A method for providing comparative counts of small particles in electron microscopy

A method for estimating the relative concentrations of particles by electron microscopy is described. The method involves the determination of the area of the field of view in the microscope binoculars, relative to a standard area, and is independent of grid type, magnification, operator or microscope. Particle counts using this method are in close agreement with counts made from electron micrographs. The method is simple, rapid and reproducible, and, for low numbers of particles, more accurate than conventional methods.     

Evaluation errors with dust measurements of laminar minerals by scanning electron microscope following German guideline VDI 3492

A number of seemingly fibrous particles are found by evaluation of dust measurements of the industrial mineral vermiculite in accordance with German Guideline VDI 3492, part 2 for “measurements of inorganic fibrous particles- SEM method.” A more thorough investigation based upon an extension of the measuring method proves the nonfibrous morphology of practically all of these particles. The basic idea of this extension is to tilt the filter holder of the electron microscope and to check the exact orientation of the particles on the filter, an examination which is necessary to determine their true length and diameter. The result indicates that only very few fibrous particles actually exist. In the case of vermiculite, these fibrous particles are rolled up and detached flakes called “scrolls.” The systematic error inherent in VDI 3492, part 2, without extension can be quantified in the case of vermiculite to have a factor > 50, that is, fibre counts obtained are more than 50 times too high.     

Automated magnification calibration in transmission electron microscopy using Fourier analysis of replica images

The magnification factor in transmission electron microscopy is not very precise, hampering for instance quantitative analysis of specimens. Calibration of the magnification is usually performed interactively using replica specimens, containing line or grating patterns with known spacing. In the present study, a procedure is described for automated magnification calibration using digital images of a line replica. This procedure is based on analysis of the power spectrum of Fourier transformed replica images, and is compared to interactive measurement in the same images. Images were used with magnification ranging from 1,000 x to 200,000 x. The automated procedure deviated on average 0.10% from interactive measurements. Especially for catalase replicas, the coefficient of variation of automated measurement was considerably smaller (average 0.28%) compared to that of interactive measurement (average 3.5%). In conclusion, calibration of the magnification in digital images from transmission electron microscopy may be performed automatically, using the procedure presented here, with high precision and accuracy.     

Complementary techniques for the characterization of thin film Ti/Nb multilayers

An aberration corrector on the probe-forming lens of a scanning TEM (STEM) equipped with an electron energy-loss spectrometer (EELS) and X-ray energy-dispersive spectrometer (XEDS) has been employed to investigate the compositional variations as a function of length scale in nanoscale Ti/Nb metallic multilayers. The composition profiles of EELS and XEDS were compared with the profiles obtained from the complementary technique of 3D atom probe tomography. At large layer widths (h≥7 nm, where h is the layer width) of Ti and Nb, XEDS composition profiles of Ti/Nb metallic multilayers are in good agreement with the EELS results. However, at reduced layer widths (h≈2 nm), profiles of EELS and atom probe exhibited similar compositional variations, whereas XEDS results have shown a marked difference. This difference in the composition profiling of the layers has been addressed with reference to the effects of beam broadening and the origin of the signals collected in these techniques. The advantage of using EELS over XEDS for these nanoscaled multilayered materials is demonstrated.     

Atomic force microscopic imaging of 30 nm chromatin fiber from partially relaxed plant chromosomes

We have developed a procedure for partially relaxing the barley metaphase chromosomes and exposing fibrous structures from the chromosomes. The observation by atomic force microscopy (AFM) showed that the fibrous structures are typically 0.5 to 1 microm long and 40 to 50 nm in diameter. In higher magnification imaging, we found the fibrous structures were composed of aligned granules and looked like "knobby fiber." These observations are consistent with previously reported features of chromatin fiber observed by AFM and scanning electron microscopy, suggesting that the structures correspond to 30 nm chromatin fibers. We observed the chromatin fiber extending straight from the periphery of the chromosomes in most cases, but fibers with different shapes, such as loop and spiral, were also observed. The procedure reported here will provide a new approach for observing the organization of chromatin fiber to higher-order structures by AFM and other high-resolution microscopy.     

