Sectioning and imaging hard mineral fibres in biological tissues

A technique is described which overcomes the problems associated with sectioning biological tissue containing hard mineral fibres. 0·2–0·5 μm thick sections were cut with a diamond knife, placed in a folding grid, conventionally stained with uranyl acetate and lead citrate and viewed at an accelerating voltage of 200 kV in the scanning transmission mode.     

Effect of microscopic parameters on EBSD spatial resolution

In this study, a quantitative approach is proposed to understand the effect of the accelerating voltage and the probe current on the physical resolution of EBSD. The accelerating voltage was varied from 5 to 30kV and probe currents of 1, 10, and 40nA were selected. The lateral, longitudinal, and depth resolutions at 10kV and 1nA were 34.5, 44.7, and 46nm for copper, respectively. When the accelerating voltage was in the range of 5-20kV, the ratio of the longitudinal to the lateral resolution was below the theoretical ratio of 2.9. Considering the channeling effect, the best physical depth resolution of 38nm was achieved at 5kV and 10nA. The physical depth resolution in an EBSD measurement is much larger due to the channel effect than that obtained without considering this effect.     

Dynamical electron diffraction simulation for non‐orthogonal crystal system by a revised real space method

In the transmission electron microscopy, a revised real space (RRS) method has been confirmed to be a more accurate dynamical electron diffraction simulation method for low‐energy electron diffraction than the conventional multislice method (CMS). However, the RRS method can be only used to calculate the dynamical electron diffraction of orthogonal crystal system. In this work, the expression of the RRS method for non‐orthogonal crystal system is derived. By taking Na₂Ti₃O₇ and Si as examples, the correctness of the derived RRS formula for non‐orthogonal crystal system is confirmed by testing the coincidence of numerical results of both sides of Schrödinger equation; moreover, the difference between the RRS method and the CMS for non‐orthogonal crystal system is compared at the accelerating voltage range from 40 to 10 kV. Our results show that the CMS method is almost the same as the RRS method for the accelerating voltage above 40 kV. However, when the accelerating voltage is further lowered to 20 kV or below, the CMS method introduces significant errors, not only for the higher‐order Laue zone diffractions, but also for zero‐order Laue zone. These indicate that the RRS method for non‐orthogonal crystal system is necessary to be used for more accurate dynamical simulation when the accelerating voltage is low. Furthermore, the reason for the increase of differences between those diffraction patterns calculated by the RRS method and the CMS method with the decrease of the accelerating voltage is discussed.     

TEM-tomography of FAU-zeolite crystals containing Pt-clusters

A method for preparing ultrathin sections (- 20 nm) of inorganic solids has been developed using ultramicrotomy of resin-embedded crystal fragments. Undamaged crystals, oriented along a crystallographic direction, could be imaged with transmission electron microscopy (TEM) at a resolution better than 0.5 nm. The true internal structure of the crystals could be investigated by imaging the second in a series of at least three consecutive ultrathin sections. Such TEM-tomography proved that Pt-ion exchanged FAU zeolite crystals, after reduction and oxidation, are occupied internally and randomly of large platinum clusters mainly in the {111-twin planes. TEM-tomography could be useful in man made nanostructures like semiconductors, epitaxial thin films, hard metal coatings, ceramics, catalysts, and biomaterials.     

Transmission electron microscopic X-ray quantitative analysis of human dentin at 200 kV accelerating voltage

Qualitative and quantitative x-ray energy dispersive spectroscopy is now used successfully to analyze many features and processes in inorganic samples. When applied to inorganic samples, however, the results are often less satisfactory due to problems of preparation of organic samples, difficulty of measuring x-rays from organic samples, damage of the sample by the electron beam, and other practical problems. In the present study we used a high voltage transmission electron microscope equipped with an energy dispersive x-ray spectrometer to examine accurate quantitative standardless analysis of thin sections of an organic sample, human dentin. Based on our experiments we found the important parameters for quantitative analysis were sample thickness and appropriate choice of model sample. Further, we show that the method of Cliff and Lorimer can be used with biological samples at 200 kV, and we show that quantitative analysis of human dentin can be carried out at 200 kV. Finally, we show that areas of human dentin can be differentiated by their morphological characteristics and x-ray analyses obtained in the transmission electron microscope.     

