Mechanisms governing secretion of proteins underlie the biologic activities and functions of human eosinophils, leukocytes of the innate immune system, involved in allergic, inflammatory, and immunoregulatory responses. In response to varied stimuli, eosinophils are recruited from the circulation into inflammatory foci, where they modulate immune responses through the release of granule-derived products. Transmission electron microscopy (TEM) is the only technique that can clearly identify and distinguish between different modes of cell secretion. In this review, we highlight the advances in understanding mechanisms of eosinophil secretion, based on TEM findings, that have been made over the past years and that have provided unprecedented insights into the functional capabilities of these cells.

We evaluated the depth resolution of annular dark-field (ADF) scanning confocal electron microscopy (SCEM) with a stage-scanning system by observation of nanoparticles. ADF-SCEM is a three-dimensional (3D) imaging technique that we recently proposed. An ADF-SCEM instrument involves a pinhole aperture before a detector for rejecting electrons from the out-of-focal plane in a specimen and an annular aperture under the specimen for collecting only scattered electrons. The stage-scanning system enables us to directly obtain optical slice images perpendicular and parallel to an optical axis at a desired position. In particular, the parallel slices visualize the elongation of nanoparticles along the optical axis, which depends on the depth resolution. ADF-SCEM effectively reduced the elongation length of the nanoparticles sufficiently to demonstrate depth sectioning, in comparison with scanning transmission electron microscopy and bright-field SCEM. The experimentally obtained length was nearly equal to the theoretically estimated one from the probe size considering the experimental conditions. Furthermore, we applied this ADF-SCEM technique to analysis of the 3D position of catalytic nanoparticles on carbon nanostructures.

Use of deep ultraviolet (DUV, below 350 nm) fluorescence opens up new possibilities in biology because it does not need external specific probes or labeling but instead allows use of the intrinsic fluorescence that exists for many biomolecules when excited in this wavelength range. Indeed, observation of label free biomolecules or active drugs ensures that the label will not modify the biolocalization or any of its properties. In the past, it has not been easy to accomplish DUV fluorescence imaging due to limited sources and to microscope optics. Two worlds were coexisting: the spectrofluorometric measurements with full spectrum information with DUV excitation, which lacked high-resolution localization, and the microscopic world with very good spatial resolution but poor spectral resolution for which the wavelength range was limited to 350 nm. To combine the advantages of both worlds, we have developed a DUV fluorescence microscope for cell biology coupled to a synchrotron beamline, providing fine tunable excitation from 180 to 600 nm and full spectrum acquired on each point of the image, to study DUV excited fluorescence emitted from nanovolumes directly inside live cells or tissue biopsies.

We present an in vitro model of human skin that, together with nonlinear optical microscopy, provides a useful system for characterizing morphological and structural changes in a living skin tissue microenvironment due to changes in oxygen status and proteolytic balance. We describe for the first time the effects of chronic oxygen deprivation on a bioengineered model of human interfollicular epidermis. Histological analysis and multiphoton imaging revealed a progressively degenerating ballooning phenotype of the keratinocytes that manifested after 48 h of hypoxic exposure. Multiphoton images of the dermal compartment revealed a decrease in collagen structural order. Immunofluorescence analysis showed changes in matrix metalloproteinase (MMP)-2 protein spatial localization in the epidermis with a shift to the basal layer, and loss of Ki67 expression in proliferative basal cells after 192 h of hypoxic exposure. Upon reoxygenation MMP-2 mRNA levels showed a biphasic response, with restoration of MMP-2 levels and localization. These results indicate that chronic oxygen deprivation causes an overall degeneration in tissue architecture, combined with an imbalance in proteolytic expression and a decrease in proliferative capacity. We propose that these tissue changes are representative of the ischemic condition and that our experimental model system is appropriate for addressing mechanisms of susceptibility to chronic wounds.