Critical-point drying versus freeze drying for scanning electron microscopy: a quantitative and qualitative study on isolated hepatocytes

Critical-point drying and freeze drying were compared both quantitatively and qualitatively as preparative procedures for scanning electron microscopy. Isolated hepatocytes were used as model cells. Nomarski differential interference contrast microscopy was used for light microscopic measurements of the hepatocytes in the unfixed, the glutaraldehyde fixed, the glutaraldehyde + OsO₄ fixed, the critical-point dried and the freeze dried states. Critical-point dried hepatocytes were found to shrink to 38% of glutaraldehyde + OsO₄ fixed volume, whereas optimal freeze dried hepatocytes (frozen in water saturated with chloroform and freeze dried at 183 K for 84 h) were found to shrink to 51% of glutaraldehyde + OsO₄ fixed volume. Transmission and scanning electron micrographs of the critical-point dried cells showed well-preserved ultrastructure and surface structure. Micrographs of the freeze dried cells showed ultrastructure destroyed by internal ice crystals and surface structure destroyed by external ice crystals. Double-fixed isolated hepatocytes were shown to swell during storage in buffer and to shrink during storage after critical-point drying. For low magnification scanning electron microscopy (up to about 3000 times) both critical-point drying and freeze drying can be used. However, for high magnification scanning electron microscopy, critical-point drying is superior to freeze drying.     

Investigations on the leaf surface ultrastructure in grapevine (Vitis vinifera L.) by scanning microscopy

Several Scanning microscopy techniques were used to investigate the leaf surface ultrastructure in the local “Razegui” grapevine cultivar (Vitis vinifera L.). Conventional scanning electron microscopy performed on glutaraldehyde‐fixed samples allowed observation of well‐preserved epidermal cells with an overlaying waxy layer. At a high magnification, the waxy layer exhibited crystalline projections in the form of horizontal and vertical platelets. Also, to avoid eventual ultrastructural alterations inherent in the use of solvents during sample preparation, fresh leaf blade samples were directly observed by environmental scanning electron microscopy. A classical image of convex living epidermal cells was observed. At 2400× magnification, epicuticular waxes exhibited a granular structure. However, high‐magnification images were not obtained with this device. The atomic force microscopy (AFM) performed on fresh leaf blade samples allowed observation of a textured surface and heterogeneous profiles attributed to epicuticular wax deposits. AFM topography images confirmed further, the presence of irregular crystalloid wax projections as multishaped platelets on the adaxial surface of grapevine leaf. SCANNING 31: 127–131, 2009. © 2009 Wiley Periodicals, Inc.     

Spot counting to locate fetal cells in maternal blood and tissue: a comparison of manual and automated microscopy

BACKGROUND: Fetal cell detection in maternal tissue requires an accurate, efficient, and reproducible microscopy method. Our objective was to compare manual scoring to a commercially available automated scanning system for the detection of chromosome signals by fluorescence in situ hybridization (FISH). METHODS: X and Y chromosome FISH signals were detected on slides of calibrated mixtures of blood, paraffin-embedded liver sections, and post-termination blood. For manual scoring (400x magnification), the number of cells located and duration of scoring were recorded. For automated scanning using the Metasystems Metafer3/Metafer4 Scanning System (200x magnification), duration of scanning, number of gallery images generated, duration of manual review of gallery images, and number of confirmed fetal cells were recorded. RESULTS: From all slides the number of target fetal cells located by manual and automated microscopy was highly correlated (r = 0.90). However, automated scanning required on average 4-fold more time than manual scoring (P < 0.0001), with an average automated scanning time of 9.7 h per slide compared with 2.4 h per slide when scored manually. CONCLUSIONS: In general, the accuracy of automated and manual microscopy is comparable, although manual scoring is more efficient because of the level of magnification necessary for automated scanning of cells, and a large number of gallery images generated by automated scanning that must then be reviewed manually. This suggests that when rapid analysis is required (i.e., clinical situations), manual microscopy is preferable. In contrast, automated scanning may have advantages over manual microscopy when time constraints are less imposed (i.e., research situations).     