Perspectives on low voltage transmission electron microscopy as applied to cell biology

Low voltage transmission electron microscopy (LVTEM) with accelerating voltages as low as 5 kV was applied to cell biology. To take advantage of the increased contrast given by LVTEM, tissue preparation was modified omitting all heavy metals such as osmium, uranium, and lead from the fixation, on block staining and counterstaining. Nonstained ultra‐thin tissue sections (40 nm thick) generated highly contrasted images. While the aspect of the cells remains similar to that obtained by conventional TEM, some new substructures were revealed. The pancreatic acinar cells granules present a heterogeneous matrix with partitions corresponding to segregation of their different secretory proteins. Microvilli display their core of microfilaments anchored to the dense top membrane. Mitochondria revealed the presence of distinct particles along their cristea membranes that may correspond to the ATP synthase complexes or oxysomes. The dense nuclear chromatin displays a honey‐comb appearance while distinct beads aligned along thin threads were seen in the dispersed chromatin. These new features revealed by LVTEM correlate with structures described or predicted through other approaches. Masking effects due to thickness of the tissue sections and to the presence of heavy metals must have prevented their observation by conventional TEM. Furthermore, the immunogold was adapted to LVTEM revealing nuclear lamin‐A at the edge of the dense chromatin ribbons. Combining cytochemistry with LVTEM brings additional advantages to this new approach in cell biology. Microsc. Res. Tech. 77:999–1004, 2014. © 2014 Wiley Periodicals, Inc.     

Low-voltage electron microscopy of polymer and organic molecular thin films

We have demonstrated the capabilities of a novel low-voltage electron microscope (LVEM) for imaging polymer and organic molecular thin films. The LVEM can operate in transmission electron microscopy, scanning transmission electron microscopy, scanning electron microscopy, and electron diffraction modes. The microscope operates at a nominal accelerating voltage of 5 kV and fits on a tabletop. A detailed discussion of the electron-sample interaction processes is presented, and the mean free path for total electron scattering was calculated to be 15 nm for organic samples at 5 kV. The total end point dose for the destruction of crystallinity at 5 kV was estimated at 5 x 10(-4) and 3.5 x 10(-2) C/cm2 for polyethylene and pentacene, respectively. These values are significantly lower than those measured at voltages greater than 100 kV. A defocus series of colloidal gold particles allowed us to estimate the experimental contrast transfer function of the microscope. Images taken of several organic materials have shown high contrast for low atomic number elements and a resolution of 2.5 nm. The materials studied here include thin films of the organic semiconductor pentacene, triblock copolymer films, single-molecule dendrimers, electrospun polymer fibers and gold nanoparticles.     

Investigation of the morphology, viability and mechanical properties of yeast cells in environmental SEM

The mechanical properties of biological cells at nanoscale may be characterized using an environmental scanning electron microscopy (ESEM) combined with a force measurement device. However, the electron beam radiation in an ESEM may damage a specimen. So far, little is known about the radiation damage to biological cells. In this work, single yeast cells were imaged using an ESEM under both high and low vacuum modes. The changes in their morphology and viability were monitored as a function of radiation time for a given beam current of 538 pA corresponding to 10 kV accelerating voltage and spot size 4. Under the two modes, the radiation damage to the morphology of yeast cells became evident after an exposure time of 3 min, but under the low vacuum mode, the damage to their morphology was more severe. However, all cells lost their viability after 5 min under the high vacuum mode with the electron beam off from an initial viability of 95+/-1%. In contrast, the viability of cells under the low vacuum mode was found to be approximately 20% after 20 min. In addition, a newly developed ESEM-based nanomanipulation technique was applied to measure the force imposed on single yeast cells and their deformation, including contact diameter and central lateral diameter for the compression of single yeast cells to a given displacement within a time frame of 1 min, and the data obtained may be used to validate mathematical modeling of the stress-strain relationship for the compression of cells in order to determine their intrinsic mechanical property parameters.     

