High resolution SEM imaging of gold nanoparticles in cells and tissues

The growing demand of gold nanoparticles in medical applications increases the need for simple and efficient characterization methods of the interaction between the nanoparticles and biological systems. Due to its nanometre resolution, modern scanning electron microscopy (SEM) offers straightforward visualization of metallic nanoparticles down to a few nanometre size, almost without any special preparation step. However, visualization of biological materials in SEM requires complicated preparation procedure, which is typically finished by metal coating needed to decrease charging artefacts and quick radiation damage of biomaterials in the course of SEM imaging. The finest conductive metal coating available is usually composed of a few nanometre size clusters, which are almost identical to the metal nanoparticles employed in medical applications. Therefore, SEM monitoring of metal nanoparticles within cells and tissues is incompatible with the conventional preparation methods. In this work, we show that charging artefacts related to non‐conductive biological specimen can be successfully eliminated by placing the uncoated biological sample on a conductive substrate. By growing the cells on glass pre‐coated with a chromium layer, we were able to observe the uptake of 10 nm gold nanoparticles inside uncoated and unstained macrophages and keratinocytes cells. Imaging in back scattered electrons allowed observation of gold nanoparticles located inside the cells, while imaging in secondary electron gave information on gold nanoparticles located on the surface of the cells. By mounting a skin cross‐section on an improved conductive holder, consisting of a silicon substrate coated with copper, we were able to observe penetration of gold nanoparticles of only 5 nm size through the skin barrier in an uncoated skin tissue. The described method offers a convenient modification in preparation procedure for biological samples to be analyzed in SEM. The method provides high conductivity without application of surface coating and requires less time and a reduced use of toxic chemicals.     

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.     

Imaging of human colon cancer cells using He-Ion scanning microscopy

Due to its low beam current and charge compensation mechanism He-Ion scanning microscopy is a very promising tool for imaging biological cells. However, to obtain relevant information, the method used for sample preparation is also critical. In this work, we have used a Carl Zeiss Orion Plus helium-ion microscope to study the effect of sample gold coating on the morphology of human colorectal adenocarcinoma Caco2 cells. The fixative glutaraldehyde was used and the selective gold coating of the samples was investigated. A comparative study with standard scanning electron microscopy is presented.     

Creating internal conductivity in dry biological SEM samples by a simple vapour treatment

Internal sample conductivity in scanning electron microscopy can be a valuable alternative to metal coating. Proton conductivity may be used for this purpose. Many solid materials with active hydrogen atoms, such as hydrogen‐ and ammonium‐salts, organic acids, and even ice, are protonic conductors or semiconductors. Here we present a method to generate proton conductivity in dry biological materials. A simple treatment with hydrogen chloride gas or hydrochloric acid vapour for a few minutes provides sufficient conductivity for many samples. After a removal of excess hydrogen chloride vapour with a vacuum desiccator, the objects may be examined in the SEM without metal coating. The use of internally conductive samples extends the range of easy‐to‐perform SEM preparation techniques. It is advantageous for material contrast imaging of uncoated samples, and it can be used in combination with metal coating to enhance conductivity on difficult samples with complex overlapping surfaces, where simple metal coating does not reliably eliminate charging problems.     

A Brief Discussion About Image Quality and SEM Methods for Quantitative Fractography of Polymer Composites

The methodology for fracture analysis of polymeric composites with scanning electron microscopes (SEM) is still under discussion. Many authors prefer to use sputter coating with a conductive material instead of applying low‐voltage (LV) or variable‐pressure (VP) methods, which preserves the original surfaces. The present work examines the effects of sputter coating with 25 nm of gold on the topography of carbon‐epoxy composites fracture surfaces, using an atomic force microscope. Also, the influence of SEM imaging parameters on fractal measurements is evaluated for the VP‐SEM and LV‐SEM methods. It was observed that topographic measurements were not significantly affected by the gold coating at tested scale. Moreover, changes on SEM setup leads to nonlinear outcome on texture parameters, such as fractal dimension and entropy values. For VP‐SEM or LV‐SEM, fractal dimension and entropy values did not present any evident relation with image quality parameters, but the resolution must be optimized with imaging setup, accompanied by charge neutralization. SCANNING 35: 196‐204, 2013. © 2012 Wiley Periodicals, Inc.     

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.     

Wear Resistance of Coated SAE 305 Aluminum Alloy Under Dry Friction Reciprocate Sliding

This work investigates the influence of two coatings on the wear resistance of the SAE 305 aluminum alloy. This material is widely used in power transmission components, and improvement in its surface properties can lead to remarkable impacts in the energy sector. The tested treatments are: (1) anodization and (2) diamond-like carbon coating. Dry friction reciprocate sliding tests using a sphere-on-plane configuration are presented. The untreated reference surface and both treatments are evaluated regarding their roughness, hardness, coefficient of friction, wear rate and morphology. The surface analyzes were carried out using confocal laser and SEM techniques. In comparison with the reference, the diamond-like carbon coating showed a significantly higher wear resistance, whereas the anodization process showed only a minor improvement.     