The year 2009 marked the centenary of the birth of Otto Scherzer, one of the early pioneers of electron microscopy. Scherzer was the originator of the famous microscopy theorem that the spherical and chromatic aberrations of rotationally symmetric electron lenses were unavoidable. In honor of this centennial occasion, we organized a special memorial symposium during Microscopy & Microanalysis 2009, which was held in Richmond, Virginia, in late July. The introductory talks of the symposium presented a fascinating mix of first-hand accounts about working with Scherzer in Darmstadt and descriptions of the correction concepts and the early corrector prototypes that emerged from his group. Placed in this historical context, the latest advances in aberration correction for scanning and fixed-beam instruments that were presented in this symposium were all the more impressive and conveyed a vivid sense of history in the making. Applications of aberration correction to a broad range of materials were also highlighted in platform and poster presentations. This special issue of Microscopy and Microanalysis contains refereed contributions from the work presented at the symposium and thus provides a representative overview of the recent emergence of aberration-corrected electron microscopy (ACEM) and some of the prospects and challenges for this burgeoning field.

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.

Bulk silicon-germanium (SiGe) alloys and two SiGe thick films (4 and 5 μm) on Si wafers were tested with the electron probe microanalyzer (EPMA) using wavelength dispersive spectrometers (WDS) for heterogeneity and composition for use as reference materials needed by the microelectronics industry. One alloy with a nominal composition of Si0.86Ge0.14 and the two thick films with nominal compositions of Si0.90Ge0.10 and Si0.75Ge0.25 on Si, evaluated for micro- and macroheterogeneity, will make good microanalysis reference materials with an overall expanded heterogeneity uncertainty of 1.1% relative or less for Ge. The bulk Ge composition in the Si0.86Ge0.14 alloy was determined to be 30.228% mass fraction Ge with an expanded uncertainty of the mean of 0.195% mass fraction. The thick films were quantified with WDS-EPMA using both the Si0.86Ge0.14 alloy and element wafers as reference materials. The Ge concentration was determined to be 22.80% mass fraction with an expanded uncertainty of the mean of 0.12% mass fraction for the Si0.90Ge0.10 wafer and 43.66% mass fraction for the Si0.75Ge0.25 wafer with an expanded uncertainty of the mean of 0.25% mass fraction. The two thick SiGe films will be issued as National Institute of Standards and Technology Reference Materials (RM 8905).

Computer vision tasks such as recognition and classification of objects and structures or image registration and retrieval can provide significant information when applied to microscopy images. Recently developed techniques for the detection and description of local features make the extraction and description of local image features that are invariant to various changes possible. The invariance and robustness of feature detection and description techniques play a key role in the design and implementation of object recognition, image registration, or image mosaicing applications. The scale-invariant feature transform (SIFT) technique is a widely used method for the detection, description, and matching of image features. In this article we present the results of our experiments regarding the repeatability of SIFT features, and to the precision of the SIFT feature matching, under image modifications specific to confocal scanning laser microscopy (CSLM). We have analyzed the behavior of SIFT while changing the pinhole aperture, photomultiplier gain, laser beam power, and electronic zoom. Our experiments, conducted on CSLM images, show that the SIFT technique is able to match detected key points between images acquired at different values of the acquisition parameters with good precision and represents a consistent tool for computer vision applications in CSLM.

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.

An electromechanical video camera coupler was developed to rotate a light microscope field of view (FOV) in real time without the need to physically rotate the stage or specimen. The device, referred to as the Camera Thetarotator, rotated microscopical views 240° to assist microscopists to orient specimens within the FOV prior to image capture. The Camera Thetarotator eliminated the effort and artifacts created when rotating photomicrographs using conventional graphics software. The Camera Thetarotator could also be used to semimanually register a dataset of histological sections for three-dimensional (3D) reconstruction by superimposing the transparent, real-time FOV to the previously captured section in the series. When compared to Fourier-based software registration, alignment of serial sections using the Camera Thetarotator was more exact, resulting in more accurate 3D reconstructions with no computer-generated null space. When software-based registration was performed after prealigning sections with the Camera Thetarotator, registration was further enhanced. The Camera Thetarotator expanded microscopical viewing and digital photomicrography and provided a novel, accurate registration method for 3D reconstruction. The Camera Thetarotator would also be useful for performing automated microscopical functions necessary for telemicroscopy, high-throughput image acquisition and analysis, and other light microscopy applications.

