With this issue, Microscopy and Microanalysis begins its eleventh year of publication. Our journal is getting better year-by-year. In 2004 this journal published more scientific articles than in any previous year. The first biological special issue, on parasites, increased the prominence of the life sciences within these pages. The growing popularity of the journal is also indicated by several letters to the editor about matters arising in certain articles. Such scientific exchanges highlight for our readership the subtleties of interpretation regarding advances in science, instrumentation, technique, and theory.

To examine new cytochemical aspects of the bacterial adhesion, a strain 41452/01 of the oral commensal Streptococcus sanguis and a wild strain of Staphylococcus aureus were grown with and without sucrose supplementation for 6 days. Osmiumtetraoxyde (OsO4), uranyl acetate (UA), ruthenium red (RR), cupromeronic blue (CB) staining with critical electrolytic concentrations (CECs), and the tannic acid–metal salt technique (TAMST) were applied for electron microscopy. Cytochemically, only RR-positive fimbriae in S. sanguis were visualized. By contrast, some types of fimbriae staining were observed in S. aureus glycocalyx: RR-positive, OsO4-positive, tannophilic and CB-positive with ceasing point at 0.3 M MgCl2. The CB staining with CEC, used for the first time for visualization of glycoproteins of bacterial glycocalyx, also reveals intacellular CB-positive substances—probably the monomeric molecules, that is, subunits forming the fimbriae via extracellular assembly. Thus, glycosylated components of the biofilm matrix can be reliably related to single cells. The visualization of intracellular components by CB with CEC enables clear distinction between S. aureus and other bacteria, which do not produce CB-positive substances. The small quantities of tannophilic substances found in S. aureus makes the use of TAMST for the same purpose difficult. The present work protocol enables, for the first time, a partial cytochemical differentiation of the bacterial glycocalyx.

The use of multifocal-plane, time-lapse recordings of living specimens has allowed investigators to visualize dynamic events both within ensembles of cells and individual cells. Recordings of such four-dimensional (4D) data from digital optical sectioning microscopy produce very large data sets. We describe a wavelet-based data compression algorithm that capitalizes on the inherent redunancies within multidimensional data to achieve higher compression levels than can be obtained from single images. The algorithm will permit remote users to roam through large 4D data sets using communication channels of modest bandwidth at high speed. This will allow animation to be used as a powerful aid to visualizing dynamic changes in three-dimensional structures.

Historically, the decapod crustacean circulatory system has been classed as open. However, recent work on the blue crab, Callinectes sapidus, suggests the circulatory system may be more complex than previously described. Corrosion casting techniques were refined and used to map the circulatory system of a variety of crab species (order: Decapoda; family: Cancridae) to determine if the complexity observed in the blue crab was present in other species. Seven arteries arose from the single chambered heart. The anterior aorta, the paired anterolateral arteries, and the paired hepatic arteries exited from the anterior aspect of the heart. The small-diameter posterior aorta exited posteriorly from the heart. Exiting from the ventral surface of the heart, the sternal artery branched to supply the legs and mouthparts of the crab. These arteries were more complex than previously described, with arterioles perfusing all areas of the body. The arterioles split into fine capillary-like vessels. Most of these capillaries were blind ending. However, in several areas (antennal gland, supraesophageal ganglion) complete capillary beds were present. After passing through the capillary-like vessels, blood drained into a series of sinuses. However, rather than being arbitrary spaces as previously described, scanning electron micrographs showed the sinuses to be distinct units. Most of the sinuses formed a series of flattened membrane-bound lacunae. This complexity may qualify the decapod crustacean circulatory system as one that is “partially closed” rather than open.

Electron microscopy has a pivotal role as an analytical tool in pharmaceutical research. However, digital image data have proven to be too large for efficient quantitative analysis. We describe here the development and application of an automated image processing (AIP) program that rapidly quantifies shape measurements of recombinant adenovirus (rAd) obtained from digitized field emission scanning electron microscope (FESEM) images. The program was written using the macro-recording features within Image-Pro® Plus software. The macro program, which is linked to a Microsoft Excel spreadsheet, consists of a series of subroutines designed to automatically measure rAd vector objects from the FESEM images. The application and utility of this macro program has enabled us to rapidly and efficiently analyze very large data sets of rAd samples while minimizing operator time.