FE‐SEM observation of swelled seaweed using hydrophilic ionic liquid; 1‐butyl‐3‐methylimidazolium tetrafluoroborate

The method to observe the exact morphology of swelled seaweed as an example of biological material by field emission scanning electron microscopy (FE‐SEM) with the aid of hydrophilic ionic liquid (IL); 1‐butyl‐3‐methylimidazolium tetrafluoroborate is reported. Seaweed was first swelled in 3.5% NaCl solution and then treated with the IL and water mixture in 1:7 weight ratios and centrifuged to remove the excess IL solution. Thus treated seaweed maintained its morphology even at high magnification and did not show drying in the FE‐SEM chamber. This observation technique might be useful for various kinds of biological materials to be observed under FE‐SEM. Microsc. Res. Tech., 2013. © 2012 Wiley Periodicals, Inc.     

Morphological investigation of various orthodontic lingual bracket slots using scanning electron microscopy and atomic force microscopy

Various labial and lingual orthodontic appliances with aesthetic materials have been developed due to an increased demand in aesthetic orthodontic treatment. However, there are few reports regarding the morphology of lingual orthodontic appliances. Therefore, this study evaluates the roughness of slot surfaces of various orthodontic lingual brackets using field emission scanning electron microscopy (FE‐SEM) and atomic force microscopy (AFM). Three types of stainless steel lingual brackets (Stealth^®, 7th Generation^®, and Clippy L^®) and one gold lingual bracket (Incognito?) with a slot size of 0.018 inches × 0.025 inches (0.457 × 0.635 mm²) were selected as representative lingual materials. Both FE‐SEM and AFM examinations showed that the Stealth^® and Clippy L^® brackets had the lowest surface roughness, while the 7th Generation^® bracket had the highest surface roughness. There was a significant difference in surface morphology between the types of lingual brackets, even when composed of the same material. The surface roughness of the bracket slot was dependent on the manufacturing process or surface polishing process rather than the fundamental properties of the bracket materials. There was no significant difference in the mean surface roughness of the slot floor between gold and stainless steel lingual brackets. These findings suggest that, although the gold lingual bracket is very expensive, it has great potential for use in patients with nickel allergy.     

Tribological studies of coated pistons sliding against cylinder liners under laboratory test conditions

The presence of coatings and surface topography play an important role in the tribological performance of sliding components. Depending on the coating used, it is possible to reduce friction and/or reduce wear. However, although there may be low friction and wear‐resistant coatings suitable for use in pistons, some coatings may hinder the tribological performance by changing the lubrication regime or by preventing additives from their intended function through chemical mechanisms. In this work, piston skirt segments extracted from a commercial aluminium alloy piston were coated with a diamond‐like carbon (DLC) coating, a graphite–resin coating or a nickel–polytetrafluoroethylene (Ni–PTFE) coating and were tribologically tested using a reciprocating laboratory test rig against commercial grey cast iron liner segments. The tribological tests used commercial synthetic motor oil at a temperature of 120 °C with a 20 mm stroke length at a reciprocating frequency of 2 Hz. Results showed that the graphite–resin coating, although it may serve as a good break‐in coating, wears rapidly. The Ni–PTFE coating showed friction reduction, whereas the DLC coating wore off quickly due to its small thickness. Furthermore, the higher hardness of the DLC coating relative to the cast iron liner surface led to pronounced changes on the liner counterface by polishing. In contrast with the uncoated piston skirt segments, all of the coatings prevented the formation of a visible tribochemical film on the cast iron surface. Copyright © 2012 John Wiley & Sons, Ltd.     

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.     

Imaging of metal-coated biological samples by scanning tunneling microscopy

A method for imaging biological samples by scanning tunneling microscopy (STM) is presented. There are two main difficulties in imaging biological samples by STM: (1) the low conductivity of biological material and (2) finding a method of reliably depositing the sample on a flat conducting surface. The first of these difficulties was solved by coating the samples with a thin film of platinum-carbon. The deposition problem was solved by a method similar to a procedure used to deposit biological molecules onto field ion microscope (FIM) tips. STM images of bacteriophage T7 and filamentous phage fd are shown. The substrate on which the samples were absorbed was atomically flat gold. The images do not show molecular detail due to the metal coating, but the gross dimensions and morphology are correct for each type of virus. Also, the surface density of virus particles increases and decreases in the way expected when the conditions of deposition are changed. These methods allow reliable and reproducible STM imaging of biological samples.     