High-voltage electron microscopy of inorganic particles in intact blood cells

Preliminary results obtained by examining intact fixed human red and white blood cells containing inorganic particles under the high-voltage transmission electron microscope are described. Iron filings and ferritin ingested in vitro by granulocytes were observed as were inorganic particles in red cells from a case of lead poisoning. The nature of the particles in red cells is discussed. A faint intracellular network was seen in normal red cells and in cells from a case of lead poisoning. It was found possible to focus the electron microscope at different planes within the cell. The optimum accelerating voltage for red cells appeared to be around 750 kV, whereas a clear image of granulocytes was obtained at voltages between 750 and 1000 kV. The first results indicate that it is possible to examine intact blood cells under the high-voltage electron microscope. Further work is in progress to determine if more information can be acquired by this technique.     

Precise and reproducible deposition of thin and ultrathin carbon films by flash evaporation of carbon yarn in high vacuum

A simple, small device and its use for reproducible flash evaporation of carbon yarn in high vacuum are characterized. Using this flash evaporator, carbon films of any thickness up to 20 nm can be deposited without spark generation under minimized photon radiation, and with an accuracy better than ±0·2 nm. The films have less background structure (imaged in phase contrast) than conventionally rod evaporated films and are therefore suitable for many kinds of thin and ultrathin carbon film applications in electron microscopy, e.g. as backing for formvar films or sections, as isolating carbon layers for autoradiography, as ultrathin films (floated from mica) for support of macromolecules to be metal shadowed and as support and cover for negative staining of various specimens by the sandwich technique.     

A linear accelerator for simulating micrometeorites

The theory, the estimated parameters, and the design features of the linear accelerator capable of accelerating charged dust particles 0.1–10 μm in diameter to velocities of 12 km/s are presented. The electrodynamical circuit of the accelerator is composed of 27 acceleration gaps, each of which is held at a potential of 20 kV. Particles are injected into the linear electrodynamical accelerator after preliminary acceleration in the linear electrostatic accelerator with an effective voltage of 145 kV. The total effective accelerating voltage is 670 kV. The total length of the accelerating sections is 3.62 m. The essential difference of this accelerator from the existing machines is that the drift tubes of the dynamical circuit are identical and that synchronism of particle motion with the voltage applied to the drift tubes is achieved by forming the accelerating voltage as a function of the particle velocity and specific charge. The measured performance data of the accelerator are presented.     

X-ray microanalysis of freeze-dried and frozen-hydrated cryosections

The elemental composition and the ultrastructure of biological cells were studied by scanning transmission electron microscopy (STEM) combined with energy dispersive X-ray microanalysis. The preparation technique involves cryofixation, cryoultramicrotomy, cryotransfer, and freeze-drying of samples. Freeze-dried cryosections 100-nm thick appeared to be appropriate for measuring the distribution of diffusible elements and water in different compartments of the cells. The lateral analytical resolution was less than 50 nm, depending on ice crystal damage and section thickness. The detection limit was in the range of 10 mmol/kg dry weight for all elements with an atomic number higher than 12; for sodium and magnesium the detection limits were about 30 and 20 mmol/kg dry weight, respectively. The darkfield intensity in STEM is linearly related to the mass thickness. Thus, it becomes possible to measure the water content in intracellular compartments by using the darkfield signal of the dry mass remaining after freeze-drying. By combining the X-ray microanalytical data expressed as dry weight concentrations with the measurements of the water content, physiologically more meaningful wet weight concentrations of elements were determined. In comparison to freeze-dried cryosections frozen-hydrated sections showed poor contrast and were very sensitive against radiation damage, resulting in mass loss. The high electron exposure required for recording X-ray spectra made reproducible microanalysis of ultrathin (about 100-nm thick) frozen-hydrated sections impossible. The mass loss could be reduced by carbon coating; however, the improvement achieved thus far is still insufficient for applications in X-ray microanalysis. Therefore, at present only bulk specimens or at least 1-μm thick sections can be used for X-ray microanalysis of frozen-hydrated biological samples.     

Electron energy-loss spectroscopy study of thin film hafnium aluminates for novel gate dielectrics

We have used conventional high‐resolution transmission electron microscopy and electron energy‐loss spectroscopy (EELS) in scanning transmission electron microscopy to investigate the microstructure and electronic structure of hafnia‐based thin films doped with small amounts (6.8 at.%) of Al grown on (001) Si. The as‐deposited film is amorphous with a very thin (～0.5 nm) interfacial SiO_x layer. The film partially crystallizes after annealing at 700 °C and the interfacial SiO₂‐like layer increases in thickness by oxygen diffusion through the Hf‐aluminate layer and oxidation of the silicon substrate. Oxygen K‐edge EELS fine‐structures are analysed for both films and interpreted in the context of the films’ microstructure. We also discuss valence electron energy‐loss spectra of these ultrathin films.     

