Regenerating titanium ventricular assist device surfaces after gold/palladium coating for scanning electron microscopy

Titanium is one of the most commonly used materials for implantable devices in humans. Scanning electron microscopy (SEM) serves as an important tool for imaging titanium surfaces and analyzing cells and other organic matter adhering to titanium implants. However, high‐vacuum SEM imaging of a nonconductive sample requires a conductive coating on the surface. A gold/palladium coating is commonly used and to date no method has been described to “clean” such gold/palladium covered surfaces for repeated experiments without etching the titanium itself. This constitutes a major problem with titanium‐based implantable devices which are very expensive and thus in short supply. Our objective was to devise a protocol to regenerate titaniumsurfaces after SEM analysis. In a series of experiments, titanium samples from implantable cardiac assist devices were coated with fibronectin, seeded with cells and then coated with gold/palladium for SEM analysis. X‐ray photoelectron spectroscopy spectra were obtained before and after five different cleaning protocols. Treatment with aqua regia (a 1:3 solution of concentrated nitric and hydrochloric acid), with or without ozonolysis, followed by sonication in soap solution and sonication in deionized water, allowed regenerating titanium surfaces to their original state. Atomic force microscopy confirmed that the established protocol did not alter the titanium microstructure. The protocol described herein is applicable to almost all titanium surfaces used in biomedical sciences and because of its short exposure time to aqua regia, will likely work for many titanium alloys as well. Microsc. Res. Tech., 2009. © 2009 Wiley‐Liss, Inc.     

3-D morphological characterization of the liver parenchyma by atomic force microscopy and by scanning electron microscopy

A comparative study of atomic force microscopy (AFM) and scanning electron microscopy (SEM) imaging of the healthy human liver parenchyma was carried out to determine the similarities and the differences. In this study, we compared the fine hepatic structures as observed by SEM and AFM. Although AFM revealed such typical hepatic structures as bile canaliculi and hepatocytes, it also showed the location of the nucleus and chromatin granules in rough relief structure, which was not visible by SEM. By contrast, SEM visualized other structures, such as microvilli, the central vein, and collagenous fibers, none of which was visualized by AFM. For better orientation and confirmation of most of the structures imaged by SEM and AFM, Congo Red-stained specimens were also examined. Amyloid deposits in the Disse's spaces were shown especially clearly in these images. The differences between the SEM and AFM images reflected the characteristics of the detection systems and methods used for sample preparation. Our results reveal that more detailed information on hepatic morphology is obtained by exploiting the advantages of both SEM and AFM.     

Identification and ultrastructural imaging of photodynamic therapy-induced microfilaments by atomic force microscopy

Atomic force microscopy (AFM) is an emerging technique for imaging biological samples at subnanometer resolution; however, the method is not widely used for cell imaging because it is limited to analysis of surface topology. In this study, we demonstrate identification and ultrastructural imaging of microfilaments using new approaches based on AFM. Photodynamic therapy (PDT) with a new chlorin-based photosensitizer DH-II-24 induced cell shrinkage, membrane blebbing, and reorganization of cytoskeletons in bladder cancer J82 cells. We investigated cytoskeletal changes using confocal microscopy and atomic force microscopy. Extracellular filaments formed by PDT were analyzed with a tandem imaging approach based on confocal microscopy and atomic force microscopy. Ultrathin filaments that were not visible by confocal microscopy were identified as microfilaments by on-stage labeling/imaging using atomic force microscopy. Furthermore, ultrastructural imaging revealed that these microfilaments had a stranded helical structure. Thus, these new approaches were useful for ultrastructural imaging of microfilaments at the molecular level, and, moreover, they may help to overcome the current limitations of fluorescence-based microscopy and atomic force microscopy in cell imaging.     

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.     

Atomic force microscopy imaging of fragments from the Martian meteorite ALH84001

A combination of scanning electron microscopy (SEM) and environmental scanning electron microscopy (ESEM) techniques, as well as atomic force microscopy (AFM) methods has been used to study fragments of the Martian meteorite ALH84001. Images of the same areas on the meteorite were obtained prior to and following gold/palladium coating by mapping the surface of the fragment using ESEM coupled with energy-dispersive X-ray analysis. Viewing of the fragments demonstrated the presence of structures, previously described as nanofossils by McKay et al . (Search for past life on Mars — possible relic biogenic activity in martian meteorite ALH84001. Science , 1996, pp. 924–930) of NASA who used SEM imaging of gold-coated meteorite samples. Careful imaging of the fragments revealed that the observed structures were not an artefact introduced by the coating procedure.     

