Mechanical scanning of the specimen in the scanning electron microscope

A scanning electron microscope (SEM) system equipped with a motor drive specimen stage fully controlled with a personal computer (PC) has been utilized for obtaining ultralow magnification SEM images. This modem motor drive stage works as a mechanical scanning device. To produce ultra-low magnification SEM images, we use a successful combination of the mechanical scanning, electronic scanning, and digital image processing techniques. This new method is extremely labor and time saving for ultra-low magnification and wide-area observation. The option of ultra-low magnification observation (while maintaining the original SEM functions and performance) is important during a scanning electron microscopy session.     

Contrast in the transmission mode of a low-voltage scanning electron microscope

The contrast thicknesses (x_k) of thin carbon and platinum films have been measured in the transmission mode of a low-voltage scanning electron microscope for apertures of 40 and 100 mrad and electron energies (E) between 1 and 30 keV. The measured values overlap with those previously measured for E (≥ 17keV) in a transmission electron microscope. Differences in the decrease of x_k with decreasing E between carbon and platinum agree with Wentzel-Kramer-Brillouin calculations of the elastic cross-sections. Knowing the value of x_k allows the exponential decrease ∝ exp(—x/x_k) in transmission with increasing mass-thickness (x = ρt) of the specimen and the increasing gain of contrast for stained biological sections with decreasing electron energy to be calculated for brightfield and darkfield modes.     

Reliability of atom column positions in a ternary system determined by quantitative high-resolution transmission electron microscopy

Employing an iterative structure refinement procedure, we have determined the atomistic structure of the Σ3 (111) grain boundary in strontium titanate (SrTiO₃) from high-resolution transmission electron microscopy (HRTEM) images. This grain boundary serves as a model system to study the effect of column occupancies on the reliability of the column positions. In this paper we introduce a method to derive confidence regions for the positions of individual atom columns at crystal defects. Based on a statistical approach we first determine the reliabilities of different types of atom columns in regions of unfaulted crystal. Next we extrapolate these reliabilities to obtain the reliabilities of individual atom columns at the grain boundary. The method accounts correctly for random errors and promises to be generally applicable provided that repetitive units of unfaulted crystal structure are contained in the HRTEM image. Under the conditions of the present study, the reliability of a column position correlates with the projected electrostatic potential of the column. Accordingly, the reliabilities of the column positions at the boundary vary with the column type: 0.008 nm for Sr–O columns, 0.014 nm for Ti columns, and 0.018 nm for O–O columns.     

The three-dimensional point spread functions of a microscope objective in image and object space

The three-dimensional point spread function (3-D PSF) of an optical system in image space is distinguished from the 3-D PSF in object space and the relation between the two 3-D PSFs is derived. By using this relation one 3-D PSF can be easily obtained from the other. The 3-D PSFs are given in a single integral expression, which can be computed numerically. The results of this study can be used in 3-D image processing for microscopy and have been applied to the analysis of the diffusion of fluorescent molecules in a 3-D porous medium.     

The application of scanning transmission electron microscopy (STEM) to the study of thin anodic films on nickel and nickel-molybdenum alloys

Anodized thin-film samples of nickel, molybedenum and a nickel-13 w/o molybdenum alloy have been analysed by scanning transmission electron microscopy, using selected area diffraction, microdiffraction and X-ray micro-analysis. Thin-film samples were obtained by ion thinning with argon and by electropolishing in acetic acid-perchloric acid. Electropolishing is the preferred technique for these studies, producing a surface with roughness on a scale of 1 nm. The anodized films on nickel and nickel 13 w/o molybdenum alloy exhibit a rugosity with a ‘particle’ size of 3 nm. The crystal structure of the films is similar to f.c.c. nickel oxide. Molybdenum enrichment was detected in the anodized alloy surfaces but there is no electron diffraction evidence for a crystalline molybdenum oxide. The electron diffraction pattern from anodized pure molybdenum suggests that the surface film is amorphous.     

The point-spread function of a confocal microscope: its measurement and use in deconvolution of 3-D data

We have measured the point-spread function (PSF) for an MRC-500 confocal scanning laser microscope using subresolution fluorescent beads. PSFs were measured for two lenses of high numerical aperture—the Zeiss plan-neofluar 63 × water immersion and Leitz plan-apo 63 × oil immersion—at three different sizes of the confocal detector aperture. The measured PSFs are fairly symmetrical, both radially and axially. In particular there is considerably less axial asymmetry than has been demonstrated in measurements of conventional (non-confocal) PSFs. Measurements of the peak width at half-maximum peak height for the minimum detector aperture gave approximately 0·23 and 0·8 μm for the radial and axial resolution respectively (4·6 and 15·9 in dimensionless optical units). This increased to 0·38 and 1·5 μm (7·5 and 29·8 in dimensionless units) for the largest detector aperture examined. The resulting optical transfer functions (OTFs) were used in an iterative, constrained deconvolution procedure to process three-dimensional confocal data sets from a biological specimen—pea root cells labelled in situ with a fluorescent probe to ribosomal genes. The deconvolution significantly improved the clarity and contrast of the data. Furthermore, the loss in resolution produced by increasing the size of the detector aperture could be restored by the deconvolution procedure. Therefore for many biological specimens which are only weakly fluorescent it may be preferable to open the detector aperture to increase the strength of the detected signal, and thus the signal-to-noise ratio, and then to restore the resolution by deconvolution.