new vessels. The objective of this study was to perform a qualitative analysis of the angiogenic response to chemical induction with dimethylbenzanthracene (DMBA) and carbamide peroxide of squamous cell carcinoma in pouches of Syrian hamsters after different periods of treatment. Twenty‐four Syrian golden hamsters, divided into three groups of eight animals each, had their right jugal pouches treated with a 5% DMBA solution three times a week and a 10% carbamide peroxide two times a week for 55, 70 and 90 days. The left pouch was considered the control. After tumor induction, five animals in each group had their pouches prepared for analysis under scanning electron microscopy and three animals for analysis under light microscopy. The control pouches showed a vascular system composed by few main vessels running parallel to the longest axis of the pouch with some branches. In the pouches submitted to tumor induction, a well‐differentiated squamous cell carcinoma was present since 55 days induction in all samples. The new vascular system showed the presence of many tortuous vessels and the majority of them were veins and capillaries. Terminal loops were extremely sinuous adopting a glomerular or corkscrew shape. These tumor vessels are different from normal vessels, presenting irregular diameters, outpouchings and constrictions. Angiogenesis of sprouting and intussusceptive kind could be identified in the tumor pouches, and they were more frequent as the tumor developed. SCANNING 31: 188–194, 2009. © 2009 Wiley Periodicals, Inc.     

A simple photographic grid system for the correlative examination of cell smears by light and scanning electron microscopy

A simple inexpensive grid system reproduced photographically on black-and-white film provides a support system that allows the same cells to be examined by light and scanning electron microscopy.     

Image comparisons of snow and ice crystals photographed by light (video) microscopy and low-temperature scanning electron microscopy

Light (video) microscopy and low-temperature scanning electron microscopy (SEM) were used to examine and record images of identical precipitated and metamorphosed snow crystals as well as glacial ice grains. Collection procedures enabled numerous samples from distant locations to be shipped to a laboratory for storage and/or observation. The frozen samples could be imaged with a video microscope in the laboratory at ambient temperatures or with the low-temperature SEM. Stereo images obtained by video microscopy or low-temperature SEM greatly increased the ease of structural interpretations. The preparation procedures that were used for low-temperature SEM did not result in sublimation or melting. However, this technique did provide far greater resolution and depth of focus over that of the video microscope. The advantage of resolution was especially evident when examining the small particles associated with rime and graupel (snow crystals encumbered with frozen water droplets), whereas the greater depth of focus provided clearer photographs of large crystals such as depth hoar, and ice. Because the SEM images contained only surface information while the video images were frequently confounded by surface and internal information, the SEM images also clarified the structural features of depth hoar crystals and ice grains. Low-temperature SEM appears to have considerable promise for future investigations of snow and ice.     

Comparative study of a block copolymer morphology by transmission electron microscopy and scanning force microscopy

Using transmission electron microscopy (TEM) and scanning force microscopy (SFM) together, it was possible to verify important structural features of a nanostructured bulk material such as the kp‐morphology in an ABC triblock copolymer. By applying suitable imaging techniques during the SFM measurements it was possible to determine the morphology without additional manipulation steps in between. In comparison, TEM investigations on this type of material usually require selective staining procedures prior to the measurement. Also electron beam damage is often encountered during TEM measurements especially if components such as poly(methacrylates) are present. In contrast, SFM measurements can be assumed not to significantly change the phase dimensions of the components.     

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.     

A quantitative evaluation of the glomerular capillary endothelium in rat and human kidneys: Utilization of vascular perfusion,freeze-cracking of tissue,and scanning electron microscopy

Modern morphological investigation requires the use of a variety of technological approaches and the employment of rigorous morphometric analysis for an adequate evaluation of the structural and ultrastructural features of a tissue or organ. The introduction of the technique of freeze-cracking of tissue to expose new surfaces has made it possible to quantitate the normal surface characteristics of the glomerular capillaries of the mammalian kidney. This report describes the techniques used for the preparation and quantitative assessment of normal glomerular endothelial morphology. The techniques of in vivo and in vitro vascular perfusion of kidneys as a method of fixation and the freeze-cracking of tissue are outlined in detail. In addition, a morphometric analysis of the endothelial surface characteristics are described and values are reported for the control rat and human kidneys from transplant donors.     

Correlative scanning electron and confocal microscopy imaging of labeled cells coated by indium‐tin‐oxide

Confocal microscopy imaging of cells allows to visualize the presence of specific antigens by using fluorescent tags or fluorescent proteins, with resolution of few hundreds of nanometers, providing their localization in a large field‐of‐view and the understanding of their cellular function. Conversely, in scanning electron microscopy (SEM), the surface morphology of cells is imaged down to nanometer scale using secondary electrons. Combining both imaging techniques have brought to the correlative light and electron microscopy, contributing to investigate the existing relationships between biological surface structures and functions. Furthermore, in SEM, backscattered electrons (BSE) can image local compositional differences, like those due to nanosized gold particles labeling cellular surface antigens. To perform SEM imaging of cells, they could be grown on conducting substrates, but obtaining images of limited quality. Alternatively, they could be rendered electrically conductive, coating them with a thin metal layer. However, when BSE are collected to detect gold‐labeled surface antigens, heavy metals cannot be used as coating material, as they would mask the BSE signal produced by the markers. Cell surface could be then coated with a thin layer of chromium, but this results in a loss of conductivity due to the fast chromium oxidation, if the samples come in contact with air. In order to overcome these major limitations, a thin layer of indium‐tin‐oxide was deposited by ion‐sputtering on gold‐decorated HeLa cells and neurons. Indium‐tin‐oxide was able to provide stable electrical conductivity and preservation of the BSE signal coming from the gold‐conjugated markers. Microsc. Res. Tech. 78:433–443, 2015. © 2015 Wiley Periodicals, Inc.     

