Backscattered electron imaging of cultured cells: application to electron probe X-ray microanalysis using a scanning electron microscope

We report a simple method to study the elemental content in cultured human adherent cells by electron probe X-ray microanalysis with scanning electron microscopy. Cells were adapted to grow on polycarbonate tissue culture cell inserts, washed with distilled water, plunge-frozen with liquid nitrogen and freeze-dried. Unstained, freeze-dried cultured cells were visualized in the secondary and backscattered electron imaging modes of scanning electron microscopy. With backscattered electron imaging it was possible to identify unequivocally major subcellular compartments, i.e. the nucleus, nucleoli and cytoplasm. X-ray microanalysis was used simultaneously to determine the elemental content in cultured cells at the cellular level. In addition, we propose some improvements to optimize backscattered electron and X-ray signal collection. Our findings demonstrate that backscattered electron imaging offers a powerful method to examine whole, freeze-dried cultured cells for scanning electron probe X-ray microanalysis.     

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.     

Electronic core level microanalyses and microscopies in multipurpose apparatus

Using an instrument equipped with two electron guns, an electron analyzer, and a Si(Li) diode detector, we developed microanalytical techniques based on inner-shell electron excitations by incident electrons and X-rays, that is, electron energy-loss spectroscopy (EELS) in the reflection mode; electron probe microanalysis (EPMA) and X-ray appearance potential spectroscopy (XAPS); electron-induced Auger electron spectroscopy (e-AES); X-ray photoelectron spectroscopy (XPS), X-ray absorption spectroscopy (XAS); X-ray induced AES (XAES), X-ray fluorescence analysis (XRF), and scanning X-ray radiography (SXR). The corresponding characteristic images (including X-ray microradiography and X-ray photoelectron microscopy) were obtained in the scanning mode. The principle of the apparatus is described. Each spectroscopy and microscopy is illustrated by an example. Their performance and limits are discussed.     

Subject index to volume 10

The scanning transmission electron microscope with a field emission electron source operated at 100 kV allows X-ray microanalysis using electron probes as small as 1 to 2 nm. Measurements of the probe in a Vacuum Generators HB-501 STEM show that spherical aberration in the objective lens controls the probe size and shape at beam convergence half-angles of 10 mrad and greater typically used for X-ray microanalysis. A virtual objective aperture eliminates X-ray contributions from the probe-forming system, but must be aligned exactly to avoid asymmetrical broadening of the probe by spherical aberration. It is estimated that 5 nm X-ray spatial resolution can be achieved in low to medium atomic number materials. Even at this resolution however, probe broadening in the specimen controls the resolution; the main limitation is one of specimen preparation and a knowledge of the final specimen thickness. Determination of composition profiles near voids, dislocations and other individual defects in thin foils also requires a knowledge of the defect depth position and deconvolution of the probe and composition profiles.     

温度对AZ31B镁合金扩散连接接头性能的影响

为实现镁合金AZ31 B的可靠连接,根据原子扩散理论,对AZ31 B镁合金进行了扩散连接工艺研究.利用VDW-15型扩散设备,对AZ31B镁合金进行了在不同连接温度条件下的扩散连接.利用金相显微镜、扫描电镜、能谱分析仪器等对扩散连接接头组织及连接界面元素成分进行了分析,测试了接头的剪切强度.结果表明,AZ31B镁合金的...     

Methods for X-ray microanalysis of epidermis: the effect of local anaesthesia

The effect of local anaesthesia on the elemental content of cells in human epidermis was studied by electron probe X-ray microanalysis. Local anaesthesia with lidocaine was given by intracutaneous injection within 1 min prior to taking a skin biopsy. Biopsies taken without local anaesthesia were used as controls. Lidocaine with or without adrenaline caused a significant increase in the concentrations of Na and Cl, and a decrease in the concentration of K in the cells of the stratum basale and the stratum spinosum, compared with the control samples. The presence of adrenalin in the anaesthetic did not change the effect of lidocaine. The effects of local anaesthesia have to be considered in planning and interpretation of clinical applications of X-ray microanalysis.     