Osmium postfixation is established as a routine procedure for transmission electron microscopy (TEM). On the one hand, this routine procedure leads to good results for TEM, but on the other hand results in blackened tissue samples that do not allow examination of any structures within the embedded tissue sample by a light microscope. Equivalent fixation results for TEM are achieved with imidazole–osmium postfixation, and with this postfixation method tissue is not blackened and can be transilluminated with point light sources. This allows easier recognition of histological details within tissue samples and makes it possible to screen embedded samples for appropriate ultrastructural processing. Jejunum is used to demonstrate the method.

The microstructure of carbon–carbon composites obtained by chemical vapor infiltration of a carbon fiber felt was comparatively studied by reflection light microscopy, transmission electron microscopy (TEM), scanning electron microscopy (SEM), atomic force microscopy (AFM), and laser scanning confocal microscopy (LSCM). Ar+ ion etching was used to reveal and distinguish structural units of the pyrolytic carbon matrix. Mechanically polished samples, polished and subsequently ion etched samples, and fractured samples were compared. The values of surface roughness and surface height after polishing or after polishing and subsequent etching determined by AFM and LSCM correlate well with the degree of texture of the matrix layers obtained by polarized light microscopy and selected area electron diffraction. The carbon matrix is composed of structural units or “cells,” which contain a carbon fiber and a sequence of several differently textured layers around each fiber. Within high-textured layers columnar grains are well recognizable using polarized reflection light microscopy and confocal microscopy. The size of depressions within high-textured carbon layers found by AFM after ion etching correlates well with the size of differently tilted domains detected by both TEM and SEM.

This work demonstrates the possibility of using the Duane–Hunt limit of the bremsstrahlung to determine E2 values of Si3N4 and AlN ceramics. The EDHL versus E0 graph demonstrates that for conductive materials, the experimental curve is parallel to the theoretical (EDHL = E0), but both curves cross in the case of insulators. The intersection points (E2 value), are 3.01 keV for Si3N4 and 2.67 keV for AlN. Imaging of ceramic grain structure at high magnification was performed to demonstrate the validity of the calculated E2 values.

Off-axis electron holography is used to measure electrostatic potential profiles across a silicon p-n junction, which has been prepared for examination in the transmission electron microscope (TEM) in two different specimen geometries using focused ion beam (FIB) milling. Results are obtained both from a conventional unbiased FIB-milled sample and using a novel sample geometry that allows a reverse bias to be applied to an FIB-milled sample in situ in the TEM. Computer simulations are fitted to the results to assess the effect of TEM specimen preparation on the charge density and the electrostatic potential in the thin sample.

Electron back scatter diffraction (EBSD) mapping and indexing has rapidly come into widespread use. However, inadequate attention has been paid to the details of the method. Many of the algorithms in current use were chosen because they were the first ones that were found to work, rather than because they were optimum. Results of systematic study are presented. It is shown that more than one method can successfully correct a sampling artifact, that there is an optimum binning ratio, that Gaussian filtering provides an alternative to “butterfly convolution,” that better alternatives for mapping image quality than those in current use are available, and that saving all the original patterns is practical and advantageous.

Principal components analysis (PCA) factor filtering is implemented for the improvement of background removal in noisy spectra. When PCA is used as a method for filtering before background removal in electron energy loss spectroscopy elemental maps, an improvement in the accuracy of the background fit with very short fitting intervals is achieved, leading to improved quality of elemental maps from noisy spectra. This opens the possibility to use shorter exposure times for elemental mapping, leading to fewer problems with, for example, drift and beam damage.

A conventional scanning electron microscope equipped with a LaB6 source has been modified to operate in a scanning transmission mode. Two detection strategies have been considered, one based on the direct collection of transmitted electrons, the other on the collection of secondary electrons resulting from the conversion of the transmitted ones. Two types of specimens have been mainly investigated: semiconductor multilayers and dopant profiles in As-implanted Si. The results show that the contrast obeys the rules of mass–thickness contrast whereas the resolution is always defined by the probe size independently of specimen thickness and beam broadening. The detection strategy may affect the bright field (light regions look brighter) or dark field (heavy regions look brighter) appearance of the image. Using a direct collection of the transmitted electrons, the contrast can be deduced from the angular distribution of transmitted electrons and their collection angles. When collecting the secondary electrons to explain the image contrast, it is also necessary to take into account the secondary yield dependence on the incidence angle of the transmitted electrons.

Following is a list of microscopy-related meetings and courses. The editors would greatly appreciate input to this list via the electronic submission form found in the MSA World-Wide Web page at http://www.msa.microscopy.com. We will gladly add hypertext links to the notice on the web and insert a listing of the meeting in the next issue of the Journal. Send comments and questions to JoAn Hudson, [email protected] or Nestor Zaluzec, [email protected].