Is microanalysis possible in the helium ion microscope?

Because the ability to perform some form of chemical microanalysis has become an essential feature for any microscope, it is necessary to investigate what options are available in the new "ORION" helium ion microscope (HIM). The HIM has the ability to visualize local variations in specimen chemistry in both the ion induced secondary electron and the Rutherford backscattered imaging modes, but this provides only limited and qualitative information. Quantitative, elementally specific, microanalysis could be performed in the HIM using secondary electron spectroscopy, Rutherford backscattered ion spectroscopy, or secondary ion mass spectroscopy, but while each of these options has promise, none of them can presently guarantee either reliable element identification or quantitative analysis across the periodic table.

A comparison of conventional Everhart-Thornley style and in-lens secondary electron detectors: a further variable in scanning electron microscopy.

The secondary electron (SE) imaging of several samples across a range of scanning electron microscopes (SEM) and SE detectors under matched operating conditions has generated a highly variable image data set. Using microanalytical conditions (10-15 kV), images from in-column SE detectors reveal the presence of surface films and contaminants that are invisible to conventional Everhart-Thornley SE detectors under the same conditions. Data from studying the effects of working distance, the image resolution derived through contrast transfer function analysis and electrostatic mirror imaging of the SE detectors in operation combine with other studies to suggest that the classically defined SE1 component can be separated from other SE components. SE images obtained by tailored mechanical design and energy-filtering will provide SE images with probe-sized resolution and dominated by surface detail currently only seen in low-voltage SEM, potentially even from thermionic-sourced columns.

Variable pressure and environmental scanning electron microscopy: imaging of biological samples.

The use of elevated gas pressures in the sample chamber of a scanning electron microscope (i.e., variable pressure SEM, or VPSEM) together with specialized electron detectors create imaging conditions that allow biological samples to be examined without any preparation. Specific operating conditions of elevated pressures combined with sample cooling (usually restricted to the environmental SEM range) can allow hydrated samples to be maintained in a pristine state for long periods of time. Dynamic processes also can be easily observed. A wider range of detector options and imaging parameters introduce greater complexity to the VPSEM operation than is present in routine SEM. The current instrumentation with field emission electron sources has nanometer-scale beam resolution (approx 1 nm) and low-voltage beam capability (0.1 kV). However, under the more extreme variable pressure conditions, useful biological sample information can be achieved by skilled operators at image resolutions to 2 to 4 nm and with primary electron beam voltages down to 1.0 kV. Imaging relating to electron charge behavior in some biological samples, generally referred to as charge contrast imaging, provides information unique to this VPSEM and environmental SEM that closely relates to luminescence imaged by confocal microscopy.

Fine structure of the mineralized teeth of the chiton Acanthopleura echinata (Mollusca: Polyplacophora).

The major lateral teeth of the chiton Acanthopleura echinata are composite structures composed of three distinct mineral zones: a posterior layer of magnetite; a thin band of lepidocrocite just anterior to this; and apatite throughout the core and anterior regions of the cusp. Biomineralization in these teeth is a matrix-mediated process, in which the minerals are deposited around fibers, with the different biominerals described as occupying architecturally discrete compartments. In this study, a range of scanning electron microscopes was utilized to undertake a detailed in situ investigation of the fine structure of the major lateral teeth. The arrangement of the organic and biomineral components of the tooth is similar throughout the three zones, having no discrete borders between them, and with crystallites of each mineral phase extending into the adjacent mineral zone. Along the posterior surface of the tooth, the organic fibers are arranged in a series of fine parallel lines, but just within the periphery their appearance takes on a "fish scale"-like pattern, reflective of the cross section of a series of units that are overlaid, and offset from each other, in adjacent rows. The units are approximately 2 microm wide and 0.6 microm thick and comprise biomineral plates separated by organic fibers. Two types of subunits make up each "fish scale": one is elongate and curved and forms a trough, in which the other, rod-like unit, is nestled. Adjacent rod and trough units are aligned into large sheets that define the fracture plane of the tooth. The alignment of the plates of rod-trough units is complex and exhibits extreme spatial variation within the tooth cusp. Close to the posterior surface the plates are essentially horizontal and lie in a lateromedial plane, while anteriorly they are almost vertical and lie in the posteroanterior plane. An understanding of the fine structure of the mineralized teeth of chitons, and of the relationship between the organic and mineral components, provides a new insight into biomineralization mechanisms and controls.

