Imaging analysis of the interface between osteoblasts and microrough surfaces of laser‐sintered titanium alloy constructs

Previous work using focused ion beam (FIB) analysis of osteoblasts on smooth and microrough Ti surfaces showed that the average cell aspect ratio and distance from the surface are greater on the rough surface. In order to better interrogate the relationship between individual cells and their substrate using multiple imaging modalities, we developed a method that tracks the same cell across confocal laser scanning microscopy (CLSM) to correlate surface microroughness with cell morphology and cytoskeleton; scanning electron microscopy (SEM) to provide higher resolution for observation of nanoroughness as well as chemical mapping via energy dispersive X‐ray spectroscopy; and transmission electron microscopy (TEM) for high‐resolution imaging. FIB was used to prepare thin sections of the cell‐material interface for TEM, or for three‐dimensional electron tomography. Cells were cultured on laser‐sintered Ti‐6Al‐4V substrates with polished or etched surfaces. Direct cell to surface attachments were observed across surfaces, though bridging across macroscale surface features occurred on rough substrates. Our results show that surface roughness, cell cytoskeleton and gross morphology can be correlated with the cell‐material cross‐sectional interface at the single cell level across multiple high‐resolution imaging modalities. This work provides a platform method for further investigating mechanisms of the cell‐material interface.     

Application of the low-loss scanning electron microscope image to integrated circuit technology part II--chemically-mechanically planarized samples.

Chemical-mechanical planarization (CMP) is a process that gives a flat surface on a silicon wafer by removing material from above a chosen level. This flat surface must then be reviewed (typically using a laser) and inspected for scratches and other topographic defects. This inspection has been done using both the atomic force microscope (AFM) and the scanning electron microscope (SEM), each of which has its own advantages and disadvantages. In this study, the low-loss electron (LLE) method in the SEM was applied to CMP samples at close to a right angle to the beam. The LLEs show shallower topographic defects more clearly than it is possible with the secondary electron (SE) imaging method. These images were then calibrated and compared with those obtained using the AFM, showing the value of both methods. It is believed that the next step is to examine such samples at a right angle to the beam in the SEM using the magnetically filtered LLE imaging method.     

Scanning electron microscopic study of immunogold-labeled human leukocytes

For many years critical point drying (CPD) has been the method of choice for preparing cells for scanning electron microscopy (SEM). Described herein is a simple, efficient, inexpensive, reproducible, and safe procedure using Peldri II, a proprietary fluorocarbon compound that is solid at room temperature and a liquid above 25°C, as a sublimation dehydrant for processing specimens for SEM. The utility of Peldri II was demonstrated in studies using leukocytes from the blood of healthy donors and patients with leukemia as well as from long-term lymphoblastoid cell lines. The application of the proposed Peldri II procedure was further documented in SEM studies in which the expression and distribution of the interleukin-2 receptor (IL-2R) on leukocyte surface membranes was imaged using colloidal gold-labeled antibodies (i.e., immunogold). When compared with current SEM preparation procedures using CPD, Peldri II is a useful alternative that is thought to offer several important advantages.     

Electron microscopy localization and characterization of functionalized composite organic-inorganic SERS nanoparticles on leukemia cells

We demonstrate the use of electron microscopy as a powerful characterization tool to identify and locate antibody-conjugated composite organic-inorganic nanoparticle (COINs) surface enhanced Raman scattering (SERS) nanoparticles on cells. U937 leukemia cells labeled with antibody CD54-conjugated COINs were characterized in their native, hydrated state using wet scanning electron microscopy (SEM) and in their dehydrated state using high-resolution SEM. In both cases, the backscattered electron (BSE) detector was used to detect and identify the silver constituents in COINs due to its high sensitivity to atomic number variations within a specimen. The imaging and analytical capabilities in the SEM were further complemented by higher resolution transmission electron microscopy (TEM) images and scanning Auger electron spectroscopy (AES) data to give reliable and high-resolution information about nanoparticles and their binding to cell surface antigens.     