Analysis of directly frozen macromolecules and tissues in the field-emission STEM

A VG Microscopes HB501 field-emission high-resolution scanning transmission electron microscope (STEM) was used to image and analyse rapidly frozen, isolated macromolecules and small organelles in tissue cryosections. Dark-field images were obtained from frozen-hydrated microtubules demonstrating that sufficient contrast is available to reveal structural information. The samples were subsequently freeze-dried in the STEM and low-dose (? 10³ e/nm²) dark-field mass maps were recorded with single electron sensitivity. Elemental analysis of individual macromolecules was achievable at high dose using parallel-detection electron energy-loss spectroscopy, albeit with some structural degradation. Detection of copper (320 atoms) in di-decameric haemocyanin molecules was easily within the limits of sensitivity. Elemental analysis of hydrated cryosections is limited by radiation damage to a resolution of approximately 1 μm². For freeze-dried sections, however, the high probe current and stable cold stage of the HB501 STEM allow energy-dispersive X-ray (EDX) microanalysis of low elemental concentrations in highly localized subcellular volumes. EDX spectra from cryosections of cerebellar cortex show that a 100-s analysis time is sufficient to quantify the calcium content of 400-nm² regions within Purkinje cell dendrites with an uncertainity of ± 2 mmol/kg dry weight, equivalent to ± 12 atoms.     

Coaxial arrangement of a scanning probe and an X-ray microscope as a novel tool for nanoscience

We report on the design and first tests of a novel instrument aimed at combining the benefits of scanning force microscopy with those of X-ray spectroscopy. For this we built an instrument combining a scanning transmission X-ray microscope with a beam-deflection atomic force microscope in a coaxial geometry. This allows to combine X-ray absorption spectroscopy and high resolution topography in-situ. When replacing the conventional scanning probe tip by a coaxially shielded tip the instrument will allow detection of the photoelectrons produced by resonant X-ray absorption. This could yield spectroscopic information with a spatial resolution approaching the values achievable with atomic force microscopy.     

Assessment of chloride secretion in human nasal epithelial cells by X-ray microanalysis

The genetic disease cystic fibrosis (CF) is due to defective epithelial chloride transport. Different treatments have been proposed that could restore chloride transport in CF patients. A new method is proposed for measuring the chloride secretion in easily accessible epithelial cells.
Fresh nasal epithelial cells were obtained by nasal brushing and made to attach to titanium grids for electron microscopy. Chloride efflux through the cystic fibrosis transmembrane regulator channel was stimulated by 20 µ m forskolin and 100 µ m isobutyl-methylxanthine (IBMX), in standard Ringer's solution (SR). Chloride efflux through the calcium-regulated channel was stimulated by 200 µ m adenosine triphosphate (ATP) in SR. The cells were rinsed after the exposure, in order to remove the experimental medium, frozen and freeze-dried. The elemental composition of the cells was determined by X-ray microanalysis.
Rinsing with distilled water or ammonium acetate appeared to cause damage to the cells, whereas rinsing with isotonic mannitol preserved the ionic composition. Stimulation of cells from healthy controls with forskolin and IBMX in a chloride-containing medium caused a significant (28 ± 6%) decrease in chloride concentration, which is indicative of net chloride efflux. In similar conditions, stimulation with ATP induced a 29 ± 5% decrease in the chloride concentration.
Stimulation of cells from CF patients with forskolin and IBMX in a chloride-containing medium caused no significant change in the intracellular chloride concentration, whereas ATP stimulation induced a response similar to that obtained in cells from healthy controls.
It is concluded that X-ray microanalysis of nasal epithelial cells may be used to determine chloride secretion in CF patients in an easily accessible cell type.     

The cryopuncher: A pneumatic cryofixation device for X-ray microanalysis of tissue specimens

A cryopunching device is described which allows cryofixation of tissue specimens by quick contact with a precooled copper surface during excision. The advantage of the cryopuncher for analytical electron microscopy of cells and tissues in defined functional states is illustrated by electron probe X-ray microanalysis of freeze-dried cryosections from rat liver and dogfish kidney. In comparison with results obtained from specimens plunged into liquid propane, cryopunching in situ results in similar preservation of morphology and remarkably improved intracellular K/Na ratio.     

