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.     

Secondary electron imaging in the variable pressure scanning electron microscope

Secondary electron imaging is not possible in the variable pressure scanning electron microscope because the mean free path of the secondaries in the gas is too short to permit them to reach the detector. This paper therefore investigates an alternative strategy for producing an image containing significant amounts of secondary electron contrast. This involves modifying the microscope by the addition of a biased electrode above the sample and then collecting a specimen current signal. This system, originally described by Farley and Shah (1988), is found to produce true secondary electron detail over a wide range of conditions.     

Stereometry as an aid to stereological analysis

Accurate measurements for the stereological analysis of rough or curved surfaces are not always possible with single images from the light microscope or the scanning electron microscope (SEM). A second image recorded to form a stereo pair provides the information about the topographic nature of the surface. This paper presents two methods by which stereo pair scanning electron micrographs can be measured to give area fractions or other geometric parameters of rough surfaces.     

Computer-generated distribution functions as an aid to the quantitative interpretation of electron micrographs of shadowed macromolecules

Using the mica-replication technique of Hall, it is possible to visualize small, metal-shadowed biological macromolecules. Consideration is given to the interpretation of micrographs obtained in this way, in order that molecular dimensions may be inferred. It is shown that the width of the shadow cast by a macromolecule can be measured precisely. A method is developed whereby the histogram of the experimentally determined shadow widths is interpreted by reference to computer-generated histograms, based upon models of assumed dimensions. Results are presented illustrating the use of this new method.     

Combined HREM and STEM microanalysis on decorated dislocation cores

Impurity precipitation at dislocation cores in silicon and germanium have been identified by lattice imaging and electron energy loss spectroscopy on a STEM. The results lead to the conclusion that oxygen segregates at dislocations with large Burgers vectors forming a crystalline phase in the form of a continuous cylinder all along the dislocation line. The ceosite phase is tentatively proposed as being this crystalline phase. Severe radiation-induced damage has been observed under the high flux employed in a STEM. This results in complete disappearance of the precipitates by desorption of oxygen at the surfaces of the thin foil after an integrated dose of 10¹⁰ C/m².     

Cryopreparation of mammalian tissue for X-ray microanalysis in STEM

A cryopreparation technique for studies of ultrastructure and distribution of diffusible elements in biological tissue is described. Electron microscopical contrast and characteristic X-ray spectra are found to be poor in completely frozen-hydrated ultrathin cryosections of fresh chemically untreated tissue. Both STEM contrast and detection of characteristic X-rays are enhanced by careful freeze-drying in the microscope. Although the ultrastructure is affected by ice crystals, intracellular compartments can be identified by STEM without staining and studied by X-ray microanalysis.     

Modification of an EM6G electron microscope for X-ray microanalysis

A dual purpose stage has been constructed for an EM6G 100 kV transmission electron microscope. With this stage the composition of thin films and bulk specimens can be determined by X-ray microanalysis. With thin films a change of specimen cartridge then enables a full analysis of crystal defects in the film to be made using tilt controls incorporated in the stage. Modifications to the stage to reduce background effects in X-ray microanalysis spectra are also described. The alternative use of this system in the bulk analysis of specimens by an X-ray fluorescence technique is also discussed.     

Secondary and backscattered electron imaging of weathered chromian spinel

Secondary (SE) and backscattered electron (BSE) imaging as well as x-ray microanalysis have demonstrated that the weathering of chromian spinel occurs as a progressive form of alteration. Numerous chemical discriminant analysis methods based on the composition of chromian spinel are used to locate valuable deposits of minerals. These methods will be misleading if the correct interpretation of the weathering of chromian spinel and the subsequent pattern of changes in its mineral chemistry are not properly assessed using scanning electron microscopy. This assessment is vital in understanding the geological processes involved and the economic potential of any indicated deposit. Minerals such as chromian spinel, pyrope garnet, and picroilmenite are considered to be highly resistant to weathering and abrasion and are therefore useful in the search for associated valuable deposits of diamond, nickel, platinum, and gold. Known as indicator minerals, they are usually present in relatively large concentrations compared with the target mineral (e.g., diamond) and form large and often subtle dispersion anomalies adjacent to the deposit. Chromian spinel has long been regarded as a stable indicator mineral; however, detailed SE and BSE imaging indicates that many of the chromian spinels that are routinely examined using scanning electron microscopes (SEM) and microprobes are extensively altered. Secondary electron and BSE imaging of weathered chromian spinel in a normal SEM provides valuable data on the form and chemical style of the alteration. Secondary electron imaging of weathered chromian spinel in the environmental SEM (ESEM) not only enhances the difference in atomic number between unaltered and altered areas but also allows high-resolution imaging of these very fine replacement textures.     