Scanning electron microscopy of some endophytic streptomycetes in snakevine--Kennedia nigricans

Soils of all types and locations have generally served as the major sources of streptomycetous bacteria. These organisms are the source of nearly 80% of the world's antibiotics. Now, it is realized that Streptomyces spp. (within the group of prokaryotic filamentous bacteria known as actinomycetes) can exist as endophytes within the interstices of some higher plants. While it is sometimes possible to isolate one or two different streptomycetes from certain plants, most plants are free of these organisms. However, the snakevine (Kennedia nigricans) of the Northern Territory of Australia has yielded at least 39 different endophytic actinomycetes (95% of them being Streptomyces spp.) Most of these isolates possessed no detectable antibiotic properties, while at least seven had antibacterial and antifungal activities. Examination of eight selected cultures by scanning electron microscopy (SEM) as well as environmental scanning electron microcopy (FEI ESEM FEG) (FEI Company, Hillsobro, Ore., USA) revealed unusual patterns, structures, and features of the spores and hyphae of these microorganisms. For instance, as revealed by ESEM FEG for the first time, it has become obvious that extremely fine hair-like structures (average 25-49 nm with gold-coated specimens) exist on the spores and hyphae of some endophytic streptomycetes. The biological purpose of these hair-like protrusions is unknown. Both SEM and ESEM FEG can be effectively used as tools in identification and elucidation of the biology of these organisms. In addition, unusual colony morphology, observed with the unaided eye can very easily be used to distinguish some of these isolates since characteristic donut and pseudo-horn shaped colonies appeared in culture.     

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.     

热沉衬底金刚石/铜复合材料表面金属化工艺研究

为了改善金刚石/铜复合材料的表面特性,采用化学镀镍与电镀金结合的方式。先通过SnCl₂溶液和PdCl₂溶液对复合材料表面进行敏化、活化等预处理,并研究预处理对后续化学镀镍层产生的影响。通过SEM（扫描电子显微镜）、EDXS（能量色散X射线光谱仪）、OM（光学显微镜）和热振、高温烘烤等实验措施数据的分析对复合镀层进行研究,研究结果表明,通过化学镀加电镀的方法在金刚石/铜复合材料表面得到了高结合力、均匀致密的镍合金镀层。     

Diesel‐lubricated ta‐C coatings

Samples coated with tetrahedral amorphous carbon were investigated in two different oscillating test setups regarding their friction and wear properties. As lubricants petrodiesel, rapeseed methyl ester biodiesel and a mixture thereof were used. Compared with a petrodiesel‐lubricated uncoated system, friction was reduced for more than 50% when a tetrahedral amorphous carbon coating was applied and biodiesel added to the lubricant. Impurities of monoglycerides are assumed to be the cause for the friction‐modifying effect. Copyright © 2013 John Wiley & Sons, Ltd.     

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.     

Scuffing Performance of Amorphous Carbon During Dry-Sliding Contact

Scuffing is a major problem that limits the life and reliability of sliding tribo-components. When scuffing occurs, friction force rises sharply and is accompanied by an increase in noise and vibration; severe wear and plastic deformation also occur on the damaged surface. Attempts have been made over the years to combat scuffing by enhancing the surface properties of the machine elements, and by methods involving lubricant formulation and coating application.

In this study, the authors evaluated the scuffing performance of an amorphous, near-frictionless carbon (NFC) coating that provides super-low friction under dry sliding conditions. The test configuration used a ball-on-flat contact in reciprocating sliding. The coating was deposited on HI3 steel. An uncoated 52100 steel ball was tested against various coated flats in room air. Compared to uncoated surfaces, the carbon coating increased the scuffing resistance of the sliding surfaces by two orders of magnitude. Microscopic analysis shows that scuffing occurred on coaled surfaces only if the coating had been completely removed. It appears that depending on coating type, the authors observed that coating failure occurs before scuffing failure by one of two distinct mechanisms: the coating failed in a brittle manner and by spoiling, or by gradual wear.     

Influence of different surface modification technologies on friction of conformal tribopair in mixed and boundary lubrication regimes

Moving machine assemblies are generally designed to operate in full film lubrication regime to ensure high efficiency and durability of components. However, it is not always possible to ensure this owing to changes in operating conditions such as load, speed, and temperature. The overall frictional losses in machines are dependent on the operating lubrication regimes (boundary, mixed or full-film). The present work is thus aimed at investigating the role of different surface modification technologies on friction of a sliding bearing/roller tribopair both in boundary and mixed lubrication regimes. A special test rig comprising of two bearings was built for the experimental studies. Tribological tests were conducted in a wide speed range to enable studies in boundary and mixed lubrication regimes. The influence of application of different surface modification technologies on both the sliding bearing and the roller surfaces on friction has been studied. The rollers used in these studies were provided with five different coatings (hard DLCs and a soft self-lubricating coating). Additionally, two uncoated rollers having different surface roughness were also studied. Uncoated bearings were used in all tribopairs except two. These two bearings were coated with DLC and phosphate coatings respectively and uncoated rollers were the mating counterparts. Friction measurements were made on the new as well as the previously run-in surfaces. It was found that the rollers with self-lubricating coating resulted in lowest boundary friction closely followed by the rollers with the hardest DLC coatings. The DLC coating applied on to the bearing showed lower boundary friction after running-in. Mixed friction has been found to be mainly dependent on the surface topography characteristics of both the original and the run-in surfaces of bearings and rollers. The harder DLC coatings and the phosphated bearing showed the lowest mixed friction due to an efficient running-in of the bearing surface.