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A fiber-optic based spectral cathodoluminescence (CL) system has been successfully installed on a variable pressure “environmental” scanning electron microscope (ESEM). The fine size of the fiber (50 μm) has been found to require careful alignment. To provide this alignment, a simple X–Y translator has been used. The aligned fiber exhibits high efficiency and the system has recorded spectra at up to 3 million “counts” per sec on strongly cathodoluminescent doped yttrium aluminium garnet (YAG) samples, at 30 kV and ˜3 nA. A resolution of 3 nm [full width half maximum (FWHM)] at 621 nm has been measured from a red laser diode spectrum, on the ESEM column. A range of uncoated materials has been measured to characterize the CL system; these materials ranged from strongly luminescent YAG to weakly luminescent polymers. Wellcharacterized doped zircons have also been investigated. These data suggest that the previously reported intrinsic peak from zircon is a consequence of high-beam currents.

Charge contrast images (CCI) of synthetic gibbsite obtained on an environmental scanning electron microscope gives information on the crystallization process. Furthermore, X-ray mapping of the same grains shows that impurities are localized during the initial stages of growth and that the resulting composition images have features similar to these observed in CCI. This suggests a possible correlation between impurity distributions and the emission detected during CCI. X-ray line profiles, simulating the spatial distribution of impurities derived from the Monte Carlo program CASINO, have been compared with experimental line profiles and give an estimate of the localization. The model suggests that a main impurity, Ca, is depleted from the solution within approximately 3–4 μm of growth.

Although Scanning Electron Microscopes (SEM) have improved greatly over the last decade the techniques usually employed to measure their performance have not changed significantly in half a century. In particular, describing the imaging performance of an SEM by a single number - its 'resolution' - provides no useful information about its real world imaging capabilities nor about any of the factors that might limit that usefulness of the SEM for tasks such as metrology. The Contrast Transfer Function (CTF) discussed here analyses the way in which the SEM processes signal components of different spatial frequencies. The resultant plot provides information on the noise limited spatial resolution limit, predicts how this will vary with noise level, and provides a powerful general diagnostic capability. This type of measurement, which has become standard practice for transmission electron microscopes, can be performed using the public domain software package IMAGE-J, is rapid, and requires only a specimen offering a broad and flat Fourier spectrum. The capabilities of this approach are demonstrated by a number of examples.

IONiSE is a Monte Carlo simulation which describes the interactions of 5-50 keV energy He+ ions with solids, and predicts the production of ion induced secondary electron (iSE) emission. Its use to determine the most probable implant depth, the maximum ion range, and the effect of straggle are presented. IONiSE has been used to numerically fit literature tabulations of iSE generation from five elements so as to derive excitation energy and mean free path parameters. By employing those parameters in IONiSE the topographic yield variation for iSE as a function of energy and the atomic number of the target has been predicted, and estimates of the individual secondary electron contributions from the incident and backscattered ions have been made. These simulations help to create a foundation for the application and the interpretation of iSE images for metrology.

The physical dimensions of nanoscale objects are an important indicator of their functionality. However, measuring feature size from a SEM image is difficult not only because of fundamental considerations, such as the nature of beam interactions and the information transfer properties of the microscope, but because the magnification of the SEM image from which a measurement will be made is completely uncalibrated and additionally is subject to local distortions and variations. Nano-gauges fabricated by electron beam lithography - one or two dimensional structures on the size scale of the objects of interest - provide a local length standards within the image field from which the relative size of features can be accurately determined. In order to provide an absolute measurement of size the dimensions of the nano-gauge structure must themselves be calibrated against some primary standard. Because there are no convenient standards of appropriate scale available we propose that this can be done using a moire fringe technique to bridge the gap between the nanoscale and common length standards such as ruled diffraction gratings.

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.

A scanning ion microscope (SIM) is analogous to a scanning electron microscope (SEM) but utilizes a beam of helium ions, with energy of 10 to 25 keV , instead of electrons. The SIM potentially offers several advantages for device critical dimension metrology as compared to the more familiar CD-SEM. These include a high brightness source which is sub-nanometer in size, an enhanced secondary electron yield, restricted beam penetration, and superior image contrast and information content. Possible problems include pervasive positive charging, ion implantation, and a lack of detailed experimental and theoretical knowledge about low energy ion interactions with solids. Comparison of line profiles across structures made by electron induced and ion induced secondary electrons show that there are some significant differences between them which arise from the different modes of interaction in the two cases. As a result the algorithms employed for line width determination will require revision in order to produce data which is consistent with CD-SEM data.

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).

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).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.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.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.