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.     

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.     

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.     

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.     

Fabrication of gold tips by chemical etching in aqua regia

We present a method to produce sharp gold tips for applications in apertureless near-field optical microscopy and spectroscopy. Thin gold wires are tapered by chemical etching in aqua regia, covered by an isooctane protective layer. Tips with apical radii of curvature of <50 nm are obtained with a 40% yield. The tip performances have been checked by shear-force imaging of amyloid fibrils samples and compared to optical fiber probes. The analysis of the tip morphology, carried out by scanning electron microscopy, shows the existence of two different etching processes occurring in bulk and at the liquid-liquid interface. A simple analytical model is presented to describe the dynamics of the tip formation at the liquid-liquid meniscus interface that fits remarkably well the experimental results in terms of tip shape and length.     

A protocol for processing the delicate larval and prepupal salivary glands of Drosophila for scanning electron microscopy

Although scanning electron microscopy (SEM) has been broadly used for the examination of fixed whole insects or their hard exoskeleton‐derived structures, including model organisms such as Drosophila, the routine use of SEM to evaluate vulnerable soft internal organs and tissues was often hampered by their fragile nature and frequent surface contamination. Here, we describe a simple four‐step protocol that allows for the reliable and reproducible preparation of the larval and prepupal salivary glands (SGs) of Drosophila for SEM devoid of any surface contamination. The steps are to: first, proteolytically digest the adhering fat body; second, use detergent washes to remove contaminating coarse tissue fragments, including sticky remnants of the fat body; third, use nonionic emulsifying polysorbate emulsifiers to remove fine contaminants from the SGs surface; and fourth, use aminopolycarboxylate‐based chelating agents to detach sessile hemocytes. Short but repeated rinses in 100 μL of a saline‐based buffer between steps ensure efficient removal of remnants removed by each treatment. After these steps, the SGs are fixed in glutaraldehyde, postfixed in osmium tetroxide, dehydrated, critically point‐dried, mounted on aluminum stubs, sputter coated with gold–palladium alloy and examined in the SEM.     

High-resolution scanning electron microscopy of immunogold-labelled cells by the use of thin plasma coating of osmium

The feasibility of plasma coating of a thin osmium layer for high‐resolution immuno‐scanning electron microscopy of cell surfaces was tested, using Drosophila embryonic motor neurones as a model system. The neuro‐muscular preparations were fixed with formaldehyde and labelled with a neurone‐specific antibody and 10 or 5 nm colloidal gold‐conjugated secondary antibodies. The specimens were post‐fixed with osmium tetroxide and freeze‐dried. Then they were coated with a 1–2 nm thick layer of osmium using a hollow cathode plasma coater. The thin and continuous coating of amorphous osmium gave good signals of gold particles and fine surface structures of neurites in backscattered electron images simultaneously. This method makes it possible to visualize the antigen distribution and the three‐dimensionally complex surface structures of cellular processes with a resolution of several nanometres.     

Rapid single‐molecule imaging in cyclic olefin copolymer channels

Rapid preparation of high quality capture surfaces is a major challenge for surface‐based single‐molecule protein binding assays. Here we introduce a simple method to activate microfluidic chambers made from cyclic olefin copolymer for single‐molecule imaging with total internal reflection fluorescence microscopy. We describe a surface coating protocol and demonstrate single‐molecule imaging in off‐the‐shelf microfluidic parts that can be activated for binding assays within a few minutes. As the first example, biotinylated protein directly captured on the neutravidin‐coated surface was detected using fluorescently labeled antibody. We then showed detection of a fusion construct containing green fluorescence protein and verified its single fluorophore behavior by observing stepwise photobleaching events. Finally, a target protein was identified in the crude cell lysate using antibody–sandwich complex formation. In all experiments, controls were completed to ensure that nonspecific binding to the surface was minimal. Based on our results, we conclude that the simple surface preparation described in this paper enables single‐molecule imaging assays without time‐consuming coating procedures. Microsc. Res. Tech. 78:309–316, 2015. © 2015 Wiley Periodicals, Inc.     

Friction and Lifetime of Laser Surface–Textured and MoS2-Coated Ti6Al4V Under Dry Reciprocating Sliding

The use of lasers for creating defined textured patterns on surfaces has steadily gained attention during the past decade. These textures can contribute to friction reduction by acting as a reservoir for lubricant, hydrodynamic bearing and trap for wear debris. In the present work, titanium alloy surfaces were textured using a nanosecond pulsed laser and subsequently coated with MoS₂. The samples were tested under dry reciprocating sliding conditions using two different oscillation amplitudes. During the test, the evolution of the coefficient of friction as a function of the number of cycles was measured until coating failure. The influence of the dimple distance on friction and lifetime was evaluated and verified by post-mortem analyses of the samples using optical, confocal and SEM microscopy as well as EDS and XPS analyses. The results show that under certain conditions, surface texturing can reduce friction, extend the lifetime of the coating and provide a progressive coating degradation until failure.     