Color cathodoluminescence display in the scanning electron microscope of deep relief surfaces

A scanning electron microscope (SEM) is a multifunctional instrument for the measurement of topographic relief on the surface of bulk specimen images. This instrument is also available to detect the physical effects induced by an electron beam into subsurface layers. Space distribution of the physical properties of measured effects in the relative microrelief is a very important problem in the SEM. We describe a method of displaying specimen information in the SEM using the color cathodoluminescence (CCL-SEM) technique nondistorted by relief influence and CCL-SEM images with composite (color and black / white ) contrast using CCL+BSEmode.     

Comparison of different preparation methods of biological samples for FIB milling and SEM investigation

When a new approach in microscopy is introduced, broad interest is attracted only when the sample preparation procedure is elaborated and the results compared with the outcome of the existing methods. In the work presented here we tested different preparation procedures for focused ion beam (FIB) milling and scanning electron microscopy (SEM) of biological samples. The digestive gland epithelium of a terrestrial crustacean was prepared in a parallel for FIB/SEM and transmission electron microscope (TEM). All samples were aldehyde-fixed but followed by different further preparation steps. The results demonstrate that the FIB/SEM samples prepared for conventional scanning electron microscopy (dried) is suited for characterization of those intracellular morphological features, which have membranous/lamellar appearance and structures with composition of different density as the rest of the cell. The FIB/SEM of dried samples did not allow unambiguous recognition of cellular organelles. However, cellular organelles can be recognized by FIB/SEM when samples are embedded in plastic as for TEM and imaged by backscattered electrons. The best results in terms of topographical contrast on FIB milled dried samples were obtained when samples were aldehyde-fixed and conductively stained with the OTOTO method (osmium tetroxide/thiocarbohydrazide/osmium tetroxide/thiocarbohydrazide/osmium tetroxide). In the work presented here we provide evidence that FIB/SEM enables both, detailed recognition of cell ultrastructure, when samples are plastic embedded as for TEM or investigation of sample surface morphology and subcellular composition, when samples are dried as for conventional SEM.     

Line profile reconstruction from simultaneously recorded secondary and backscattered electron signals

We propose a reconstruction method of surface morphology using a combination of secondary and back-scattered electron signals from the scanning electron microscope (SEM). Compared with multiple-detector methods, the proposed system requires only conventional secondary and backscattered electron detectors for a line profile reconstruction in one direction. This method is an application of genetic algorithms to the measurement of surface morphology in SEM. We use the chi-square distribution of the reconstruction error as the objective function within a scheme to minimize the number of vertices in the reconstructed surface profile. (The reconstruction error is the relative difference between the calculated and experimental data.) To evaluate the efficacy of our method, a surface profile is successfully reconstructed from a pair of line scans across the center of a latex particle.     

A new HPF specimen carrier adapter for the use of high‐pressure freezing with cryoscanning electron microscope: two applications: stearic acid organization in a hydroxypropyl methylcellulose matrix and mice myocardium

Cryogenic transmission electron microscopy of high‐pressure freezing (HPF) samples is a well‐established technique for the analysis of liquid containing specimens. This technique enables observation without removing water or other volatile components. The HPF technique is less used in scanning electron microscopy (SEM) due to the lack of a suitable HPF specimen carrier adapter. The traditional SEM cryotransfer system (PP3000T Quorum Laughton, East Sussex, UK; Alto Gatan, Pleasanton, CA, USA) usually uses nitrogen slush. Unfortunately, and unlike HPF, nitrogen slush produces water crystal artefacts. So, we propose a new HPF specimen carrier adapter for sample transfer from HPF system to cryogenic‐scanning electronic microscope (Cryo‐SEM). The new transfer system is validated using technical two applications, a stearic acid in hydroxypropyl methylcellulose solution and mice myocardium. Preservation of samples is suitable in both cases. Cryo‐SEM examination of HPF samples enables a good correlation between acid stearic liquid concentration and acid stearic occupation surface (only for homogeneous solution). For biological samples as myocardium, cytoplasmic structures of cardiomyocyte are easily recognized with adequate preservation of organelle contacts and inner cell organization. We expect this new HPF specimen carrier adapter would enable more SEM‐studies using HPF.     