Object-marking,a bridge between light and analytical electron microscopy for particles characterization

In problems of air or water pollution, or in evaluation of exposure to pathogenic dusts from biological specimens ( e.g., asbestos fibers) some typical particles can be used as tracers. As far as they have sufficiently typical properties (shape, color, anisotropy,…) particles are rapidly recognized and quantified by light microscopy (LM), even if they are very scarce or dispersed among large amounts of unsignificant ones. For accurate characterization, analytical electron microscopy (scanning or transmission) is required, but cannot be efficiently applied for low concentrations of particles. A technique using a high precision object-marker under LM has been developed in the past by Jedwab (1975) to be used with SEM, but there is actually no equivalent for TEM. Such a technique is proposed in this paper. Its major interest resides in the greater amount of analytical data available for one single particle (high magnification morphology, crystallographic structure, chemistry). Practical results were obtained with asbestos fibers and bodies recovered from biological specimens, but the technique can be extended to many other problems concerning micron-sized particles.     

Application of the focused ion beam technique in aerosol science: detailed investigation of selected, airborne particles

The focused ion beam technique was used to fabricate transmission electron microscope lamellas of selected, micrometre‐sized airborne particles. Particles were sampled from ambient air on Nuclepore polycarbonate filters and analysed with an environmental scanning electron microscope. A large number of particles between 0.6 and 10 µm in diameter (projected optical equivalent diameter) were detected and analysed using computer‐controlled scanning electron microscopy. From the resulting dataset, where the chemistry, morphology and position of each individual particle are stored, two particles were selected for a more detailed investigation. For that purpose, the particle‐loaded filter was transferred from the environmental scanning electron microscope to the focused ion beam, where lamellas of the selected particles were fabricated. The definition of a custom coordinate system enabled the relocation of the particles after the transfer. The lamellas were finally analysed with an analytical transmission electron microscope. Internal structure and elemental distribution maps of the interior of the particles provided additional information about the particles, which helped to assign the particles to their sources. The combination of computer‐controlled scanning electron microscopy, focused ion beam and transmission electron microscopy offers new possibilities for characterizing airborne particles in great detail, eventually enabling a detailed source apportionment of specific particles. The particle of interest can be selected from a large dataset (e.g. based on chemistry and/or morphology) and then investigated in more detail in the transmission electron microscope.     

Erosion of Low-Carbon Steel by Coal Particles

The erosion of low-carbon steel, in the annealed and water-quenched condition, by coal particles, has been studied as a function of particle size, velocity, and angle of impingement. It was found that both unicrostructures, pearlitic and martensitic, showed a maximum erosion at an angle of impingement of between 20 and 30°. Under all condition of testing, the martensitic structure showed the higher rate of erosion. The coal particles were found to fracture on impact with the steel surfaces. The amount of fracture was found to increase will increased velocity and particle size. Erosion rate was found to increase with decreasing particle size and was related to particle velocity (V) through a power law relationship of the form: E = kVⁿ, where the exponent (n) has valves between 2.6 and 3.7. Scanning electron microscopy studies indicated that only the harder mineral constituents in the coal, such as pyrite and quartz, were responsible for the erosion of the steel.     

Active pixel sensor array as a detector for electron microscopy

A new high-resolution recording device for transmission electron microscopy (TEM) is urgently needed. Neither film nor CCD cameras are systems that allow for efficient 3-D high-resolution particle reconstruction. We tested an active pixel sensor (APS) array as a replacement device at 200, 300, and 400 keV using a JEOL JEM-2000 FX II and a JEM-4000 EX electron microscope. For this experiment, we used an APS prototype with an area of 64 x 64 pixels of 20 microm x 20 microm pixel pitch. Single-electron events were measured by using very low beam intensity. The histogram of the incident electron energy deposited in the sensor shows a Landau distribution at low energies, as well as unexpected events at higher absorbed energies. After careful study, we concluded that backscattering in the silicon substrate and re-entering the sensitive epitaxial layer a second time with much lower speed caused the unexpected events. Exhaustive simulation experiments confirmed the existence of these back-scattered electrons. For the APS to be usable, the back-scattered electron events must be eliminated, perhaps by thinning the substrate to less than 30 microm. By using experimental data taken with an APS chip with a standard silicon substrate (300 microm) and adjusting the results to take into account the effect of a thinned silicon substrate (30 microm), we found an estimate of the signal-to-noise ratio for a back-thinned detector in the energy range of 200-400 keV was about 10:1 and an estimate for the spatial resolution was about 10 microm.     