Correlative high-resolution morphologic analysis of the three-dimensional organization of human chromosomes

A correlative morphologic analysis was carried out on isolated metaphase chromosomes by means of field emission in-lens scanning electron microscopy (FEISEM) and atomic force microscopy (AFM). Whereas FEISEM provides ultra-high resolution power and allows the surface analysis of biological structures free of any conductive coating, the AFM allows imaging of biological specimens in ambient as well as in physiologic conditions. The analysis of the same samples was made possible by the use of electrical conductive and light transparent ITO glass as specimen holder. Further preparation of the specimen specific for the instrumentation was not required. Both techniques show a high correlation of the respective morphologic information, improving their reciprocal biological significance. In particular, the biological coat represents a barrier for surface morphologic analysis of chromosome spreads and it is sensitive to protease treatment. The chemical removal of this layer permits high-resolution imaging of the chromatid fibers but at the same time alters the chromosomal dimension after rehydration. The high-resolution level, necessary to obtain a precise physical mapping of the genome that the new instruments such as FEISEM and AFM could offer, requires homogeneously cleaned samples with a high grade of reproducibility. A correlative microscopical approach that utilizes completely different physical probes provides complementary useful information for the understanding of the biological, chemical, and physical characteristics of the samples and can be applied to optimize the chromosome preparations for further improvement of the knowledge about spatial genome organization.     

Scanning electron microscopy studies of protein-functionalized atomic force microscopy cantilever tips

Protein-functionalized atomic force microscopy (AFM) tips have been used to investigate the interaction of individual ligand-receptor complexes. Herein we present results from scanning electron microscopy (SEM) studies of protein-functionalized AFM cantilever tips. The goals of this study were (1) to examine the surface morphology of protein-coated AFM tips and (2) to determine the stability of the coated tips. Based on SEM images, we found that bovine serum albumin (BSA) in solution spontaneously adsorbed onto the surface of silicon nitride cantilevers, forming a uniform protein layer over the surface. Additional protein layers deposited over the initial BSA-coated surface did not significantly alter the surface morphology. However, we found that avidin-functionalized tips were contaminated with debris after a series of force measurements with biotinylated agarose beads. The bound debris presumably originated from the transfer of material from the agarose bead. This observation is consistent with the observed deterioration of functional activity as measured in ligand-receptor binding force experiments.     

Sample preparation procedures for biological atomic force microscopy

Since the late 1980s, atomic force microscopy (AFM) has been increasingly used in biological sciences and it is now established as a versatile tool to address the structure, properties and functions of biological specimens. AFM is unique in that it provides three-dimensional images of biological structures, including biomolecules, lipid films, 2D protein crystals and cells, under physiological conditions and with unprecedented resolution. A crucial prerequisite for successful, reliable biological AFM is that the samples need to be well attached to a solid substrate using appropriate, nondestructive methods. In this review, we discuss common techniques for immobilizing biological specimens for AFM studies.     

Toward a better modulus at shallow indentations—Enhanced tip and sample characterization for quantitative atomic force microscopy

Approximations of the geometry of indenting probes, particularly when using shallow indentations on soft materials, can lead to the erroneous reporting of mechanical data in atomic force microscopy (AFM). Scanning electron microscopy (SEM) identified a marked change in geometry toward the tip apex where the conical probe assumes a near linear flat-punch geometry. Polydimethylsiloxane (PDMS) is a ubiquitous elastomer within the materials and biological sciences. Its elastic modulus is widely characterized but the data are dispersed and can display orders of magnitude disparity. Herein, we compare the moduli gathered from a range of analytical techniques and relate these to the molecular architecture identified with AFM. We present a simple method that considers sub-100 nm indentations of PDMS using the Hertz and Sneddon contact mechanics models, and how this could be used to improve the output of shallow indentations on similarly soft materials, such as polymers or cells.     

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).     

