Micro-computed tomography with the highly intense, monochromatic X rays produced by the synchrotron is a superior method to nondestructively measure the local absorption in three-dimensional space. Because biological tissues and cells consist mainly of water as the surrounding medium, higher absorbing agents have to be incorporated into the structures of interest. Even without X-ray optics such as refractive lens, one can uncover the stain distribution with the spatial resolution of about 1 μm. Incorporating the stain at selected cell compartments, for example, binding to the RNA/DNA, their density distribution becomes quantified. In this communication, we demonstrate that tomograms obtained at the beamlines BW2 and W2 (HASYLAB at DESY, Hamburg, Germany) and 4S (SLS, Villigen, Switzerland) clearly show that the RNA/DNA-stained HEK 293 cell clusters have a core of high density and a peripheral part of lower density, which correlate with results of optical microscopy. The inner part of the clusters is associated with nonvital cells as the result of insufficient oxygen and nutrition supply. This necrotic part is surrounded by (6 ± 1) layers of vital cells.

The design and construction of a double-hexapole aberration corrector has made it possible to build the prototype of a spherical-aberration corrected transmission electron microscope dedicated to high-resolution imaging on the atomic scale. The corrected instrument, a Philips CM200 FEG ST, has an information limit of better than 0.13 nm, and the spherical aberration can be varied within wide limits, even to negative values. The aberration measurement and the corrector control provide instrument alignments stable enough for materials science investigations. Analysis of the contrast transfer with the possibility of tunable spherical aberration has revealed new imaging modes: high-resolution amplitude contrast, extension of the point resolution to the information limit, and enhanced image intensity modulation for negative phase contrast. In particular, through the combination of small negative spherical aberration and small overfocus, the latter mode provides the high-resolution imaging of weakly scattering atom columns, such as oxygen, in the vicinity of strongly scattering atom columns. This article reviews further lens aberration theory, the principle of aberration correction through multipole lenses, aspects for practical work, and materials science applications.

This issue of Microscopy and Microanalysis contains contributions presented at the Frontiers of Electron Microscopy in Materials Science (FEMMS) meeting held in Kasteel Vaalsbroek, The Netherlands, on September 25–30, 2005. Tenth in the series of biennial conferences, the meeting focused on the latest developments in the field of advanced instrumentation and application of electron microscopy in materials science. The international character of this series of conferences was once again emphasized by the presence of over 140 delegates whose interests include academia, national laboratories, and industry from 16 countries representing all areas of the globe.

On September 1, 2006, the Microscopy and Microanalysis electronic submission and review website went live. Our publisher, Cambridge University Press, has teamed with ScholarOne to provide this service. From now on all manuscript submissions to this journal must be made electronically through the following website:

http://mc.manuscriptcentral.com/mam

Editor in Chief Editor, Microanalysis: Charles E. Lyman

Editor, Biological Applications: Robert L. Price

Editor, Materials Applications: David J. Smith

Editor, Materials Applications: Elizabeth Dickey

Editor, Light and Scanning Probe Microscopies: Brian Herman

Editor, Biological Applications: Heide Schatten

Calendar Editor Book/Review Editor: JoAn Hudson

Special Section Editor: James N. Turner

Expo Editor: William T. Gunning III

Proceedings Editor: Stuart McKernan

Extended abstract of a paper presented at Microscopy and Microanalysis 2006 in Chicago, Illinois, USA, July 30 – August 3, 2006

In 1956 Duncumb and Cosslett developed the first imaging method capable of showing the location of elements in a solid with a spatial resolution of a micrometer. This technique was dubbed “X-ray mapping” probably because a separate image was used to show the presence or absence of each element within the field of view. Since its first demonstration, X-ray mapping has become one of the most popular and useful methods of X-ray microanalysis. It has been widely applied in areas of biology, chemistry, physics, geology, environmental science, and materials science.

We at Oxford Instruments were interested to read the article published in Microscopy and Microanalysis by Newbury (2005). Although our own views differ from those of Dr. Newbury in some respects, we do agree that this is an important matter. If this article generates an interest in the ability of a system and a user to accurately identify elements in a spectrum and encourages people to assess the quality of the qualitative analysis provided by a microanalysis system, then we believe this article has served a very useful purpose. After all, accurate element identification is the core requirement for a microanalysis system.

