Morphologic and cathodoluminescence studies of diamond films by scanning electron microscopy

Using recent papers on scanning electron microscopy (SEM) of chemical vapor deposition (CVD) diamond films, two analytical applications of the SEM are discussed: the morphologic investigations (secondary electron emission mode) and the recognition of impurities and defects [cathodoluminescence (CL) mode]. Studies of CVD diamond films by SEM demonstrate that the morphologies of these films are affected by synthesis conditions, especially by substrate temperature, methane concentration, and total pressure in the reactor. CL spectra and images are useful tools for clarifying the relationship between emission centers and different types of defects generated during the process of diamond crystal growth. The paper shows that the investigations of the morphology, crystallinity, local CL emission, as well as the surface distribution of CL spectra on CVD diamond films by SEM led to the correlative information for quality estimation of films in comparison with natural diamond.     

Quantitative and reliable in vitro method combining scanning electron microscopy and image analysis for the screening of osteotropic modulators

The increased generation and up-regulated activity of bone resorbing cells (osteoclasts) play a part in the impairment of bone remodeling in many bone diseases. Numerous drugs (bisphosphonates, calcitonin, selective estrogen receptor modulators) have been proposed to inhibit this increased osteoclastic activity. In this report, we describe a pit resorption assay quantified by scanning electron microscopy coupled with image analysis. Total rabbit bone cells with large numbers of osteoclasts were cultured on dentin slices. The whole surface of the dentin slice was scanned and both the number of resorption pits and the total resorbed surface area were measured. Resorption pits appeared at 48 h and increased gradually up to 96 h. Despite the observation of a strong correlation between the total resorption area and the number of pits, we suggest that area measurement is the most relevant marker for osteoclastic activity. Osteotropic factors stimulating or inhibiting osteoclastic activity were used to test the variations in resorption activity as measured with our method. This reproducible and sensitive quantitative method is a valuable tool for screening for osteoclastic inhibitors and, more generally, for investigating bone modulators.     

A fast method to study the secretory activity of neuroendocrine cells at the ultrastructural level

Cryo field emission scanning electron microscopy (cryo-FE-SEM) is a versatile technique that allows the investigation of the three-dimensional organization of cells at the ultrastructural level over a wide range of magnifications. Unfortunately, cryopreparation of the specimens for this technique remains cumbersome, in particular because ice crystal formation must be prevented during freezing. Here we report that a light prefixation with glutaraldehyde and incubation in glycerol as cryoprotectant or a high-pressure freezing approach are both excellent procedures for cryopreparation of animal cells to be used in combination with cryo-FE-SEM. Using the proopiomelanocortin-producing intermediate pituitary melanotrope cells of Xenopus laevis as a physiologically inducible neuroendocrine system, we compared the ultrastructural characteristics of inactive and hyperactive neuroendocrine cells. The overall quality of the ultrastructural images was comparable for the two cryopreparation procedures, although some fine structures were better conserved using high-pressure freezing. Melanotrope cells in a secretory inactive state contained numerous storage granules and a poorly developed endoplasmic reticulum (ER), while large amounts of rough ER were present in hyperactive cells. Thus, the cryo-FE-SEM approach described here allows a fast ultrastructural study on the secretory activity of neuroendocrine cells.     

Theory experiment comparison of the electron backscattering factor from solids at low electron energy (250-5,000 eV)

The electron backscattering factor was measured from 24 different elements at low primary beam energy (250-5,000 eV). The results were compared with Monte Carlo simulations from a variety of freely available programs and an in-house developed program. The results suggest that a thin film of oxide can modify the backscattering factor at low primary energy. In addition, a number of problems have been identified with the freely available programs.     

Image signal-to-noise ratio estimation using Shape-Preserving Piecewise Cubic Hermite Autoregressive Moving Average model

We propose to cascade the Shape-Preserving Piecewise Cubic Hermite model with the Autoregressive Moving Average (ARMA) interpolator; we call this technique the Shape-Preserving Piecewise Cubic Hermite Autoregressive Moving Average (SP2CHARMA) model. In a few test cases involving different images, this model is found to deliver an optimum solution for signal to noise ratio (SNR) estimation problems under different noise environments. The performance of the proposed estimator is compared with two existing methods: the autoregressive-based and autoregressive moving average estimators. Being more robust with noise, the SP2CHARMA estimator has efficiency that is significantly greater than those of the two methods.     