Charge-related problems associated with X-ray microanalysis in the variable pressure scanning electron microscope at low pressures.

In variable pressure scanning electron microscopy (VPSEM) the current data suggests that considerable caution is required in the interpretation of X-ray data from nonconductive samples, depending on the operating conditions. This article reviews some of the documented approaches and presents data that illustrate the nature and magnitude of the effects of charge above, on, and in the sample on the detected X-ray emissions from the sample and from elsewhere within the VPSEM specimen chamber. The collection of reliable and reproducible X-ray data has been found to require relatively high specimen chamber gas pressures, at the upper end of or beyond the available pressures for most VPSEMs. It is also shown that sample characteristics, including composition, strongly influence local charge effects, which can significantly affect the primary electron landing energy and consequently the resultant emitted X-ray signal under low pressure environments.

Possibility of charge contrast imaging of polymeric materials.

Preliminary results illustrate the possibility of charge contrast imaging (CCI) of polymeric materials. Possible CCI images of low-density polyethylene and polyvinyl chloride reveal details that may aid in the characterization of the microstructure of polymeric materials. These pictures were obtained with a Hitachi S-3000N variable pressure scanning electron microscope with the environmental secondary electron detector (ESED).

Characterization of the macrophages associated with the tunica vasculosa lentis of the rat eye.

PURPOSE: The tunica vasculosa lentis (TVL) is a transient vascular network surrounding the developing lens that regresses prenatally in humans and postnatally in rodents. Macrophage-like cells, sometimes referred to as hyalocytes, have been postulated to play a role in the regression of this vascular system; however, the precise identity of these cells is still unclear. The aim of the present investigation was to provide phenotypic data on these cells in combination with their three-dimensional distribution on the lens surface during the period when regression of the TVL is taking place. To this end the authors have used a novel combination of silver-enhanced immunogold labeling and environmental scanning electron microscopy (ESEM). METHODS: The eyes of Wistar rats at various pre- and postnatal ages (E20, postnatal days [D]0, 2, 5, 7, and 10) were studied by either conventional scanning electron microscopy (SEM) or ESEM. The latter was used to study specimens that had been incubated with various antileukocyte monoclonal antibodies (ED1, ED2, Ox6, Ox42), followed by immunolabeling with gold-conjugated secondary antibody visualized by silver enhancement. RESULTS: Conventional SEM of the developing lens revealed a pattern of radial and interconnecting vessels in the TVL similar to previous studies. In addition, large numbers of cells with the morphologic characteristics of macrophages were noted on the lens surface closely associated with the vessels. The gradual attenuation and regression of vessels was noted over the course of the time period investigated. Immunolabeled specimens examined by ESEM revealed that most of the macrophage-like cells were indeed ED1(+) and ED2(+) (both pan-macrophage markers) and MHC class II(-) (Ox6) and CD11b/18(-) (Ox42): a phenotype characteristic of macrophages. This phenotype altered little between E20 and D10. CONCLUSIONS: The cells surrounding the developing lens that are postulated to play a role in regression of the TVL have the morphologic and immunophenotypic characteristics of resident tissue macrophages similar to those previously identified in the adult rodent uveal tract and the vitreous (hyalocytes). This phenotype differs from that of dendritic cells and microglia; however, it is postulated that lens-associated macrophages are ideally located to act as a source of retinal microglia after completion of their role in TVL regression.

Three-dimensional reconstruction of microstructures in gibbsite using charge contrast images.

An iterative process of serial sectioning and charge contrast imaging has been applied to gibbsite particles embedded in epoxy resin. The features observed in the two-dimensional sections have been reconstructed into three-dimensional (3-D) objects, so that the volumetric relationships in the microstructure are visualised. The 3-D objects confirm that the image detail present in charge contrast images of gibbsite is related to the microstructure and the processes occurring during the crystallisation of gibbsite. Speculative explanations for what these microstructures may represent are presented.