We have implemented and tested a new automatic method for the montage synthesis and three-dimensional (3D) reconstruction of large tissue volumes from confocal laser scanning microscopy data (CLSM). This method relies on maximization of the phase correlation between adjacent images. It was tested on a large specimen (a murine heart) that was cut into a number of individual sections with thickness appropriate for CLSM. The sections were scanned horizontally (in-plane) and vertically (perpendicular to the optical planes) to produce “tiles” of a 3D volume. Phase correlation maximization was applied to the montage synthesis of in-plane tiles and 3D alignment of optical slices within a given physical section. The performance of the new method is evaluated.

Extended abstract of a paper presented at Microscopy and Microanalysis 2006 in Chicago, Illinois, USA, July 30 – August 3, 2005

I am pleased to respond to the letter from Simon Burgess commenting on my article, “Misidentification of Major Constituents by Automatic Qualitative Energy Dispersive X-ray Microanalysis: A Problem That Threatens the Credibility of the Analytical Community” (Newbury, 2005). First, I am grateful for the words of support for my article, even if inevitably qualified, expressed in Mr. Burgess's letter. In response to his specific comments (in boldface), I offer the following:

This review traces the development of X-ray mapping from its beginning 50 years ago through current analysis procedures that can reveal otherwise obscure elemental distributions and associations. X-ray mapping or compositional imaging of elemental distributions is one of the major capabilities of electron beam microanalysis because it frees the operator from the necessity of making decisions about which image features contain elements of interest. Elements in unexpected locations, or in unexpected association with other elements, may be found easily without operator bias as to where to locate the electron probe for data collection. X-ray mapping in the SEM or EPMA may be applied to bulk specimens at a spatial resolution of about 1 μm. X-ray mapping of thin specimens in the TEM or STEM may be accomplished at a spatial resolution ranging from 2 to 100 nm, depending on specimen thickness and the microscope. Although mapping has traditionally been considered a qualitative technique, recent developments demonstrate the quantitative capabilities of X-ray mapping techniques. Moreover, the long-desired ability to collect and store an entire spectrum at every pixel is now a reality, and methods for mining these data are rapidly being developed.

The aim of this study was to examine the suitability of digital image analysis, using the KS400 software system, for the morphometric evaluation of the tissue response after prosthesis implantation in an animal model. Twenty-four female pigs aged 10 weeks were implanted with infrarenal Dacron® prostheses for 14, 21, 28, and 116 days. Following the explantation and investigation of the neointima region, the expression of beta-1-integrin, the proliferation rate by means of Ki-67 positive cells, and the intima thickness were evaluated as exemplary parameters of the tissue response after implantation. Frozen tissue sections were immunohistochemically stained and subsequently examined using computer-aided image analysis. A maximum expression of 32.9% was observed for beta-1-integrin 14 days after implantation, gradually declining over time to 9.8% after 116 days. The proliferation rate was found to be 19% on day 14, increasing to 39% on day 21 with a subsequent gradual decline to 5% after 116 days. The intima thickness increased from 189.9 μm on day 14 to 1228.0 μm on day 116. In conclusion, digital image analysis was found to be an efficient and reproducible method for the morphometric evaluation of a peri-prosthetic tissue response.

A novel approach for nanoscale imaging and characterization of the orientation dependence of electromechanical properties—vector piezoresponse force microscopy (Vector PFM)—is described. The relationship between local electromechanical response, polarization, piezoelectric constants, and crystallographic orientation is analyzed in detail. The image formation mechanism in vector PFM is discussed. Conditions for complete three-dimensional (3D) reconstruction of the electromechanical response vector and evaluation of the piezoelectric constants from PFM data are set forth. The developed approach can be applied to crystallographic orientation imaging in piezoelectric materials with a spatial resolution below 10 nm. Several approaches for data representation in 2D-PFM and 3D-PFM are presented. The potential of vector PFM for molecular orientation imaging in macroscopically disordered piezoelectric polymers and biological systems is discussed.

Editorial Board

Editorial Board Representatives from Affiliated Societies

Founding Editor

Aberration correctors using hexapole fields have proven useful to correct for the spherical aberration in electron microscopy. We investigate the limits of the present design for the hexapole corrector with respect to minimum probe size for the scanning transmission electron microscope and discuss several ways in which the design could be improved by rather small and incremental design changes for the next generation of advanced probe-forming systems equipped with a gun monochromator.