Real imaging and size values of Saccharomyces cerevisiae cells with comparable contrast tuning to two environmental scanning electron microscopy modes

The combined application of electron microscopy (EM) is frequently used for the microstructural investigation of biological specimens and plays two important roles in the quantification and in gaining an improved understanding of biological phenomena by making use of the highest resolution capability provided by EM. The possibility of imaging wet specimens in their "native" states in the environmental scanning electron microscope (ESEM) at high resolution and large depth of focus in real time is discussed in this paper. It is demonstrated here that new features can be discovered by the elimination of even the least hazardous approaches in some preparation techniques, that destroy the samples. Since the analysis conditions may influence the morphology and the extreme surface sensitivity of living biological systems, the results obtained from the same cultured cell with two different ESEM modes (Lvac mode and wet mode) were compared. This offers new opportunities compared with ESEM-wet/Lvac-mode imaging, since wet-mode imaging involves a real contrast and gives an indication of the changes in cell morphology and structure required for cell viability. In this study, wet-mode imaging was optimized using the unique ability of cell quantities for microcharacterization in situ giving very fine features of topological effects. Accordingly, the progress is reported by comparing the results of these two modes, which demonstrate interesting application details. In general, the functional comparisons have revealed that the fresh unprocessed Saccharomyces cerevisiae cells (ESEM-wet mode) were essentially unaltered with improved and minimal specimen preparation timescales, and the optimal cell viability degree was visualized and also measured quantitatively while the cell size remained unchanged with continuous images.     

Surface damage induced by FIB milling and imaging of biological samples is controllable

Focused ion beam (FIB) techniques are among the most important tools for the nanostructuring of surfaces. We used the FIB/SEM (scanning electron microscope) for milling and imaging of digestive gland cells. The aim of our study was to document the interactions of FIB with the surface of the biological sample during FIB investigation, to identify the classes of artifacts, and to test procedures that could induce the quality of FIB milled sections by reducing the artifacts. The digestive gland cells were prepared for conventional SEM. During FIB/SEM operation we induced and enhanced artifacts. The results show that FIB operation on biological tissue affected the area of the sample where ion beam was rastering. We describe the FIB-induced surface major artifacts as a melting-like effect, sweating-like effect, morphological deformations, and gallium (Ga(+)) implantation. The FIB induced surface artifacts caused by incident Ga(+) ions were reduced by the application of a protective platinum strip on the surface exposed to the beam and by a suitable selection of operation protocol. We recommend the same sample preparation methods, FIB protocol for milling and imaging to be used also for other biological samples.     

An electron beam lithography and digital image acquisition system for scanning electron microscopes

A low‐cost microcontroller based control and data acquisition unit for digital image recording of scanning electron microscope (SEM) images and scanning electron microscope based electron beam lithography (EBL) is described. The developed microcontroller low‐level embedded software incorporates major time critical functions for image acquisition and electron beam lithography and makes the unit an intelligent module which communicates via USB with the main computer. The system allows recording of images with up to 4096 × 4096 pixel size, different scan modes, controllable dwell time, synchronization with main power frequency, and other user controllable functions. The electron beam can be arbitrary positioned with 12‐bit precision in both dimensions and this is used to extend the scanning electron microscope capabilities for electron beam lithography. Hardware and software details of the system are given to allow its easy duplication. Performance of the system is discussed and exemplary results are presented.     

Quasi-cleavage fracture planes in spheroidized A533B steel

Electron backscattered diffraction (EBSD) has been used to acquire crystal orientation information around unusual microcracks induced by tensile deformation of notched specimens of spheroidized A533B steel. This unusual fracture mode has been called quasi‐cleavage and occurs at relatively low temperatures with fracture energies below that of the upper shelf. EBSD measurements on sectioned samples showed that the quasi‐cleavage cracks were intragranular. A two‐dimensional analysis technique was used in which EBSD measured crystal orientations were combined with secondary electron imaging to obtain the trace of the crack facet on the section plane. The measurements revealed that the observed crack facets were consistent with crack propagation along the {001} and {011} planes.