Reciprocating Sliding Wear Performance of Hard Coating on Modified Titanium Alloy Surfaces

The high strength, low weight, and outstanding corrosion resistance properties possessed by titanium alloys have led to a wide range of successful applications in aerospace, automotive, and chemical industries and in power generation. Titanium alloys are characterized by poor wear resistance properties and their utilization has been excessive in nontribological applications. Surface texturing is a well-known and effective means of surface modification to improve the tribological properties of sliding surfaces. In the present work, modification of titanium alloy surfaces (Ti6Al4V) was done by lapping and laser surface texturing. The wear-resistant coating, AlCrN, was applied over the modified titanium alloy surfaces, with and without a chromium interlayer. Linear reciprocating sliding wear tests were performed with ball-on-flat contact geometry to evaluate the tribological performance of the coated alloy. The tests were performed under different normal loads for a period of 105 cycles at a frequency of 5 Hz. The friction force between the contact pair and displacement of the ball were simultaneously observed using a force transducer and laser displacement sensor. Optical microscopy was used to quantify the wear volume by measuring the wear scar diameter on both the specimen and the counterbody. Scanning electron microscopy (SEM) was employed to study the morphology of the wear scar. The characteristic behavior of the AlCrN coating such as bonding strength, wear volume, wear rate, and coefficient of friction with the chromium interlayer was evaluated and compared with the coating directly applied over the substrate. The coating on the textured surface, with the chromium interlayer showed better tribological performance.     

The effect varied scanning electron microscopy desiccation techniques has on demineralized dentin

The study objective was to assess (a) the effect of a rubbing‐application of ethylenediaminetetraacetic acid (EDTA) or citric acid (CA) has on the ultrastructure of surface dentin and (b) the effect of two scanning electron microscopy (SEM) desiccation preparation techniques have on the collagen surface produced. Treatment regions on proximal root surfaces of extracted human teeth were root planned to expose dentin. Cotton pellets soaked in either 30% CA or 24% EDTA solution were rubbed on the treatment region then processed for SEM using one of two desiccation techniques, that is, (a) critically point dried from liquid CO₂ (control) or (b) air‐dried from tetramethylsilane (experimental). Specimens were coated with gold/palladium and viewed/photographed with an SEM. Specimens of the control groups displayed tufted fibrils (CA > EDTA) with many dentin tubules being partially obscured by overhanging fibrils. Air‐dried specimens of both treatment groups displayed a flat intact monolayer devoid of a matted meshwork of fibrous collagen. Discrete fibril “sprigs,” emanating from the surface monolayer, were characteristic of the EDTA group only. The rubbing‐application of EDTA on dentin produces a tufted fibril surface somewhat similar to that produced by CA. Air‐drying desiccation of both resulted in marked distortion with fibril collapse/coalescence of the tufted collagen matrix.     

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.     

Tribological Impact of SiC Encapsulation of Released Polycrystalline Silicon Microstructures

A method for coating released polysilicon microstructures with thin, uniform and conformal coatings of SiC derived from the single source precursor, 1,3-disilabutane (DSB) has been developed. This coating method has been successfully applied to micromechanical test devices which allow evaluation of friction and wear properties of the coating. Here, data on the coefficient of static friction of SiC coatings produced from DSB is presented. Also, a comparative wear study for devices which have been oxidized, treated with an anti-adhesion coating, and SiC coated is shown. Wear is examined by scanning electron microscopy (SEM) on devices which have been cycled repetitively under a nominal load. It is found that the application of a few nanometers-thin SiC coating provides exceptional wear resistance as well as significant reduction in friction on the microscale.     

Structure analysis of sputter-coated and ion-beam sputter-coated films: a comparative study

Gold, platinum and tungsten films were deposited by low energy input (7 mA, 450 V), or high deposition rate (80 mA, 1500 V), diode sputter coating and by ion beam sputter coating. Film structures on Formvar coated grids and on the surface of coated erythrocytes, resin embedded, sectioned, and recorded at high magnification in a TEM were compared using computer-assisted measurements and analysis of film thickness and grain size. The average grain size of the thinnest gold and platinum films was relatively independent of the mode or rate of deposition but as the film thickness increased, significant differences in grain size and film structure were observed. Thick platinum or gold films deposited by low energy input sputter coating contained large grain size and electron transparent cracks; however, more even films with narrower cracks but larger grain size were produced at high deposition rates. Ion beam sputter coated gold had relatively large grain size in 10 nm thick films, but beyond this thickness the grains coalesced to form a continuous film. Platinum films deposited by ion beam sputter coating were even and free of electron transparent cracks and had a very small grain size (1–2 nm), which was relatively independent of the film thickness. Tungsten deposition either by low energy input or ion beam sputter coating resulted in fine grained even films which were free of electron transparent cracks. Such films remained granular in substructure and had a grain size of about 1 nm which was relatively independent of film thickness. Tungsten films produced at high deposition rates were of poorer quality. We conclude that thick diode sputter coated platinum and gold films are best deposited at high deposition rates provided the specimens are not heat sensitive, the improvement in film structure being more significant than the slight increase in grain size. Thick diode or ion beam sputter coated gold films should be suitable for low resolution SEM, and thin discontinuous gold films for medium resolution SEM. Diode sputter coated platinum should be suitable for medium resolution SEM and ion beam sputter coated platinum for medium and some high resolution SEM. 1–5 nm thick tungsten films, deposited by low energy input or ion beam sputter coating should be suitable for high resolution SEM, particularly where contrast is of less importance than resolution.     