Applications of medium-voltage STEM for the 3-D study of organelles within very thick sections

Scanning transmission electron microscopy at 300 kV enables the visualization of nucleolar silver-stained structures within thick sections (3–8 μm) of Epon-embedded cells at high tilt angles (–50°; + 50°). Thick sections coated with gold particles were used to determine the best conditions for obtaining images with high contrast and good resolution. For a 6-μm-thick section the values of thinning and shrinkage under the beam are 35 to 10%, respectively. At the electron density used in these experiments (100e^—/Å₂/s) it is estimated that these modifications of the section stabilized in less than 10 min. The broadening of the beam through the section was measured and calculations indicated that the subsequent resolution reached 100 nm for objects localized near the lower side of 4-μm-thick sections with a spot-size of 5·6 nm. Comparing the same biological samples, viewed alternately in CTEM and STEM, demonstrated that images obtained in STEM have a better resolution and contrast for sections thicker than 3 μm. Therefore, the visualization of densely stained structures, observed through very thick sections in the STEM mode, will be very useful in the near future for microtomographic reconstruction of cellular organelles.     

Multiple scattering effects of MeV electrons in very thick amorphous specimens

Multiple scattering has an important influence on the analysis of microns-thick specimens with MeV electrons. In this paper, we report on effects of multiple scattering of MeV electrons on electron transmission and imaging of tilted and thick amorphous film specimens by experiment and theoretical analysis. Electron transmission for microns-thick epoxy-resin and SiO₂ specimens calculated by the multiple elastic-scattering theory is in good agreement with measurements in the ultrahigh voltage electron microscope (ultra-HVEM) at Osaka University. Electron transmission and electron energy are then presented in an approximate power law. The bright-field ultra-HVEM images of gold particles on the top or bottom surfaces of 5 and 15 μm thick specimens further illustrate the effect of multiple scattering on image quality. The observed top‐bottom effect for the very thick specimens appears to be mainly caused by multiple elastic scattering. With increase in the accelerating voltage from 1 to 2 MV, image blurring, contrast, the signal-to-noise ratio, and the top‐bottom effect are improved because of reduction in the influence of multiple scattering. However, the effect of specimen thickness on image blurring is shown to be stronger than that of accelerating voltage. At the 2 MV accelerating voltage, the 100 nm gold particle can be imaged with less blurring of ∼4 nm when located at the bottom surface of a 15 μm thick epoxy-resin specimen.     

An experimental method for calibration of the plasmon mean free path

Transmission electron microscopy specimens in the form of elongated, conical needles were made using a dual‐beam focused ion beam system, allowing the specimen thickness to be geometrically determined for a range of thickness values. From the same samples electron energy loss maps were acquired and the plasmon mean free path (λ) for inelastic scattering was determined experimentally from the measured values of specimen thickness. To test the method λ was determined for Ni (174 ± 17 nm), α‐Al₂O₃ (143 ± 14 nm), Si (199 ± 20 nm) and amorphous SiO₂ (238 ± 12 nm), and compared both to experimental values of λ taken from the literature and to calculated values. The calculated values of λ significantly underestimate the true sample thickness for high accelerating voltages (300 kV) and large collection angles. A linear dependence of λ on thickness was confirmed for t/λ < 0.5–0.6, but this method also provides an approach for calibrating λ at sample thicknesses for which multiple scattering occurs, thus expanding the thickness range over which electron energy loss spectroscopy can be used to determine the absolute sample thickness (t/λ > 0.6). The experimental method proposed in this contribution offers a means to calibrate λ for any type of material or phase that can be milled using a focused ion beam system.     