An integrated approach to the study of living cells by atomic force microscopy

We describe a technique for studying living cells with the atomic force microscope (AFM) in tapping mode using a thermostated, controlled-environment culture system. We also describe the integration of the AFM with bright field, epifluorescence and surface interference microscopy, achieving the highest level of integration for the AFM thus far described. We succeeded in the continuous, long-term imaging of relatively flat but very fragile cytoplasmic regions of COS cells at a lateral resolution of about 70 nm and a vertical resolution of about 3 nm. In addition, we demonstrate the applicability of our technology for continuous force volume imaging of cultured vertebrate cells.
The hybrid instrument we describe can be used to collect simultaneously a diverse variety of physical, chemical and morphological data on living vertebrate cells. The integration of light microscopy with AFM and steady-state culture methods for vertebrate cells represents a new approach for studies in cell biology and physiology.     

Structural and functional alterations of cellular components as revealed by electron microscopy

Scanning (SEM) and transmission electron microscopy (TEM) are two fundamental microscopic techniques widely applied in biological research for the study of ultrastructural cell components. With these methods, especially TEM, it is possible to detect and quantify the morphological and ultrastructural parameters of intracellular organelles (mitochondria, Golgi apparatus, lysosomes, peroxisomes, endosomes, endoplasmic reticulum, cytoskeleton, nucleus, etc.) in normal and pathological conditions. The study of intracellular vesicle compartmentalization is raising even more interest in the light of the importance of intracellular localization of mediators of the signaling in eliciting different biological responses. The study of the morphology of some intracellular organelles can supply information on the bio‐energetic status of the cells. TEM has also a pivotal role in the determination of different types of programmed cell death. In fact, the visualization of autophagosomes and autophagolysosomes is essential to determine the occurrence of autophagy (and also to discriminate micro‐autophagy from macro‐autophagy), while the presence of fragmented nuclei and surface blebbing is characteristic of apoptosis. SEM is particularly useful for the study of the morphological features of the cells and, therefore, can shed light, for instance, on cell–cell interactions. After a brief introduction on the basic principles of the main electron microscopy methods, the article describes some cell components with the aim to demonstrate the huge role of the ultrastructural analysis played in the knowledge of the relationship between function and structure of the biological objects. Microsc. Res. Tech., 76:1057–1069, 2013. © 2013 Wiley Periodicals, Inc.     

Correlative light and electron microscopy for the analysis of cell division

Correlative light and electron microscopy (CLEM) has recently gained increasing attention, because it enables the acquisition of dynamic as well as ultrastructural information about subcellular processes. It is the power of combining the two imaging modalities that gives additional information as compared to using the imaging techniques separately. Here, we briefly summarize two CLEM approaches for the analysis of cells in mitosis and cytokinesis.     

Influence of nanostructure composition on its morphometric characterization by different techniques

Morphometric characterization of nanoparticles is crucial to determine their biological effects and to obtain a formulation pattern. Determining the best technique requires knowledge of the particles being analyzed, the intended application of the particles, and the limitations of the techniques being considered. The aim of this article was to present transmission (TEM) and scanning (SEM) electron microscopy protocols for the analysis of two different nanostructures, namely polymeric nanoemulsion and poly(lactic‐co‐glycolic acid) (PLGA) nanoparticles, and to compare these results with conventional dynamic light scattering (DLS) measurements. The mean hydrodynamic diameter, the polydispersity index, and zeta potential of the nanostructures of polymeric nanoemulsion were 370.5 ± 0.8 nm, 0.133 ± 0.01, and ?36.1 ± 0.15 mV, respectively, and for PLGA nanoparticles were 246.79 ± 5.03 nm, 0.096 ± 0.025, and ?4.94 ± 0.86 mV, respectively. TEM analysis of polymeric nanoemulsion revealed a mean diameter of 374 ± 117 nm. SEM analysis showed a mean diameter of 368 ± 69 nm prior to gold coating and 448 ± 70 nm after gold coating. PLGA nanoparticles had a diameter of 131 ± 41.18 nm in TEM and 193 ± 101 nm in SEM. Morphologically, in TEM analysis, the polymeric nanoemulsions were spherical, with variable electron density, very few showing an electron‐dense core and others an electron‐dense surface. PLGA nanoparticles were round, with an electron‐lucent core and electron‐dense surface. In SEM, polymeric nanoemulsions were also spherical with a rough surface, and PLGA nanoparticles were round with a smooth surface. The results show that the “gold standards” for morphometric characterization of polymeric nanoemulsion and PLGA nanoparticles were, respectively, SEM without gold coating and TEM with negative staining. Microsc. Res. Tech. 77:691–696, 2014. © 2014 Wiley Periodicals, Inc.     