Quantitative X-ray imaging of labelled molecules in tissues and cells

Using cisplatin as a model system, we have been able to demonstrate the feasibility of studying the cellular and subcellular distribution of a labelled molecule containing a single atom of platinum per molecule in bone marrow. An X-ray imaging system consisting of a microcomputer, a 4pi system and a software package was interfaced with an electron microscope enabling the computer to control the beam movements as well as receive signals from the STEM and EDS X-ray detectors. X-ray imaging is useful for both tissue and samples in which the population of cells is not homogeneous. Imaging permits elemental distributions to be measured throughout the sample and not in just randomly selected areas as previously done in X-ray microanalysis. Images are created for not only the element labelling the molecule of interest but also other specified elements present.
Three types of maps for imaging labelled molecules are compared and discussed. When the original (collected) data are mapped, the elements of interest are obscured by the continuum. The maps calculated using an internal standard give a concentration distribution on the basis of volume (mmol L⁻¹ of packed cells). The maps calculated using the continuum normalization method according to Hall produces concentration distribution on the basis of mass (mmol kg⁻¹ dry weight). By recalculating using the 'Peak' or 'Hall' method the continuum problem is removed yielding quantitative images of the intracellular distribution of labelled molecules present in low concentrations.     

A procedure to prepare cultured cells in suspension for electron probe X-ray microanalysis: application to scanning and transmission electron microscopy

We describe a simple procedure to prepare cultured cells in suspension to analyse elemental content at the cellular level by electron probe X-ray microanalysis. Cells cultured in suspension were deposited onto polycarbonate tissue, culture plate well inserts, centrifuged at low g , washed to remove the extracellular medium, cryofixed and freeze-dried, and analysed in the scanning mode of a scanning electron microscope. We tested the effect of different washing solutions (150 m m ammonium acetate, 300 m m sucrose, and distilled water) on the elemental content of cultured cells in suspension. The results demonstrated that distilled water was the best washing solution to prepare cultured cells. In addition, the low Na content, high K content and high K/Na ratio of the cells indicated that this procedure, based on the centrifugation at low g followed by cryopreparation, constitutes a satisfactory method to prepare cultured cells in suspension. We also investigated the effects of different accelerating voltages on X-ray signal collection. The results showed that moderate accelerating voltages, i.e. 10–11 kV, should be used to analyse whole cells in the scanning mode of the scanning electron microscope. We show that this method of preparation makes it possible to prepare cryosections of the cultured cells, thus permitting analysis of the elemental content at the subcellular level, i.e. nucleus, cytoplasm and mitochondria, using a scanning transmission electron microscope.     

Frontiers in electron probe microanalysis: application to cell physiology

The application of electron probe microanalysis techniques, using X-ray and electron energy loss instruments, to problems in cell physiology is reviewed. The details of the special methodological requirements for the analysis of cryosections at high spatial resolution in an analytical electron microscope are discussed together with a comprehensive review of data obtained on major organ systems and cell types.     

Crystal structure determination by direct inversion of dynamical microdiffraction patterns

The intensity at points where coherent convergent-beam transmission diffraction discs overlap is shown to be described by interference between elements of the same row but different columns of the dynamical scattering matrix for an axial orientation. These intensities are used as the basis for an exact, nonperturbative inversion of the multiple electron scattering problem, allowing crystal structure factors to be obtained directly from the intensities of multiply scattered Bragg beams. Eigenvectors of the structure matrix are obtained using coherent CBED patterns from many crystal orientations. Unique eigenvalues are obtained from these patterns recorded at two accelerating voltages. The inevitable variation in electron probe position at different crystal tilts is considered. The analysis applies to centrosymmetric crystals with anomalous absorption, to centrosymmetric projections of acentric crystals and to acentric crystals if the mean absorption potential only is included. The method would allow the direct synthesis of charge-density maps of unknown crystal structures at high resolution from multiple scattering data, using a scanning transmission electron microscope (STEM). The resolution of this map may be much higher than the first-order d -spacing; however, the STEM need only be capable of resolving this first-order spacing. Such a charge-density map provides fractional atomic coordinates and the identification of atomic species (as in X-ray crystallography) from microcrystalline samples and other multiphase inorganic materials for which large single crystals cannot be obtained or X-ray powder patterns obtained or analysed. In summary, we solve the inversion problem of quantum mechanics for the case of electron scattering from a periodic potential, described by the nonrelativistic Schrödinger equation, in which the scattering is given as a function of some parameter, and the potential sought.     

The measurement of atomic number and composition in an SEM using backscattered detectors

The atomic number dependence of electron backscattering can be used as the basis of a microanalysis technique. The operating procedures and condition for quantitative measurements of specimen atomic number are outlined and an expression relating the accuracy of composition to the atomic number sensitivity has been derived. Some measurements of the spatial resolution of backscattered electron microanalysis are also presented and compared with the resolution of X-ray microanalysis. Although the range of application of this technique is limited, where it can be applied it has the following advantages: (i) higher spatial resolution than X-ray microanalysis for bulk specimens; (ii) very rapid measurement; (iii) can be applied to compounds of low atomic number elements, (e.g. borides, carbides, nitrides, etc.); (iv) specimen preparation is often relatively straightforward.     