Comparative atomic force and scanning electron microscopy: an investigation on fenestrated endothelial cells in vitro

Rat liver sinusoidal endothelial cells (LEC) contain fenestrae, which are clustered in sieve plates. Fenestrae control the exchange of fluids, solutes and particles between the sinusoidal blood and the space of Disse, which at its back side is flanked by the microvillous surface of the parenchymal cells. The surface of LEC can optimally be imaged by scanning electron microscopy (SEM), and SEM images can be used to study dynamic changes in fenestrae by comparing fixed specimens subjected to different experimental conditions. Unfortunately, the SEM allows only investigation of fixed, dried and coated specimens. Recently, the use of atomic force microscopy (AFM) was introduced for analysing the cell surface, independent of complicated preparation techniques. We used the AFM for the investigation of cultured LEC surfaces and the study of morphological changes of fenestrae. SEM served as a conventional reference.
AFM images of LEC show structures that correlate well with SEM images. Dried-coated, dried-uncoated and wet-fixed LEC show a central bulging nucleus and flat fenestrated cellular processes. It was also possible to obtain height information which is not available in SEM. After treatment with ethanol or serotonin the diameters of fenestrae increased (+6%) and decreased (−15%), respectively. The same alterations of fenestrae could be distinguished by measuring AFM images of dried-coated, dried-uncoated and wet-fixed LEC. Comparison of dried-coated (SEM) and wet-fixed (AFM) fenestrae indicated a mean shrinkage of 20% in SEM preparations. In conclusion, high-resolution imaging with AFM of the cell surface of cultured LEC can be performed on dried-coated, dried-uncoated and wet-fixed LEC, which was hitherto only possible with fixed, dried and coated preparations in SEM and transmission electron microscopy (TEM).     

Cell surface distribution of fibronectin in cultures of fibroblasts and bladder derived epithelium: SEM-immunogold localization compared to immunoperoxidase and immunofluorescence

Expression of cell surface fibronectin in cultures of untransformed fibroblasts is well documented, but little is known of its presence and distribution in cultured epithelial cells. Using species monospecific anti-fibronectin antibodies, the distribution of fibronectin in untransformed fibroblasts and in normal and neoplastic bladder epithelial cells was characterized by indirect labelling experiments using immunogold scanning electron microscopy (SEM). The surface matrix of fibronectin expressed in rodent and human fibroblast cell lines was demonstrated with ease by SEM of gold-tagged second antibodies. However, no fibronectin could be detected on any of the mouse and human bladder epithelium-derived cells studied in single or in mixed epithelial-fibroblast cultures. These SEM-immunogold observations were compared to and confirmed by immunofluorescence and immunoperoxidase microscopy. Immunofluorescence and SEM localization of the fibronectin in the extracellular matrix presented similar distribution patterns but the higher resolution of the SEM provided a more detailed analysis.     

Imaging the cell surface: argon sputtering to expose inner cell structures

Established microscopies such as Scanning Electron Microscopy (SEM) and more recent developments such as Atomic Force Microscopy (AFM) and X-ray Photo-Electron Emission spectroMicroscopy (X-PEEM) can only image the sample surface. We present an argon sputtering method able to progressively expose inner cell structures without apparent damage. By varying the sputtering time, the structure of cell cytoskeleton, vesicles, mitochondria, nuclear membrane, and nucleoli can be imaged. We compared images obtained with confocal fluorescence microscopy, transmission electron microscopy (TEM), SEM, and X-PEEM on similar samples after argon sputtering, then confirmed the similarity of reference intracellular structures, including cytoskeleton fibers, cell-cell and cell-substrate adhesion structures, and secretory vesicles. We conclude that the sputtering method is a new valuable tool for surface sensitive microscopies.     