Quantitative electron probe microanalysis of nitrogen

Quantitative electron probe microanalysis of nitrogen has been performed on 18 nitride specimens in the voltage range of 4–30 kV. A conventional lead stearate and a new W/Si multilayer analyzer crystal (2d = 5.98 nm) were used simultaneously. The net peak count rates on the latter crystal were approximately 2.8 times higher than on the lead stearate crystal. In addition, significant improvements in the peak to background ratios were achieved, which proved critical when dealing with difficult nitrides such as ZrN, Nb₂N, and Mo₂N. A most conspicuous feature of the new crystal, however, is its effective suppression of higher-order reflections in the wavelength range of N-Kα. As a result, backgrounds are free of interfering lines and spectral artifacts, which facilitates accurate background determination. Two procedures for the analysis of nitrogen in the presence of titanium are also discussed. One procedure is based on the assumption that the area–peak factor (APF, integral k-ratio/peak k-ratio) of the N-Kα peak in Ti–N compounds relative to a nitrogen standard (Cr₂N) has a fixed value which can be used in order to separate the Ti-_ll peak from the N-Kα peak. Extensive tests on a number of compositions in the Ti–N system have shown that it is possible to do quantitative analysis of nitrogen in Ti–N compounds with a relative accuracy of better than 5%. For very low levels of nitrogen (below 15 at%) the APF method was found unsuitable. In such cases, however, the well-known multiple least-squares digital-fitting techniques appeared to do an excellent job. In order to account for the effects of peak shape alterations in the N-Kα peak, all nitrogen spectra were recorded in integral fashion and compared with those of the Cr₂N standard. The shape alterations for the N-Kα peak were much smaller than for B-Kα and C-Kα radiations: The observed values for the area–peak factor relative to Cr₂N differed less than 5% from unity. The present work has resulted in a database containing 144 integral k-ratios for N-Kα and 149 peak k-ratios for the x-ray lines of the metal partners in the nitrides. Our own Gaussian ?(pz) matrix correction procedure “PROZA,” used in conjunction with a set of improved mass absorption coefficients, yielded excellent results for the nitrogen data: An average value of k_calc/K_meas of 1.0051 and a relative root mean square deviation of 3.99%.     

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.     

Studies of supported metal catalysts using high resolution secondary electron imaging in a STEM

An additional technique for use in the characterization of catalysts by electron microscopy is presented. High resolution secondary electron images obtained in a VG HB501 scanning transmission electron microscope have been used to study the surface topography of catalysts consisting of small metal particles on high surface area carbon supports. Surface features down to nanometre dimensions can be seen, allowing the examination of micropores in the support as well as larger pore structures. The results are compared with pore size distributions determined by gas adsorption methods, and are shown to yield valuable additional information. In addition, the method in principle allows examination of the locations of small metal catalyst particles on the support.     

Position-sensitive diffractive imaging in STEM by an automated chaining diffraction algorithm

The diffractive imaging process used for retrieval of an aberration-free exit-wave function of a complex-valued object is optimized with a newly developed automated chaining diffraction (ACD) algorithm. Our algorithm enables automatic recovery of the amplitude and phase of the complex-valued objects with diffraction-limited resolution, starting from selected-area electron diffraction (SAED) patterns recorded from partially overlapping regions in STEM/CTEM. Based on a ‘differential map’ (DM) approach, the ACD algorithm meets very general requirements and, similar to ‘hybrid input–output’ (HIO) algorithm, can be applied to non-periodic, real or complex structures. In contrast to many other algorithms, it is not limited by the object's finite size or tight object support. Wide-field-of-view reconstructions for the complex-object-wave amplitude and phase made with ACD algorithm from SAED patterns down to sub-Angström resolution show the potential of diffractive imaging for quantitative analysis of functional materials at different length scales in terms of absorption and scattering mechanisms. The method can be applied also for imaging magnetic properties of samples by the electron or neutron microscopy and/or imaging of non-periodic objects with X-ray microscopy.     

Surface microanalysis by reflection electron energy-loss spectroscopy

Several basic physical concepts of applying eq. I_k = IσN^xt to surface microanalysis by reflection electron energy-loss spectroscopy (REELS) are clarified. Here I^k and I are the integrated intensities of the core ionization edge and the low loss part, σ is the scattering cross section of element x with atomic concentration N^x, and t is the specimen thickness. The reflected inelastic electrons are found to be distributed almost symmetrically around the Bragg sports and can be reasonably described by a Lorentzian function. EELS microanalysis can be performed by using the diffracted sports. The ω correction, arising from the angular contributions of the neighbouring spots into the spectrometer collecting aperture, is required to be considered.     

Biological applications of electron probe microanalysis in france

The contribution of French science to the field of biological X-ray microanalysis is reviewed. The main analytical microscopy centers are listed, and their methods and main results are summarized.     

Complementary optical and electron microanalysis of high-temperature superconductors

Scanning electron microscopy coupled to quantitative energy-dispersive x-ray spectroscopy provides an excellent tool for determining the compositions of various cuprate superconducting phases in the course of processing from insulator to superconductor. When coupled with a new room temperature optical microscopy technique for the identification of individual superconducting grains, it provides a complete picture of the processed state of the material. The techniques are complementary. Two different materials were examined with both optical and electron microscopy: (1) a Tl-Sr-Ca-Cu-O system superconducting material doped with Hg and (2) a metallic, but not superconducting La-Sr-Cu-O system material. The results and the complementary nature of optical and electron microscopy in assessing the stoichiometry and homogeneity of these materials are described.     

Auger microanalysis in a conventional scanning electron microscope

We have coupled an Auger spectrometer to a conventional scanning electron microscope. Specimens were examined without an ultrahigh vacuum by maintaining a high partial pressure of argon in the vicinity of the specimen. It was possible to examine the specimens by means of both Auger and x-ray microanalysis. Sputtering profiles could be recorded by removing surface layers by ion bombardment. Examples of the application of the method are drawn from studies of surface layers on metals, and are compared with XRMA results.