In this study, we discuss a method for cross-sectional thin specimen preparation from a specific site using a combination of a focused ion beam (FIB) system and an intermediate voltage transmission electron microscope (TEM). A FIB-TEM compatible specimen holder was newly developed for the method. The thinning of the specimen using the FIB system and the observation of inside structure of the ion milled area in a TEM to localize a specific site were alternately carried out. The TEM fitted with both scanning transmitted electron detector and secondary electron detector enabled us to localize the specific site in a halfway milled specimen with the positional accuracy of better than 0.1 µm. The method was applied to the characterization of a precipitate in a steel. A submicron large precipitate was thinned exactly at its center for the characterization by a high-resolution electron microscopy and an elemental mapping.

In this report, we describe the distribution of the platelet-derived growth factor receptor α (PDGFRα) by immunolocalization in the embryonic day 10.5 mouse heart and defects in heart development associated with the absence of this receptor in the Patch mouse. The Patch mouse is a naturally occurring mutant that has been accepted as a model for determining the role of the PDGFRα in early cardiac development. Even though other genetic defects exist in this naturally occurring mutant, most defects associated with cardiac development are believed to be a result of the absence of this receptor. Gross morphological defects including improper septation of the outflow tract, dysmorphic shape of the heart, and lack of trabecular development are similar to those that have been previously described. Many of these defects have been attributed to the failure of a subset of non-neuronal neural crest cells to properly migrate into the region of the developing outflow tract. In these studies, we have also used confocal scanning laser and transmission electron microscopy to describe and compare the organization and differentiation of the cytoskeletal proteins actin and myosin in littermate control and Patch mouse hearts. Cytoskeletal organization of the cardiac myocytes in Patch mouse hearts has not previously been described. In most cardiac myocytes of Patch mice, actin was found only on the periphery of the cells, and the organization of actin, myosin, and precursor Z-band material into distinct myofibrils was greatly reduced.

Complementary to our first paper on the origin of threefold contrast on SiC high resolution transmission electron microscopy (HRTEM) images, we now provide an example of threefold contrast produced by a stacking layer sequence which corresponds to one unit cell of the 9R polytype.

Autofluorescence, also known as adventitious fluorescence or background fluorescence, often poses a significant problem in many applications of fluorescence microscopy. It contributes to unwanted noise and can overwhelm the desired signal. Particularly difficult samples to image include many pathology specimens that have been processed using crosslinking fixatives (typically formaldehyde). This procedure dramatically increase the autofluorescence level, leading to bright, broad spectrum emissions, particularly from connective tissue components. Unprocessed plant tissue and neuronal tissue also have extremely high levels of endogenous autofluorescence that can make many convenient labeling strategies, including most green fluorescent protein (GFP) labels, extremely problematic. Various solutions have been proposed for the reduction or elimination of autofluorescence. These include using narrow bandpass emission filters to try to isolate the desired fluorescence signal, the use of labels that can be excited at wavelengths that are much less likely to induce autofluorescence (moving the excitation towards the NIR is effective), and post-processing aldehyde-fixed samples with such reagents as sodium borohydride or toluidine blue to chemically suppress the autofluorescence signal. However, in many cases, these approaches are either infeasible or ineffective. Spectral imaging, that is, the acquisition of a high-resolution optical spectrum at every pixel of an image, offers another approach to elimination of the contribution of autofluorescence. This has recently become possible due to the development of a number of technologies that allow the collection of spectral datasets from fluorescently labeled samples using fluorescence microscopy. Such technologies include liquid crystal tunable filters (LCTFs) and Fourier transform imaging spectrometers, both of which are now commercially available.

