This special issue of Microscopy and Microanalysis explores quantitative electron diffraction from nonbiological materials. Jim Turner and I have put many hours of work into bringing it together, and we thank the authors for their fine contributions. The articles cover a wide range of materials and techniques, from convergent-beam electron diffraction (CBED) to the new Kohler SAD mode, as well as the use of direct methods, the study of diffuse elastic scattering from defects, strain measurement, and multiwavelength methods. We were sorry that we could not obtain recent work using the precession electron diffraction camera by our deadline, but readers should be aware of that promising method also.

Microscopy and Microanalysis has made significant strides forward over the past year, and I would like to comment on two of these. First, the Institute for Scientific Information (ISI) ranked this journal third among the nine microscopy journals it indexes. The ranking was in terms of ISI's Impact Factor, which tracks the number of citations to papers published in the journal. A strong Impact Factor indicates that information in the journal is of interest to other workers in the field. Second, the National Library of Medicine (NLM) has selected Microscopy and Microanalysis to be indexed in MEDLINE (PubMed), beginning with the first issue of 2003. As any biologist will tell you, this listing is essential for the electronic visibility of papers in the fast-moving world of life sciences research. I thank Editorial Board member Dave Piston for his efforts in writing the initial letter of application to the NLM.

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Over the years, I have read with great interest several articles and book chapters about historical aspects of electron microscopy. The report by F. Haguenau et al. about the key events in the history of electron microscopy (Microscopy and Microanalysis, Vol. 9, No. 2, April 2003, pp. 96–138) is, to my knowledge, by far the most comprehensive article ever published. The authors did a superb job in first listing chronologically the development of electron optics and instrumentation for the years from 1897 to 2002, and then describing major applications in physics and materials science, as well as life sciences.

Elastin-mimetic block copolymers were produced by genetic engineering. Genetically driven synthesis permitted control of the final physiochemical characteristics of the block copolymers. We designed BB and BAB block copolymers in which the A-block was hydrophilic and the B-block was hydrophobic. By designing the copolymers in this manner, it was proposed that they would self-assemble into micellar aggregates that, at high concentration, would form thermoreversible hydrogels. To analyze the three-dimensional fine surface morphology of the copolymers, to the resolution level of a few nanometers, we employed cryo-HRSEM. This method provided vast expanses of the specimen in its frozen hydrated state for survey. In our initial cryo-HRSEM studies, we observed the protein filaments and micelles surrounded by lakes of vitreous ice. Upon examination at low and intermediate magnifications, there was an extensive honeycomb-like filamentous network. To delineate the fine morphology of the hydrogel network at high magnification and to greater depths, we cryoetched away unbound water from the sample surface, in high vacuum, prior to chromium deposition. By using this technique, we were able to visualize for characterization purposes the fine fibril networks formed from the micellar aggregates over the surface of the hydrogel.

Spectral imaging in the scanning electron microscope (SEM) equipped with an energy-dispersive X-ray (EDX) analyzer has the potential to be a powerful tool for chemical phase identification, but the large data sets have, in the past, proved too large to efficiently analyze. In the present work, we describe the application of a new automated, unbiased, multivariate statistical analysis technique to very large X-ray spectral image data sets. The method, based in part on principal components analysis, returns physically accurate (all positive) component spectra and images in a few minutes on a standard personal computer. The efficacy of the technique for microanalysis is illustrated by the analysis of complex multi-phase materials, particulates, a diffusion couple, and a single-pixel-detection problem.

The electron backscatter coefficient (η) and the related correction factor for X-ray intensity (R) are both strongly dependent on atomic number and although quite good data are available for pure elements, the derivation of values for compounds is problematic. This issue is addressed by Donovan, Pingitore, and Westphal (Microscopy and Microanalysis, Vol. 9, No. 3, June 2003, pp. 202–215), in which an “electron fraction” averaging method is advocated as an improvement on “traditional” mass fraction averaging, which is known to be only an approximation. (The difference, according to Table 3, is only significant, however, for samples containing heavy elements such as Pb and Th.) New thinking on this topic is welcome, but I believe this proposal should be treated with caution pending more rigorous testing.

Accurate low-order structure factors for copper metal have been measured by quantitative convergent beam electron diffraction (QCBED). The standard deviation of the measured structure factors is equal to or smaller than the most accurate measurement by any other method, including X-ray single crystal Pendellösung, Bragg γ-ray diffraction, and high-energy electron diffraction. The electron structure factor for the (440) reflection was used to determine the Debye-Waller (DW) factor. The local heating of the specimen by the electron beam is determined to be 5 K under the current illumination conditions. The low-order structure factors for copper measured by different methods are compared and discussed. The new data set is used to test band theory and to obtain a charge density map. The charge deformation map shows a charge surplus between the atoms and agrees fairly well with the simple model of copper 2+ ions at the atomic sites in a sea of free uniformly distributed electrons.