Surface roughness of preparations for backscattered electron-scanning electron microscopy: the image differences and their Monte Carlo simulation

Patterns and levels of mineralisation in the biological hard tissues have been studied using the backscattered electron (BSE) mode in the scanning electron microscope (SEM). To prevent gross topographic detail overwhelming changes in signal from composition, samples are embedded in polymethylmethacrylate (PMMA) and a flat block surface produced by polishing or micromilling. This study was undertaken to establish the degree of residual topography achieved in these finishing processes. A sample of human rib was embedded in PMMA and prepared, as for examination in the SEM, by polishing on graded abrasives and pre- and, finally, ultramilling. After each preparation step, the block face was imaged using a confocal reflection microscope surface mapping facility. The recorded topographies were used in a Monte Carlo simulation to model the surface interface and thus, for each of the sample preparation techniques, to calculate predicted variations in BSE signal. The latter were compared with experimental data derived under standard operating conditions in the SEM. Micromilling produced block faces with typical peak-trough relief of 80 nm, while hand polishing left occasional scratches 1.5 microns deep with a general undulation of 150-250 nm. Monte Carlo simulations of a rough surface of bone using these data predicted that additional contrast levels of 5% could be expected from micromilled surfaces and > 10% for hand polished samples of bone. Thus, micromilling is the best preparation method for bone, since this tissue develops a collagen orientation-related relief on polishing, which may be largely responsible for the (incorrect) supposition that lamellation in bone is related to changes in net degree of mineralisation.     

Investigation of morphological change of green tea polysaccharides by SEM and AFM

The objective of this study is to investigate the morphological structure and its change of green tea polysaccharides (GTPS) before and after enzyme reaction by scanning electron microscope (SEM) and atomic force microscope (AFM). Before enzyme reaction, with the novel sample preparation method SEM images of GTPS have obtained many branches and network structures. After enzyme reaction, the morphological structure of GTPS changed, and surface roughness increased. The microstructure of GTPS from SEM with the novel sample preparation method was in accordance with the results from AFM with the tapping mode. The results indicate that the novel sample preparation of GTPS for SEM is a simple, feasible, and reliable method for observing the surface morphology.     

Detection of CD133 (prominin‐1) in a human hepatoblastoma cell line (HuH‐6 clone 5)

We examined CD133 distribution in a human hepatoblastoma cell line (HuH‐6 clone 5). We directly observed the cultured cells on a pressure‐resistant thin film (silicon nitride thin film) in a buffer solution by using the newly developed atmospheric scanning electron microscope (ASEM), which features an open sample dish with a silicon nitride thin film window at its base, through which the scanning electron microscope beam scans samples in solution, from below. The ASEM enabled observation of the ventral cell surface, which could not be observed using standard SEM. However, observation of the dorsal cell surface was difficult with the ASEM. Therefore, we developed a new method to observe the dorsal side of cells by using Aclar® plastic film. In this method, cells are cultured on Aclar plastic film and the dorsal side of cells is in contact with the thin silicon nitride film of the ASEM dish. A preliminary study using the ASEM showed that CD133 was mainly localized in membrane ruffles in the peripheral regions of the cell. Standard transmission electron microscopy and scanning electron microscopy revealed that CD133 was preferentially concentrated in a complex structure comprising filopodia and the leading edge of lamellipodia. We also observed co‐localization of CD133 with F‐actin. An antibody against CD133 decreased cell migration. Methyl‐β‐cyclodextrin treatment decreased cell adhesion as well as lamellipodium and filopodium formation. A decrease in the cholesterol level may perturb CD133 membrane localization. The results suggest that CD133 membrane localization plays a role in tumor cell adhesion and migration. Microsc. Res. Tech. 76:844–852, 2013. © 2013 Wiley Periodicals, Inc.     