Aperture contrast in thick amorphous specimens using scanning transmission electron microscopy

The contrast observed in thick amorphous specimens using a scanning transmission electron microscope (STEM) can be considerably improved by the use of an optimum collector aperture angle. The size of this angle can be calculated by considering the variation of electron current transmitted through the specimen as a function both of the specimen thickness and of the angle of collection subtended at the specimen. Typically these calculations predict optimum angles to be several times the half-width of the elastic scattering distribution, often 10(-1) rad or more. Observations of biological sections of up to 2 micron in thickness using scanning attachments of commercial transmission microscopes have verifie these results at beam voltages of 50, 100 and 200 kV. Wide angle convergent beam diffraction patterns were used to give accurate values of the effective angles represented by the various collector apertures. Once the linearity of the detector-amplifier system had been established, operation in a line modulation mode enabled quantitative measurements to be made of the image contrast. Such measurements also offer a quick effective method of comparing electron beam penetrations.     

The precise measurement of the thickness of ultrathin sections by a ‘re-sectioned’ section technique

The thickness of ultrathin tissue sections embedded in Epon-Araldite and cut with a diamond knife was measured by re-sectioning and electron microscopic examination of the section profiles. A secondary section mounted on a Formvar-coated slot grid provided enough normally cut segments (seven to seventeen) for measurements giving a precise estimate of mean thickness, comparable to that obtainable by interference microscopy (±2.3% or less for grey to dark gold sections). The standard deviation of section thickness within sections was never more than 5 nm, corresponding to a coefficient of variation of 6.5% or less for sections more than 48 nm thick. This suggests that variation in section thickness, within sections, may be less than has been supposed, so that quantitative work may be based on thickness measurements made over a limited representative area. A silver interference colour was associated with sections 49–60 nm thick.     

Imaging of radiation-sensitive samples in transmission electron microscopes equipped with Zernike phase plates

We have optimized a bright-field transmission electron microscope for imaging of high-resolution radiation-sensitive materials by calculating the imaging dose n(0) needed to obtain a signal-to-noise ratio (SNR)=5. Installing a Zernike phase plate (ZP) decreases the dose needed to detect single atoms by as much as a factor of two at 300 kV. For imaging larger objects, such as Gaussian objects with full-width at half-maximum larger than 0.15 nm, ZP appears more efficient in reducing the imaging dose than correcting for spherical aberration. The imaging dose n(0) does not decrease with extending of chromatic resolution limit by reducing chromatic aberration, using high accelerating potential (U(0)=300 kV), because the image contrast increases slower than the reciprocal of detection radius. However, reducing chromatic aberration would allow accelerating potential to be reduced leading to imaging doses below 10 e(-)/A(2) for a single iodine atom when a CS-corrector and a ZP are used together. Our simulations indicate that, in addition to microscope hardware, optimization is heavily dependent on the nature of the specimen under investigation.     

The effect of some instrument operating conditions on the x-ray microanalysis of particles in the environmental scanning electron microscope

The objective of this investigation was to evaluate the practical effects of electron beam broadening in the environmental scanning electron microscope (ESEM) on particle x-ray microanalysis and to determine some of the optimum operating conditions for this type of analysis. Four sets of experiments were conducted using a Faraday cage and particles of copper, glass, cassiterite, andrutile. The accelerating voltage and chamber pressure varied from 20 to 10 kV and from 665–66 Pa (5.0 to 0.5 torr), respectively. The standard gaseous secondary electron detectors (GSED) and the long environmental secondary dectectors (ESD) for the ESEM were evaluated at different working distances. The effect of these parameters on the presence of artifact peaks was evaluated. The particles were mounted on carbon tape on an aluminum specimen mount and were analyzed individually and as a mixture. Substrate peaks were present in almost all of the spectra. The presence of neighboring particle peaks and the number of counts in these depended upon the operating conditions. In general, few of these peaks were observed with the long ESD detector at 19 mm working distance and at low chamber pressures. More peaks and counts were observed with a deviation from these conditions. The most neighboring peaks and counts were obtained with the GSED detector at 21.5 mm working distance, 10 kV accelerating voltage, and 665 Pa (5.0 torr) chamber pressure. The results of these experiments support the idea that the optimum instrumental operating conditions for EDS analysis in the ESEM occur by minimizing the gas path length and the chamber water vapor pressure, and by maximizing the accelerating voltage. The results suggest that the analyst can expect x-ray counts from the mounting materials. These tests strongly support the recommendation of the manufacturer to use the long ESD detector and a 19 mm working distance for EDS analysis. The results of these experiments indicate that neighboring particles millimeters from the target may contribute x-ray counts to the spectrum.