What is cryogenic high resolution (in-lens) scanning electron microscopy (HRSEM), and do I need it? Structural cell biologists and bioorganic chemists will find this newly developed imaging mode to be an accurate and useful research tool for molecular level investigations in the hydrated state.

The environmental cell device discussed in this paper provides a modest low-vacuum scanning electron microscopy (SEM) capability to a standard SEM without requiring additional pumping. This environmental cell confines a volume of low vacuum in contact with the sample surface using a container that has an aperture for admitting the primary electron beam. The aperture is large enough to permit a limited field of view of the sample, and small enough to limit the outflow of gas into the SEM chamber to that which can be accommodated by the standard SEM pumping system. This environmental cell also functions as a gaseous detector device.

It is not easy to understand how the electron microscopes and electron microscope techniques that we know today developed from the primitive ideas of the first microscopists of the 1930s. Newcomers to the subject in particular, their time almost fully occupied with grasping practical methods and modern computing techniques, can rarely devote much attention to the history of their subject. For some, however, this is a source of frustration: If a guide to the principal stages in the development of the subject and to the main actors and their publications were available, they would find the time to study it.

Abnormal cilia were demonstrated in the lining epithelial cells of three cases of intraspinal endodermal (bronchogenic) cyst. The changes comprised a wide spectrum of ultrastructural abnormalities, including (a) cilia with abnormal axonemal microtubules, (b) swollen cilia, (c) compound cilia with or without excessive ciliary matrix, (d) naked cilia without limiting membrane, and (e) intracytoplasmic cilia and aggregates of microtubules. Of these, compound cilia and swollen cilia were most common. Cilia with dynein arm deficiency were not observed. Ciliary abnormalities found in the present study were very similar to those described in the bronchial epithelium of various diseases. The present findings suggest that the lining epithelium of intraspinal endodermal cyst shares similar ciliogenesis and susceptibility to abnormal ciliary formation as that of the bronchial epithelium.

High-resolution transmission electron microscopy (HR-TEM) has been used as the ultimate method of thickness measurement for thin films. The appearance of phase contrast interference patterns in HR-TEM images has long been confused as the appearance of a crystal lattice by nonspecialists. Relatively easy to interpret crystal lattice images are now directly observed with the introduction of annular dark-field detectors for scanning TEM (STEM). With the recent development of reliable lattice image processing software that creates crystal structure images from phase contrast data, HR-TEM can also provide crystal lattice images. The resolution of both methods has been steadily improved reaching now into the sub-Ångstrom region. Improvements in electron lens and image analysis software are increasing the spatial resolution of both methods. Optimum resolution for STEM requires that the probe beam be highly localized. In STEM, beam localization is enhanced by selection of the correct aperture. When STEM measurement is done using a highly localized probe beam, HR-TEM and STEM measurement of the thickness of silicon oxynitride films agree within experimental error. In this article, the optimum conditions for HR-TEM and STEM measurement are discussed along with a method for repeatable film thickness determination. The impact of sample thickness is also discussed. The key result in this article is the proposal of a reproducible method for film thickness determination.

We employed multiphoton laser scanning microscopy (MPLSM) to image changes in mitochondrial distribution in living rhesus monkey embryos. This method of imaging does not impair development; thus, the same specimen can be visualized multiple times at various developmental stages. Not only does this increase the amount of information that can be gathered on a single specimen but it permits the correlation of early events with subsequent development in the same specimen. Here we demonstrate the utility of MPLSM for determining changes in mitochondrial organization at various developmental stages and show that rhesus zygotes possess a distinct accumulation of mitochondria between the pronuclei prior to syngamy. We present evidence that suggests that this pronuclear accumulation may be positively correlated with development to the blastocyst stage—in the same embryo—thereby illustrating how MPLSM can be used to correlate cellular dynamics of primate oocytes and early embryos with their developmental potential. Understanding the relationship between mitochondrial distribution and the subsequent development of mammalian embryos, particularly primates, will increase our ability to improve embryo culture technologies, including those used for human assisted reproduction.

Optical Imaging Association Members

A new single-tilt technique for performing TEM stereomicroscopy of strain fields in crystalline materials has been developed. The technique is a weak beam technique that involves changing the value of g and/or sg while tilting across a set of Kikuchi bands. The primary benefit of the technique is it can be used with single-tilt TEM specimen holders including many specialty holders such as in situ straining, heating, and cooling holders. Standard stereo-TEM techniques are almost always limited to holders allowing two degrees of rotational freedom (i.e., double-tilt or tilt/rotation holders). An additional benefit of the new technique is that it eliminates the need to focus with the specimen height control. These advantages make it useful for stereo viewing or for quantitative stereomicroscopy provided necessary consideration is given to errors that may result from the technique.