Sample preparation and microscopical investigation techniques for metal and ceramics containing graphite and graphene‐like layered particles

Scanning electron microscopy (SEM) techniques are widely used in microstructural investigations of materials since it can provide surface morphology, topography, and chemical information. However, it is important to use correct imaging and sample preparation techniques to reveal the microstructures of materials composed of components with different polishing characteristics such as grey cast iron, graphene platelets (GPLs)‐added SiAlON composite, SiC and B₄C ceramics containing graphite or graphene‐like layered particles. In this study, all microstructural details of gray cast iron were successfully revealed by using argon ion beam milling as an alternative to the standard sample preparation method for cast irons, that is, mechanical polishing followed by chemical etching. The in‐lens secondary electron (I‐L‐SE) image was clearly displayed on the surface details of the graphites that could not be revealed by backscattered electron (BSE) and Everhart–Thornley secondary electron (E‐T SE) images. Mechanical polishing leads to pull‐out of GPLs from SiAlON surface, whereas argon ion beam milling preserved the GPLs and resulted in smooth surface. Grain and grain boundaries of polycrystalline SiC and B₄C were easily revealed by using I‐L SE image in the SEM after only mechanical polishing without any etching process. While the BSE and E‐T SE images did not clearly show the residual graphites in the microstructure, their distribution in the B₄C matrix was fully revealed in the I‐L SE image.     

A specimen carrier, storage disc system for scanning electron microscopy (SEM): evaluation of stainless steel as a substratum for cell culture in vitro

A method of sample preparation for scanning electron microscopy (SEM) studies based on the use of stainless steel discs as a cell culture substratum is described in detail. A number of different cell lines were grown on stainless steel, and the growth patterns and biocompatibility of cells cultured on stainless steel were compared to identical cells cultured on aluminium, glass and plastic substrata. Stainless steel provides cells with an excellent growth surface which allows these cells to retain their normal growth characteristics and appearance. The non-toxic stainless steel discs can be manipulated through any combination of fixatives and organic solvents. The discs have been incorporated into a versatile system of sample preparation for SEM.     

Use of microwave fixation in the preparation of cell cultures for observation with the scanning electron microscope

This paper describes a method for primary fixation of cultured cells for scanning (SEM) and transmission (TEM) electron microscopy using microwaves alone. This method of fixation takes 8 seconds and is therefore quicker and less expensive than conventional fixation techniques. The preservation of cell morphology is excellent and cultures of mammalian immune system cells and peripheral nervous tissue have been examined using this fixation.     

Use of sputter coating to prepare whole mounts of cytoskeletons for transmission and high-resolution scanning and scanning transmission electron microscopy

This paper describes the use of sputter coating to prepare detergent-extracted cytoskeletons for observation by scanning (SEM), scanning transmission (STEM), inverted contrast STEM, and transmission (TEM) electron microscopy. Sputtered coats of 1–2 nm of platinum or tungsten provide both an adequate secondary electron signal for SEM and good contrast for STEM and TEM. At the same time, the grain size of the coating is sufficiently fine to be just at (platinum) or below (tungsten) the limit of resolution for SEM and STEM. In TEM, the granular structure of platinum coats is resolved, and platinum decoration artifacts are observed on the surface of structures. The platinum is deposited as small islands with a periodic distribution that may reveal information about the underlying molecular structure. This method produces samples that are similar in appearance to replicas prepared by low-angle rotary shadowing with platinum and carbon. However, the sputter-coating method is easier to use; more widely available to investigators; and compatible with SEM, STEM, and TEM. It may also be combined with immunogold and other labeling methods. While TEM provides the highest resolution images of sputter-coated cytoskeletons, it also damages the specimens owing to heating in the beam. In SEM and STEM cytoskeletons are stable and the resolution is adequate to resolve individual microfilaments. The best single method for visualizing cytoskeletons is inverted contrast STEM, which images both the metal-coated cytoskeletal structures and electron-dense material within the nucleus and cytoplasm as white against a dark background. STEM and TEM were both suitable for visualizing colloidal gold particles in immunolabeled samples.     

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.