Quantitative evaluation of autoradiographs by X-ray spectroscopy.

Measurements of mass of developed silver in autoradiograms have been made using X-ray microanalysis. This procedure has been applied to a scanning electron microscopic study of protease-induced cell stimulation in 3H-thymidine-labelled bladder tissues in vitro. Differences in silver concentrations over individual nuclei were demonstrated by X-ray analysis and quantitative data was obtained showing an increased rate of 3H-thymidine incorporation in protease-treated cultures. The initial evidence indicates that X-ray microanalysis could provide a potentially useful quantitative procedure for autoradiography.     

X-ray microanalysis of wet biological specimens in the environmental scanning electron microscope. 1. Reduction of specimen distance under different atmospheric conditions

X-ray microanalysis of non-biological and biological specimens was carried out in the environmental scanning electron microscope (ESEM) under different conditions of specimen distance (the distance travelled by the electron probe within the specimen chamber) and chamber atmosphere. Using both water vapour and argon atmospheres, it was shown that reduction in specimen distance had no effect on atmospheric gas X-ray signal in either case. Unlike water vapour, increased levels of argon (up to 10 torr) caused a marked depression of specimen P/B ratios, with a decrease in both characteristic and background (continuum) counts. These effects in argon were not altered by reduction in specimen distance. Specimen distance was important in relation to beam skirting and elemental analysis. With an extended assembly (short specimen distance), beam skirting in a water-vapour atmosphere was much reduced – leading to enhanced element detectability in a discrete biological specimen (Anabaena cyclindrica).     

The preparation and X-ray microanalysis of bulk frozen hydrated vacuolate plant tissue

A series of modifications have been devised which allow the peak to background ratio X-ray analytical method to be used more effectively to measure elemental concentrations in large vacuolate plant cells. Planar, frozen-hydrated fracture faces of bulk plant tissue are coated with a thin film of evaporated chromium, which prevents surface charging. Provided the film is sufficiently thin, c. 5–10 nm, there is no attenuation of the electron beam and only a small absorption of soft X-rays. The chromium makes a small but measurable contribution to the spectral background and suitable corrections may be made to the quantitative results. An improved back-scattered imaging system is described, which helps to overcome the problem of spurious X-ray signals from rough surfaces. The microscope column has been modified to permit a continuous readout of beam current, sensu stricta, during X-ray microanalysis and to allow rapid exchange of the electron gun assembly during low temperature operation. Calculations are given relating the size of the X-ray interactive volume to electron penetration and X-ray emission in both frozen hydrated and frozen dried cells. The problems of X-ray microanalysis are discussed in relation to the highly vacuolate cells found in most mature plant tissues and an example given of the distribution of four major cations in tobacco leaves.     

Multiple imaging techniques demonstrate the manipulation of surfaces to reduce bacterial contamination and corrosion

Surface imaging techniques were combined to determine appropriate manipulation of technologically important surfaces for commercial applications. The complementarity of the microscopy methods, scanning electron microscopy, electron probe microanalysis and atomic force microscopy assessed and correlated form and function of the surface modifications. Stainless steel disks (1 cm in diameter) were laser‐cut from the same sheets of stainless steel and treated by electropolishing or left untreated for controls. Each treatment was analysed separately using each technique. First, the disks were examined by visual inspection and electron probe microanalysis for surface characteristics and elemental composition, respectively. Aliquots of bacterial suspensions (saline rinses of poultry carcasses from a commercial broiler processing plant) were then diluted in broth and monitored for growth by spectrophotometry. Stainless steel disks (1 cm in diameter) were added and the cultures were grown to sufficient density to allow attachment of bacterial cells to test surfaces. Relative differences in the surface morphology shown by atomic force microscopy, including Z ranges, roughness and other measurements, corresponded by treatment with the differences in reduction of bacterial counts shown by scanning electron microscopy. A model of wet‐processing conditions tested the effects of corrosive treatment of surfaces. Less bacterial attachment occurred after corrosive treatment on controls and electropolished samples. Electropolishing significantly reduced bacterial numbers and the effects of corrosive action compared to the controls. Thus, the multiple imaging techniques showed that engineered changes on stainless steel surfaces improved the resistance of the surface finish to bacterial attachment, biofilm formation, and corrosive action.