We have used a two-step casting procedure to create replicas of fibrillar collagen. A negative replica or mold is first created using either Mercox casting resin or a prepolymerized methacrylate resin that was originally described by Murakami. In the second step, a positive replica is made from the first using a solution of polystyrene or polyurethane to coat the methacrylate cast. The resulting replica is removed from the methacrylate cast. Results indicate the highest level of spatial resolution is obtained using a modified Murakami resin for the negative cast and polystyrene for the positive replica. The 29-nm-wide repeat banding structure present on collagen fibrils could be clearly identified in both the methacrylate cast and in the polystyrene positive replica of the methacrylate cast. Thus high-resolution replicas can be obtained. Such replicas may be useful to imprint inplantable devices with biologically relevant textures to improve tissue integration or for the development of textured surfaces for the in vitro expansion of cultured cells.

This study provides a quantitative validation of qualitative automated three-dimensional (3-D) analysis methods reported earlier. It demonstrates the applicability and quantitative accuracy of our method to detect, characterize, and count Feulgen stained cell nuclei in two tissues (hippocampus and testes). A laser-scanned confocal light microscope was used to record 3-D images i which our algorithms automatically identified individual nuclei from the optical sections given an estimate of minimum nuclear size. The hippocampal data sets were also manually counted independently by five trained observers using the STERECON 3-D image reconstruction system. The automated and manual counts were compared. A nucleus-by-nucleus comparison of the manual and automated counts verified that the automated analysis was accurate and reproducible, and permitted additional quantitative analyses not available from manual methods. The algorithms also identified subpopulations of nuclei within the hippocampal samples, and haploid and diploid nuclei in the testes. Our methods were shown to be repeatable, accurate, and more consistent than manual counting.

This study provides a quantitative validation of qualitative automated three-dimensional (3-D) analysis methods reported earlier. It demonstrates the applicability and quantitative accuracy of our method to detect, characterize, and count Feulgen stained cell nuclei in two tissues (hippocampus and testes). A laser-scanned confocal light microscope was used to record 3-D images i which our algorithms automatically identified individual nuclei from the optical sections given an estimate of minimum nuclear size. The hippocampal data sets were also manually counted independently by five trained observers using the STERECON 3-D image reconstruction system. The automated and manual counts were compared. A nucleus-by-nucleus comparison of the manual and automated counts verified that the automated analysis was accurate and reproducible, and permitted additional quantitative analyses not available from manual methods. The algorithms also identified subpopulations of nuclei within the hippocampal samples, and haploid and diploid nuclei in the testes. Our methods were shown to be repeatable, accurate, and more consistent than manual counting.

The effectiveness of applying a high-frequency, low-energy, reactive gas plasma for the removal of hydrocarbon contamination from specimens and components for electron microscopy has been investigated with a variety of analytical techniques. Transmission electron microscopy (TEM) analysis of specimens that have been plasma cleaned shows an elimination of the carbonaceous contamination from the specimen. With extended cleaning times the removal of existing carbon contamination debris due to previously conducted microanalysis is shown. Following plasma cleaning, specimens may be examined in the electron microscope for several hours without exhibiting evidence of recontamination. The effectiveness of plasma cleaning is not limited to applications for TEM specimens. Scanning electron microscopy (SEM) specimens that have been plasma cleaned likewise show an elimination of carbonaceous contamination. Furthermore, other electron microscopy parts and accessories, such as aperture strips, specimen clamping rings, and Wehnelts, among others, can benefit from plasma cleaning.

We report here a specific type of edge strength anisotropy observed in field emission scanning electron microscope (FESEM) images. The images show weaker edge gradients in the scanning direction and hence these edges frequently go undetected. Direct application of edge detection algorithms to images with nondistinct edges, such as powder particles, show strong bias to edges perpendicular to the scanning direction. Edge orientation polarograms obtained from these images always show strong fictitious particle orientation in the scanning direction. In this work, we discuss an edge-sharpening algorithm that corrects for this bias and results in relatively more accurate and consistent edge orientation information.

In June of 1995, the first conference on Fluorescent Microscopy and Fluorescent Probes was held in the beautiful city of Prague in the Czech Republic and the proceedings of that meeting were published by Plenum Press in 1996 (Fluorescence Microscopy and Fluorescent Probes, Vol. 1, edited by Jan Slavík). Based on the success of the first conference, a second conference was held two years later again in Prague, and this book is the proceedings of that meeting.