Cryo-high-resolution scanning electron microscopy was used to analyze the conformation of fibronectin fibrils formed in human skin fibroblast cultures or in a cell-free system by treating soluble plasma fibronectin with guanidine. Structurally, fibrils assembled in the cell-free system and in culture were similar. Assembly of both fibrillar networks involves interactions with the III1 and amino terminal repeats of fibronectin; their conformations consist of either smooth surfaces or patches of smooth surfaces and nodules randomly spaced along the fibril. The random distribution of these two conformations in fibrils indicates that fibronectin fibrils are capable of undergoing localized conformational changes. The nodules may be discrete domains of 3 to 4 type III repeats, as they can be labeled with the monoclonal antibody IST-2 to the III13-14 repeats in fibronectin and are found in 160 kDa and 85 kDa fragments of fibronectin that only contain type III repeats. In our study, smooth regions of fibrils were never recognized by the IST-2 antibody, suggesting that the epitope in the III13-14 repeats is masked in these regions. These results demonstrate that fibronectin fibrils are flexible and certain epitopes of fibronectin may be buried, or exposed, depending on the conformation of the fibril. They also show that fibrils assembled in cell-free conditions can be a powerful tool for studying fibril formation.

The examination of archival pathology specimens of human small intestine by light microscopy, field emission scanning electron microscopy (FESEM), and confocal scanning laser microscopy using fluorescent in situ hybridization (FISH) techniques was undertaken to better understand the epidemiology of Giardia. Giardia trophozoites were tentatively identified in the light microscope after hematoxylin and eosin (H&E) staining. The organisms were adherent to the intestinal epithelium where they were also associated with strands of mucus within the lumen. Fluorochrome-conjugated antisense oligonucleotide probes, developed for the 16S rRNA of Giardia lamblia and Giardia muris, were used in FISH experiments with confocal scanning laser microscopy. Positive identification of trophozoites could be obtained with the G. lamblia probe, but not with the G. muris probe. FESEM examination of serial sections adjacent to FISH-stained sections revealed trophozoites characterized by their morphological features. The 16S rDNA probes specifically distinguished different species of Giardia, but whether multiple infections can occur within an individual host could not be determined.

Sea urchin embryos have served as a model system for the investigation of many concepts in developmental biology. Their gastrulation consists of two stages; primary invagination, where part of the epithelium invaginates into the blastocoel, and secondary invagination, where the archenteron elongates to completely traverse the blastocoel. Primary invagination involves proliferation of cells within the vegetal plate during primary invagination, but until recently, it was assumed that the larval gastrointestinal (GI) tract developed without further involution of epithelial cells. To investigate rigorously the contribution of epithelial cell involution during archenteron and GI tract development in the sea urchin, Lytechinus variegatus, we developed a new method for cell tracking based on two-photon excited photorelease of caged fluorophores. Single-cell embryos were injected with caged dye and two-photon excitation uncaging was employed to mark small groups of cells throughout gastrulation. Two-photon excitation allowed for noninvasive, three-dimensionally resolved uncaging inside living cells with minimal biological damage. Cellular involution into the archenteron was observed throughout primary and secondary invagination, and the larval intestine was formed by further involution of cells following secondary invagination, which is inconsistent with the traditional model of sea urchin gastrulation. Further, as two-photon excitation microscopy becomes accessible to many researchers, the novel techniques described here will be broadly applicable to development of other invertebrate and vertebrate embryos.

In this report, we describe the implementation of an angle-gating mechanism into a reflection optical microscope for imaging through a turbid medium. A significant improvement in image resolution and contrast is obtained when a polarizing annular objective is used to image a reflective test bar through various turbid media. The experimental results confirm that the improvement is caused by the efficient suppression of diffused photons scattered from the turbid medium when a polarized annular objective is employed as an angular gate.

Three-dimensional (3D) grain sizes and size distribution functions (SDFs) are generally obtained by converting the 2D or ID grain sizes to 3D with the methods based on a spherical grain shape model (S-model), which can produce large systematic errors. In this report, a new polyhedral grain model (P-model) is developed and experimentally checked with the directly measured 3D grain sizes. The results show that the systematic error originating from the S-model can be corrected by using the new polyhedral grain model.

We have observed that a slight crystal tilt away from the zone axis paradoxically decreases the dynamical extinction length, as if the electron-crystal interaction increases. This effect disappears at larger tilt angles. Using the simplified channeling theory, this behavior is explained.

Electron microscopy recently lost one of its outstanding pioneers when John Reisner passed away on June 18 of this year. We who have known and respected John, pause here to remind ourselves of his many contributions to our field and to visualize for later generations the times and social forces that brought forth such a remarkable human being. He was remarkable because of his courageous and skillful management of problems arising from a severe case of poliomyelitis. He was remarkable because of his family's determination to help him reach his full potential. He did in fact lead American development of the electron microscope at RCA from 1950 to 1970 and served (E)MSA in several roles from 1950 on until very near the end of his life, including as President of the Society.

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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 Barbara Reine, [email protected] or Nestor Zaluzec, [email protected].