The depth profile of thin film layers on bulk substrate, avoiding the cross-sectioning of samples, is commonly performed by techniques such as X-ray photoelectron spectroscopy (XPS), Auger electron spectroscopy (AES), and secondary ion mass spectroscopy (SIMS). Techniques based on X-ray emission intensity measurements by energy dispersive spectroscopy (EDS), with conventional matrix or ZAF correction, are normally applied to cross-sectioned samples. This article compares XPS with surface X-ray intensity measurements by EDS, carried out with a more realistic X-ray generation and absorption model, known as the ϕ(ρ Z) model. The ϕ(ρ Z) approach has been adopted together with Monte Carlo simulation for the proper selection of SEM accelerating voltages, in conjunction with the analysis of SEM morphological images for thin film density correction. The method discussed hereafter and compared with the XPS technique, has advantages of higher lateral resolution, non-destructive elemental analyses, morphological visualization, low cost, and faster performance. This methodology has been followed to verify the layered structure of SnO2/metal-based gas sensors. X-ray intensities were measured using an EDS ultra-thin window detector. Two different porous layers, 25-nm thick of SnO2 and 10-nm thick of Cu, were detected, showing better agreement with their nominal thickness compared to results obtained using XPS measurements where porosity affects XPS data. If confirmed to be reliable and as effective as XPS depth profiling, this technique may be adopted for process quality control purposes.

A high performance hyperspectral imager with high light throughput suitable for microscopy and analytical imaging was built and tested. The imager utilizes phenomenon of optical activity. The new technique provides a continuous spectral range of several hundreds of nanometers starting in deep UV. Similar spectral range starting in the near IR is also achievable.

A performance of a low cost implementation of the new technology is presented. The imager has a form of a microscope adaptor, which is inserted between the microscope and a low-cost 8-bit CCD camera. The resulting instrument is simple, robust, and highly compact. The imager module is placed in-line to the microscope imaging system and does not introduce observable image aberrations. Advantageously, the imager is transparent to conventional imaging operations, thus with the imager in-place there is no need for reconfiguration of the microscope to switch between conventional and hyperspectral video/digital imaging modes.

The presented spectral imager answers the need for a practical, sensitive, compact, and affordable imaging spectrometer. The instrument is well suited for a broad range of applications requiring rapid parallel acquisition of highly resolved concurrent spatial and spectral information such as high throughput screening, biochip analysis, remote sensing or nondestructive semiconductor testing.

Both scanning electron microscopy (SEM) and contact mode imaging via atomic force microscopy (AFM) have been utilized to elucidate the ultrastructure of mung bean seed surfaces. The results indicate: 1) that AFM is useful in the examination of seed surface ultrastructure ex-vaccuo without the need for additional complex preparative procedures; and 2) that both the cotyledon and seed coat of different strains of mung beans bear specific ultrastructural details unique to each strain. To our knowledge, these are the first AFM images of seed surfaces.

As part of the Microbeam Analysis Society (MAS) symposium marking 50 years of electron microprobe analysis, this article reviews the important advances made over the decades to the automation of data collection and computerized analysis of data from the electron microprobe. Out of many innovations that contributed to the advance of microprobe automation, we have chosen to focus on a few developments that the authors feel represent the major trends in advancement of the “state of the art” of this instrumentation. After providing brief summaries of the three generations of advances in the hardware and software of automation systems, several key applications developments are described, followed by our prediction of which current developments may impact the future automation of the microprobe.

Confocal Laser Scanning Microscopy (CLSM) is being used to visualize folliculogenesis and presumed apoptotic events in whole neonatal mouse/rat ovaries. Our laboratory has previously demonstrated that apoptosis can be visualized in 8-9 day whole mouse embryos, 12 day fetal mouse eyes or 12-15 fetal rat limbs stained with the fluorescent lysosomal stain, LysoTracker Red (LT). The observed LT staining has been correlated with Nile blue staining (lysosomes and phagocytosis), TUNEL staining (DNA breaks) and apoptotic tissue morphology in rat and mouse developmental systems. This increased LT staining intensity appeared to be associated with acidic tissue, lysosomes, phagocytosis and dying cells. Here we describe recent progress using LT to evaluate the apoptotic process in whole ovaries. We are currently optimizing sample preparation and confocal machine operation to improve the image quality. to increase depth of visualization, ovaries derived from 10-45 day old mice/rats are fixed with an aldehyde and then made transparent by MEOH dehydration followed by benzyl alcohol/benzyl aldehyde (BABB) clearing. Such whole ovaries or tissue sections are viewed by CLSM using 568-wavelength laser light to obtain images of oocytes and follicles. Follicles showing gross morphologic changes typical of atresia were also characterized by intense LT staining of granulosa cells. This increased LT staining intensity appeared to be associated with acidic tissue and dying cells contained within the follicle.