Electron beam-induced thickening of the protective oxide layer around Fe nanoparticles

There are many circumstances in science where the process of measuring the properties of a system alters the system. An imaging process can exert an inadvertent effect on the object being observed. Consequently, what we observe does not necessarily represent what had been present before the observation. Normally, this effect can be ignored if the consequence of such a change is believed not to be significant. The expansion of nanostructured materials has made high-resolution transmission electron microscopy one of the indispensable tools for probing the characteristics of nanomaterials. Modification of nanoparticles by the electron beam during their imaging has been widely noticed and this is generally believed to be due to electron beam-induced heating effect, defect formation in the particles, charging of the particle, or excitation of surrounding gases. However, an explicit experimental identification of which process dominates is often very hard to establish. We report the thickening of native oxide layer on iron nanoparticle under electron beam irradiation. Based on atomic level imaging, electron diffraction, and computer simulation, we have direct evidence that the protecting oxide layer formed on Fe nanoparticle at room temperature in air or oxygen continues to grow during an electron beam bombardment in the vacuum system typical of most TEM systems. Typically, the oxide layer increases from approximately 3 to approximately 6 nm following approximately 1h electron beam exposure typically with an electron flux of 7 x 10(5)nm(-2)s(-1) and an vacuum of approximately 3 x 10(-5)Pa. Partial illumination of a nanoparticle and observation of the shell thickening conclusively demonstrates that many of the mechanisms postulated to explain such processes are not occurring to a significant extent. The observed growth is not related to the electron beam-induced heating of the nanoparticle, or residual oxygen ionization, or establishment of an electrical field, rather it is related to electron beam-facilitated mass transport across the oxide layer (a defect-related process). The growth follows a parabolic growth law.     

Occurrence of melting during the solid particle erosion of copper

Copper single crystals were eroded by glass particles at a velocity of 133 m s^?1 and an incidence angle of 30°. After erosion, surfaces and debris were examined by electron microscopy. Clear evidence was obtained for localized melting of the surface layers; this is associated with embedded fragments of erodant. The most likely cause of melting appears to be the smearing of a thin layer of copper between the erodant particle and the embedded fragments. Under such conditions the layer may melt if the rate of heating during impact is sufficiently larger than the rate of thermal conduction through the low conductivity embedded and impacting glass erodant. The implication of these results for other studies and the erosion of two-phase microstructures is discussed.     

Atomic force and scanning electron microscopy of atmospheric particles

Atomic force microscopy (AFM) and scanning electron microscopy with energy dispersive spectroscopy (SEM-EDS) have been used for both morphological and elemental mass analysis study of atmospheric particles. As part of the geometrical particle analysis, and in addition to the traditional height profile measurement of individual particles, AFM was used to measure the volume relative to the projection area for each particle separately, providing a particle shape model. The element identification was done by the EDS analysis, and the element mass content was calculated based on laboratory calibration with particles of known composition. The SEM-EDS mass measurements from two samples collected at 150 and 500 m above the surface of the Mediterranean Sea were found to be similar to mass calculations derived from the AFM volume measurements. The AFM results show that the volume of most of the aerosols that were identified as soluble marine sulfate and nitrate aerosol particles can be better estimated using cylindrical shapes than spherical or conical geometry.     

A comparative study of colloidal particles as imaging standards for microscopy

Colloidal particles have long been used as imaging standards for electron microscopy and, more recently, for scanning probe microscopy. We have analysed gold, polystyrene and silica colloidal particles by both transmission electron microscopy and atomic/scanning force microscopy in an attempt to determine if any can be truly used as 'standards' of shape and/or size. From the transmission electron micrographs, we have obtained precise information of the particle circumference and mean diameter. By comparing the ratio of these to the value for π, we obtained a measure of the sphericity of the particles. We have also shadowed the particles with metal at a known angle and have analysed the shadow length to determine the particles' heights and shapes. The height information obtained from the shadow length data collected from the transmission electron micrographs was then compared with that obtained by atomic/scanning force microscopy. Our results show that cleaned (washed) silica or polystyrene particles closely approach true spheres. In the case of gold particles, height data obtained from shadow lengths analysed in transmission electron micrographs show good agreement with that obtained from the atomic/scanning force microscopy images even without washing. However, the gold particles often deviate from sphericity. Based upon both the shape and the physical properties of the colloidal particles, silica would be the best choice as a standard. We also have noticed that metal shadowing of colloidal particle samples used for atomic/scanning force microscopy offers an advantage which we call a 'nanoscale metric' visible in the image directly at each particle site. This information can be important if one wishes to use samples prepared from colloidal particles simply and reliably to determine the probe shape for scanning probe microscopy from image deconvolution/restoration methods or as a calibration sample.     