Preservation of protein fluorescence in embedded human dendritic cells for targeted 3D light and electron microscopy

In this study, we present a correlative microscopy workflow to combine detailed 3D fluorescence light microscopy data with ultrastructural information gained by 3D focused ion beam assisted scanning electron microscopy. The workflow is based on an optimized high pressure freezing/freeze substitution protocol that preserves good ultrastructural detail along with retaining the fluorescence signal in the resin embedded specimens. Consequently, cellular structures of interest can readily be identified and imaged by state of the art 3D confocal fluorescence microscopy and are precisely referenced with respect to an imprinted coordinate system on the surface of the resin block. This allows precise guidance of the focused ion beam assisted scanning electron microscopy and limits the volume to be imaged to the structure of interest. This, in turn, minimizes the total acquisition time necessary to conduct the time consuming ultrastructural scanning electron microscope imaging while eliminating the risk to miss parts of the target structure. We illustrate the value of this workflow for targeting virus compartments, which are formed in HIV‐pulsed mature human dendritic cells.     

Implications of foliar epidermal micromorphology using light and scanning electron microscopy: A useful tool in taxonomy of Korean irises

The genus Iris L., comprising approximately 210 species, is one of the most species-rich genera in the family Iridaceae. In this study, the first comprehensive leaf micromorphological characters of Korean irises were studied using light and scanning electron microscopy. Our objective was to evaluate the foliar micromorphological characteristics (namely epidermal cells, stomata types, and guard cell size) of Korean Iris taxa in a systematic context. All the investigated Korean Iris taxa had amphistomatic or hypostomatic leaves with anomocytic stomatal complexes. Guard cell length varied among species, ranging from 24.8 μm (I. rossii) to 56.0 μm (I. domestica). Although the presence of papillae on the outer periclinal wall is not of taxonomic significance, leaf margin pattern, guard cell size, and sunken stomata type were useful for species-level identification of Korean Iris species. The occurrence of polymorphic stomatal types was reported here for the first time, and the correlation between genome size and epidermal guard cell length was discussed.     

Exploring the structure of a hydrogen cyanide polymer by electron spin resonance and scanning force microscopy

Aqueous solutions of potassium cyanide and ammonium hydroxide are known to yield a heterogeneous cyanide polymer, containing paramagnetic sites and biologically significant substructures including polypeptides. Here, such solutions were used to prepare various samples of polymer for study by X-band and W-band electron spin resonance (ESR), scanning electron microscopy (SEM), and scanning force microscopy (SFM). Elemental composition of a typical sample of the polymer was C-35.2%, N-38.47%, 0-14.51%, and H-4.13%, exposing the polymer to 6M HCl hydrolyzed portions of the polymer and released glycine and traces of other amino acids. The X-band ESR spectra consist of a single slightly asymmetric line centered at g = 2.003; spin concentration measurements made at X-band using a nitroxide radical standard yield approximate radical concentrations of 10(18) spins/gm. W-band ESR indicates the presence of a single rhombic paramagnetic site with g(x) = 2.0025, g(y) = 2.0030, and g(z) = 2.0048 and the possibility of small 14N hyperfine splittings. The ESR spin echo studies yield a longitudinal relaxation time, Tl of 75 microS and a short-phase memory relaxation time, Tm, of about 300 nS. Scanning electron microscopy studies of the polymer show that it is made of ellipsoidal particles about one micron in size. The particles tend to clump together when suspended in aqueous solution. The particles disperse and dissolve in dimethyl sulfoxide (DMSO); when these solutions dry on microscope slides, optical microscopy shows a branched island morphology for the polymer. This morphology is reminiscent of snowflakes and is identified as dendritic. Phase contrast SFM of the dendritic arms show a striking segregation and ordering of various components of the polymer. Paramagnetic sites are conserved in the series of steps leading to dendritic structures.     

A routine flat embedding method for electron microscopy of microorganisms allowing selection and precisely orientated sectioning of single cells by light microscopy

A simple method is described to embed material in resin, in the form of microscope slides, to observe it with high resolution light microscopy, to select, orient and section it for TEM. This method can be applied to many kinds of material but is particularly useful for the study of rare or tiny plant or animal microorganisms from field or culture. A diamond scriber, translucent hydrosoluble resin release agent, translucent and smooth resin stubs and a longitudinally perforated block-holder for ultramicrotome are the specific tools of this method.