Yeast cells represent a powerful model system in cell biology mainly due to their amenability to genetic manipulations. Increasingly, studies focus on mutant genes resulting in alterations of cellular structures and organelles. To ascertain the phenotypic changes involved, it is often desirable to use the resolving power of electron microscopy. In contrast to higher eukaryotic cells, yeast cells are particularly difficult to preserve mainly due to the presence of a thick cell wall that acts as a barrier against diffusion of fixatives. Although several procedures are targeted to overcome these difficulties, none of them have become established as a standard procedure. As a consequence, electron microscopy is still not used routinely as a tool in yeast cell biology. This prompted us to develop an easy-to-follow protocol for yeast transmission electron microscopy that should be useful in all cases where membrane integrity and organelle morphology is emphasized. One means of making the yeast cytoplasm more attainable to fixation and staining solutions is by enzymatic digestion of the cell wall. Following this approach, we were able to reliably preserve yeast cells and their cellular organelles. Enzymatic treatment with zymolyase 20T to partially remove the yeast cell wall allowed the fixation, preservation, and visualization of the yeast cytoplasm revealing detailed ultrastructure. The advancement of this technique is demonstrated with mitochondria as a model organelle. Our studies on various yeast mutants clearly show the power of the enzymatic digestion technique in visualizing subtle changes of membrane structure and organelle morphology.

Quantitative X-ray microanalysis requires the use of many fundamental constants related to the interaction of the electron beam with the sample. The current state of our knowledge of such constants in the particular areas of electron stopping power, X-ray ionization cross-sections, X-ray fluorescence yield, and the electron backscattering yield, is examined. It is found that, in every case, the quality and quantity of data available is poor, and that there are major gaps remaining to be filled.

Fluorescent recovery from photobleaching coupled with confocal microscopy was explored as a potential high-resolution method of imaging the distribution of enzyme activity in single living cardiac myocytes without relying on steady state measurements of fluorescence. On a fundamental level, much remains to be determined regarding how local conditions within a cell affect metabolism. Many studies have suggested that energy metabolism in muscle cells cannot be accurately described assuming a homogenous system of enzymatic reactions [1,2]. The autofluorescence of NADH has been used in many studies as a quantitative assay of mitochondrial energy metabolism [3,4], but studies of steady state fluorescence cannot distinguish between changes in energy production or utilization.

In this study conditions were created where the fluorescent recovery of a probe would be solely dependent on cellular enzymatic activity (Enzyme Dependent Fluorescence Recovery after Photobleaching (ED-FRAP)). Experiments examining the inherent fluorescence of NADH (351nm excitation, 450 nm emission) were conducted on small droplets (less than 325 μm diameter) containing NADH alone, in droplets containing an enzyme system capable of synthesis of NADH (Figure 1A) and on isolated rabbit cardiac myocytes (Figure 1D). Photobleaching of the entire cell or droplet eliminated diffusion or bulk transport of NADH from non-bleached regions. Droplets containing NADH alone did not recover, while droplets containing enzyme were shown to recover exponentially (Figure 1B) with a rate constant of fluorescent recovery (kf) that was proportional to enzyme concentration (Figure 1C).

Errors in quantitative electron microprobe analysis arise from many sources including those associated with sampling, specimen preparation, instrument operation, data collection, and analysis. The relative magnitudes of some of these factors are assessed for a sample of NiAl used to demonstrate important concerns in the analysis of even a relatively simple system measured under standard operating conditions. The results presented are intended to serve more as a guideline for developing an analytical strategy than as a detailed error propagation model that includes all possible sources of variability and inaccuracy. The use of a variety of tools to assess errors is demonstrated. It is also shown that, as sample characteristics depart from those under which many of the quantitative methods were developed, errors can increase significantly.