The following papers are samples of ongoing Portuguese contributions to the field of microscopy research. They were first communicated at the 43rd Annual Meeting of the Portuguese Society for Microscopy (Sociedade Portuguesa de Microscopia, SPMicros) held in Porto, October 30–31, 2008, and, together with most of the other communications, were published as extended abstracts in the Proceedings of the meeting (Microsc. Microanal. 16, Suppl. S3). This was the second time that the abstracts of the SPMicros Annual Meetings were made available worldwide through our collaborations with Microscopy and Microanalysis; although throughout its long history, the society has always kept Portuguese and English records of the communications either in abstract books or as journal supplements.

Electron-beam-induced carbon film deposition has long been recognized as a side effect of scanning electron microscopy. To characterize the nature of this type of contamination, silicon wafers were subjected to prolonged exposure to 15 kV electron beam energy with a probe current of ∼300 pA. Using Raman spectroscopy, the deposited coating was identified as an amorphous carbon film with an estimated crystallite size of 125 Å. Using atomic force microscopy, the cross-sectional profile of the coating was found to be raised and textured, indicative of the beam raster pattern. A map of the Raman intensity across the coating showed increased intensity along the edges and at the corner of the film. The intensity profile was in excess of that which could be explained by thickness alone. The enhancement was found to correspond with a modeled local field enhancement induced by the coating boundary and showed that the deposited carbon coating generated a localized disturbance in the opto-electrical properties of the substrate, which is compared and contrasted with Raman edge enhancement that is produced by surface structure in silicon.

Otto Scherzer was one of the pioneers of theoretical electron optics. He was coauthor of the first comprehensive book on electron optics and was the first to understand that round electron lenses could not be combined to correct aberrations, as is the case in light optics. He subsequently was the first to describe several alternative means to correct spherical and chromatic aberration of electron lenses. These ideas were put into practice by his laboratory and students at Darmstadt and their successors, leading to the fully corrected electron microscopes now in operation.

The morphology of the osteocyte lacuno-canalicular system at the bone-biomaterial implant-interface has not been fully investigated. In this study, the resin-cast scanning electron microscopy technique was used, for the first time, to image the lacuno-canalicular network within neoformed bone around bioactive glass (BG) particles implanted in rat tibia bone marrow. The most salient finding was that the osteocyte canaliculi pass through the calcium-phosphorus layer formed at the bone-BG interface and reach the silica-rich layer of the reacted BG.

The aim was to characterize the external structure, roughness, and absolute depth profile (ADP) of fluorotic enamel compared with healthy enamel. Eighty extracted human molars were classified into four groups [TFI: 0, control (C); 1–3, mild (MI); 4–5, moderate (MO); 6–9, severe fluorosis (S)] according to the Thylstrup-Fejerskov Index (TFI). All samples were analyzed by atomic force microscopy.

The mean values of enamel surface roughness (ESR) in nm were: Group C, 92.6; Group MI, 188.8; Group MO, 246.9; and Group S, 532.2. The mean values of absolute depth profile in nm were: C, 1,065.7; MI, 2,360.7; MO, 2,536.7; and S, 6,146.2. The differences between mean ESR and mean ADP among groups were statistically significant (p < 0.05). This structural study confirms at the nanometer level that there is a positive association between fluorosis severity, ESR, and ADP, and there is an association with the clinical findings of fluorosis measured by TFI as well.

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.

A high quality X-ray spectrum image of a 3.3 μm diameter sphere of K411 glass resting on a copper substrate was collected at 25 keV. The same sample configuration was modeled using the NISTMonte Monte Carlo simulation of electron and X-ray transport as is integrated into the quantitative X-ray microanalysis software package DTSA-II. The distribution of measured and simulated X-ray intensity compare favorably for all the major lines present in the spectra. The simulation is further examined to investigate the influence of angle-of-incidence, sample thickness, and sample diameter on the generated and measured X-ray intensity. The distribution of generated X-rays is seen to deviate significantly from a naive model which assumes that the distribution of generated X-rays is similar to bulk within the volume they share in common. It is demonstrated that the angle at which the electron beam strikes the sample has nonnegligible consequences. It is also demonstrated that within the volume that the bulk and particle share in common that electrons, which have exited and later reentered the particle volume, generate a significant fraction of the X-rays. Any general model of X-ray generation in particles must take into account the lateral spread of the scattered electron beam.