Traditional coating techniques in scanning electron microscopy compared to uncoated charge compensator technology: Looking at human blood fibrin networks with the ZEISS ULTRA Plus FEG‐SEM

Scanning electron microscopy (SEM) studies surface morphology. Biological material needs to be coated to render the material conductive, and gold coating is traditionally used, although other coating material like carbon and ruthenium vapors may also be used. With modern SEM technology (e.g., ZEISS ULTRA Plus FEG‐SEM), we are able to work at very low kilovolts and also view fine surface structure in much better detail than with previous older technology. Some machines also allow for the study of uncoated material, although this is usually not done with biological material. This study focuses on surface clarity by comparing gold, ruthenium vapor, and carbon coating techniques for biological material. Human fibrin networks are used as example. Uncoated specimens are also viewed with a ZEISS ULTRA Plus FEG‐SEM because of its unique nitrogen charge compensator, and here, the first micrographs for uncoated human fibrin networks versus carbon, gold, and ruthenium coating are shown. We conclude that gold coating for biological material is not preferable with the latest SEM machines, as this method forms gold islands on top of the biological material and therefore produces a false surface morphology. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.     

Factors which influence light microscopic visualization of biological material in sections prepared for electron microscopy

Epoxy-embedded biological material, sectioned for conventional or high-voltage electron microscopy, can be visualized within the section with good contrast and detail by phase-contrast or dark-field light microscopy. The (phase) contrast of such material is not substantially influenced by the type of embedding resin or section support substrate. It is, however, influenced by the type of fixation, by heavy metal (uranyl and lead) staining and by the section thickness. After screening ultrathin and semithin sections for content with the light microscope, one need stain and examine only those grids containing sections of interest. This approach eliminates the need to screen sections with the electron microscope and, in some cases, the need to stain non-useful sections. This time-saving procedure is particularly useful for studies requiring ultrastructural examination of a selected area or structure which is large enough to be visualized with the light microscope but which comprises only a small volume of the embedded material.     

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.     

The structure of proteoglycan aggregate determined by atomic force microscopy

Proteoglycan aggregate is the major extracellular matrix component in cartilage, comprising about 18% of the dry weight of hyaline cartilage. The proteoglycan aggregate is the major substance in cartilage which resists compression in the joint. The purpose of this study was to utilize the newly developed imaging technique, Atomic force Microscopy (AFM), to visualize the ultrastructure of proteoglycan aggregates. The proteoglycan aggregate molecules were imaged in air using the tapping mode of the AFM. The images illustrated the ultrastructure of the aggregates, especially the individual proteoglycan and the core hyaluronic acid. In addition to the length and width of each molecule, the height of the proteoglycan aggregates and the individual proteoglycans could be directly measured. The images of the ultrastructures of proteoglycan aggregates visualized from the AFM are comparable with those using conventional electron microscopy approaches. Nevertheless, the sample preparation for AFM imaging does not involve fixation, staining, coating, and other routine procedures required for traditional electron microscopy imaging. Thus, this technique could be a simple alternative approach for future analysis of proteoglycan aggregate and its assembly.     

The use of rhodamine 6G and fluorescence microscopy in the evaluation of phospholipid-based polymeric biomaterials

A technique is described that allows the staining and subsequent visualization of polymers that contain the phosphorylcholine (PC) group. These materials are useful as bulk materials or coatings for the fabrication of medical devices. The staining method employs rhodamine 6G, which can be simply and rapidly applied to the polymer coating and imaged using fluorescence microscopy. The specificity of the staining for the PC polymers makes this technique suitable for the evaluation of a wide range of substrates and provides qualitative information on coating uniformity, coverage and morphology. It can be used to examine the durability of, and defects in, the coating. Statistical analysis of the fluorescent intensity by measuring the pixel value during imaging can allow for the method to be used as a quality control tool.