Quantitative detection of gold nanoparticles on individual, unstained cancer cells by scanning electron microscopy

Gold nanoparticles are rapidly emerging for use in biomedical applications. Characterization of the interaction and delivery of nanoparticles to cells through microscopy is important. Scanning electron microscopes have the intrinsic resolution to visualize gold nanoparticles on cells. A novel sample preparation protocol was developed to enable imaging of cells and gold nanoparticles with a conventional below lens scanning electron microscopes. The negative influence of 'charging' on the quality of scanning electron microscopes' images could be limited by deposition of biological cells on a conductive (gold) surface. The novel protocol enabled high-resolution scanning electron microscopes' imaging of small clusters and individual gold nanoparticles on uncoated cell surfaces. Gold nanoparticles could be counted on cancer cells with automated routines.     

Performance results of MEMS coated with a conformal DLC

A MEMS electrostatic lateral output motor has been successfully coated with a diamond like carbon (DLC) coating to protect against wear. Experiments were performed to characterize coating chemistry and performance. Friction results from accelerated screening tests using a miniature, lightly loaded ball on flat tribometer showed that the DLC coating maintained low friction longer compared to uncoated silicon. DLC on DLC experiments showed the lowest friction, and those that were run in 30% RH showed a much longer lifetime than ones run in dry air. Uniformity of DLC coverage on MEMS was verified by Auger electron spectroscopy (AES), microRaman spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM) and energy dispersive X-ray spectroscopy (EDS). Micrographs revealed that there is about a 3:1 ratio of DLC coating for a line of sight deposition region to a non line of sight deposition region. DLC coated MEMS outperformed uncoated MEMS by 16X in air and 300X in vacuum, albeit performance in vacuum was lower than in air. A very clear difference in wear debris was seen between devices run in air and in vacuum. Cylindrical rolls were dominant in the devices that were run in air and platelets were dominant on devices run in vacuum. Ultimately, the DLC coating was found to greatly improve performance over uncoated MEMS.     

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.     

Relating contact conditions to abrasive wear

Damage caused by particles within rolling/sliding contacts can severely reduce the operational life of machinery such as roller bearings, gears and pumps.Abrasive wear of spherical roller thrust bearings has been studied using a stylus apparatus and scanning electron microscopy (SEM). Both a standard bearing and a bearing with rollers coated with metal mixed amorphous carbon (Me-C:H) were studied. The SEM measurements were performed systematically across the contact surfaces so that surfaces with gradually different contact situations could be examined. These measurements were compared to the measured wear depth of the components of the roller bearing. Also, the calculated contact conditions in terms of creep, contact size and surface separation have been related to the observed wear pattern at various locations.To attempt to understand the wear behaviour of the bearing with coated rollers, the coating as well as the material content of the surfaces were examined using both SEM and energy dispersive X-ray spectrometry (EDS). This revealed that the coating did not flake off but rather was scratched off.It is possible to link the abrasive wear behaviour to the contact conditions. It is crucial to understand this relationship when building a simulation model of abrasive wear.     

Bacterial biofilm on successful and failed orthodontic mini‐implants—a scanning electron microscopy study

Mini‐implants have been extensively used in Orthodontics as temporary bone anchorage devices. However, early failure of mini‐implants due to mobility might occur and the colonization of their surfaces by pathogenic bacteria has been referred to as one of the contributing factors. In this study, scanning electron microscopy (SEM) was used to assess the presence of microorganisms adhered to the surface of mini‐implants that failed due to loss of stability. Twelve self‐drilling titanium mini‐implants (1.6 mm diameter × 9.0 mm long) were collected from 12 patients undergoing orthodontic treatment—7 successful and 5 failed mini‐implants. The mean time of permanence in the mouth was 15.8 and 2.4 months for successful and failed mini‐implants, respectively. The devices were placed in the maxilla and/or mandible and removed by the same surgeon and were processed for SEM analysis of the presence of microorganisms on their surfaces (head, transmucosal profile, and body). Extensive bacterial colonization on mini‐implant head and transmucosal profile was observed in all successful and failed mini‐implants. None of the failed mini‐implants exhibited bacteria on its body and only one mini‐implant belonging to the successful (stable) group exhibited bacteria on its body. The results did not suggest a relationship between failure and presence of bacterial colonies on mini‐implant surfaces. Microsc. Res. Tech. 78:1112–1116, 2015. © 2015 Wiley Periodicals, Inc.