Analysis of submicron particles by scanning electron microscopy-energy-dispersive X-ray spectrometry-accuracy of size measurement

Scanning electron microscopy combined with energy-dispersive x-ray spectrometry (SEM-EDXS) is widely used for particle analysis. In the case of submicron particles, especially for particles that are smaller than 300 nm, the measured particle size is influenced by specimen preparation, SEM operating parameters, the mean atomic number of the particles, and the threshold value used for binarization. The use of uncoated particles on a conductive substrate and image acquisition using an in-lens detector are recommended for precise morphologic results in this size range.     

Evaluation of quantitative procedures for X-ray microanalysis of environmental particles

Scanning electron microscopy combined with energy-dispersive X-ray spectrometry is particularly suited to characterizing morphology and elemental composition of individual microparticles. Although not straightforward, quantitative X-ray microanalysis of low-Z-containing particles is achievable using atmospheric thin-window X-ray detectors. A critical aspect of light element analysis is the choice of substrate material. In this work, particles were deposited on specially developed boron substrates. Three case studies were investigated successively in the order of increasing difficulty. Firstly, hundreds of calcium carbonate (CaCO(3)) particles ranging in size from 0.3 to 10 microm were analyzed. Three quantitative procedures were tested: the "k-ratio" method, conventional ZAF correction, and Monte Carlo simulations. Average relative errors obtained by the reverse Monte Carlo quantitative program named CASINO were better than 2.5 wt %, carbon included. Secondly, further evaluation was carried out on a finely crushed biotite mineral, containing more than nine elements. Finally, airborne particulate matter, consisting of a complex heterogeneous mixture of particles, was investigated. By applying the Monte Carlo quantitative procedure, the observed particles were easily classified into particle types. Pure compounds (e.g., CaSO(4).2H(2)O, SiO(2), CaCO(3), etc) were directly assigned according to stoichiometry. In some cases (marine-derived particles), a partial reactivity of atmospheric particles was demonstrated by quantitative analysis.     

A porous carbon film for the collection of atmospheric aerosol for transmission electron microscopy

This paper describes a method of direct sampling of fine urban aerosol using porous carbon films, to increase collection efficiency and retain the structural integrity of the aerosol. A nuclepore membrane filter was coated with carbon and washed with chloroform to leave a clean porous carbon film on a 200-mesh transmission electron microscope copper grid. The grid was attached to a clean nuclepore filter using carbon cement. Sampling was conducted for 1 h at 1.9 L min⁻¹ flow rate. The porous carbon grid with sample was then assayed using transmission electron microscopy, and negatives were suitable for image analysis. The collection of fine airborne particles using this method is appropriate for electron microscopy, with a minimum of interference to the structure of collected particles.     

Microstructural and mechanical properties of nanometric magnesium oxide particulate-reinforced aluminum matrix composites produced by powder metallurgy method

In this research, aluminum alloy (A356.1) matrix composites reinforced with 1.5, 2.5 and 5 Vol.% nanoscale MgO particles were fabricated via powder metallurgy method. Pure atomized aluminum powder with an average particle size of 1μm and MgO particulate with an average particle size between 60 to 80 nm were used. The specimens were pressed by Cold Isostatic Press machine (CIP), and were subsequently sintered at various sintering temperatures, viz. 575, 600 and 625°C. Optimum amount of reinforcement and sintering temperature were determined by evaluating the density, microstructure and mechanical properties of composites. The composites were characterized by scanning electron microscopy (SEM) and X-ray diffraction (XRD). Hardness and compression tests were carried out in order to identify mechanical properties. Reinforcing the Al matrix alloy with MgO particles improved the hardness and compressive strength of the alloy to a maximum value of 44 BHN and 288 MPa, respectively. The most improved compressive strength was obtained with the specimen including 2.5% of MgO sintered at 625°C. According to the experiments, a sintering temperature of 625°C showed better results than other temperatures. A good distribution of the dispersed MgO particulates in the matrix alloy was achieved.