The design of the microvasculature of cerebellum and nontegmental rhombencephalic areas was studied in eight adult Acipenser ruthenus L. by scanning electron microscopy of vascular corrosion casts and three-dimensional morphometry. Gross vascularization was described and diameters and total branching angles of parent and daughter vessels of randomly selected arterial and capillary bifurcations (respectively, venous mergings) were measured. With diameters ranging from 15.9 ± 1.9 μm (cerebellum; mean ± S.D.) to 15.9 ± 1.7 mm (nontegmental rhombencephalon; mean ± S.D.) capillaries in Acipenser were significantly (p ≥ .05) smaller than in cyclostomes (18–20 μm) but significantly thicker than in higher vertebrates and men (6–8 μm). With the exception of the area ratio β (i.e., sum of squared daugther diameters divided by squared diameter of parent vessel) of the venular mergings in the nontegmental rhombencephalon, no significant differences (p ≥ .05) existed between the two brain areas. Data showed that arteriolar and capillary bifurcations and venular mergings are optimally designed in respect to diameters of parent vessel to daughter vessels and to branching (merging) angles. Quantitative data are discussed both in respect to methodical pitfalls and the optimality principles possibly underlying the design of vascular bifurcations/mergings in selected brain areas of a nonteleost primitive actinopterygian fish.

Initial results from an ultrahigh-vacuum (UHV) third-order spherical aberration (Cs) corrector for a dedicated scanning transmission electron microscopy, installed at the National Institute for Materials Science, Tsukuba, Japan, are presented here. The Cs corrector is of the dual hexapole type. It is UHV compatible and was installed on a UHV column. The Ronchigram obtained showed an extension of the sweet spot area, indicating a successful correction of the third-order spherical aberration Cs. The power spectrum of an image demonstrated that the resolution achieved was 0.1 nm. A first trial of the direct measurement of the fifth-order spherical aberration C5 was also attempted on the basis of a Ronchigram fringe measurement.

Performing reflection-mode (backscatter-mode) confocal microscopy on cells growing on reflective substrates gives images that have improved contrast and are more easily interpreted than standard reflection-mode confocal micrographs (Keith et al., 1998). However, a number of factors degrade the quality of images taken with the highest-resolution microscope objectives in this technique. We here describe modifications to reflection-enhanced backscatter confocal microscopy that (partially) overcome these factors. With these modifications of the technique, it is possible to visualize structures the size—and refractility—of individual microtubules in intact cells. Additionally, we demonstrate that this technique, in common with fluorescence techniques such as standing wave widefield fluorescence microscopy and 4-Pi confocal microscopy, offers improved resolution in the Z-direction.

Electron-excited X-ray mapping is a key operational mode of the scanning electron microscope (SEM) equipped with energy dispersive X-ray spectrometry (EDS). The popularity of X-ray mapping persists despite the significant time penalty due to the relatively low output count rates, typically less than 25 kHz, that can be processed with the conventional EDS. The silicon drift detector (SDD) uses the same measurement physics, but modifications to the detector structure permit operation at a factor of 5–10 times higher than conventional EDS for the same resolution. Output count rates as high as 500 kHz can be achieved with 217 eV energy resolution (at MnKα). Such extraordinarily high count rates make possible X-ray mapping through the method of X-ray spectrum imaging, in which a complete spectrum is captured at each pixel of the scan. Useful compositional data can be captured in less than 200 s with a pixel density of 160 × 120. Applications to alloy and rock microstructures, ultrapure materials with rare inclusions, and aggregate particles with complex chemistry illustrate new approaches to characterization made practical by high-speed X-ray mapping with the SDD.Note: The Siegbahn notation for characteristic X-rays is commonly used in the field of electron beam X-ray spectrometry and will be used in this article. The equivalent IUPAC notation is indicated in parentheses at the first use.In this article, the following arbitrary definitions will be used when referring to concentration (C) ranges: major: C > 0.1 (10 wt%), minor: 0.01 ≤ C ≤ 0.1 (1–10 wt%), and trace: C < 0.01 (1 wt%).