Acrylic resin mixtures are commonly used to study microscopic sections of biological specimens, giving the advantage of good morphological preservation. Existing embedding protocols, however, are... Show moreAcrylic resin mixtures are commonly used to study microscopic sections of biological specimens, giving the advantage of good morphological preservation. Existing embedding protocols, however, are suitable for tissue blocks, not exceeding 1 mm in thickness. We have developed a protocol to embed larger specimens (up to 2 cm 3) in Technovit 8100. This medium allowed us to perform classic histological (trichrome), silver, as well as immunohistochemical staining, needed for multi-signal detection at high-resolution imaging to reconstruct a three-dimensional interpretation of a serially sectioned muscle. The technique was applied to reconstruct the semitendinosus muscle of a fetal pig, 44 days post conception, featuring connective tissue, intramuscular nerves, blood vessels, and muscle fibre types. For the reconstruction, a technique was used that enabled us to insert high-resolution images of histological details into low-resolution images of the entire muscle. (c) 2005 Wiley-Liss, Inc. Show less
Nowadays, the development of experimental procedures for the determination of the secondary structure of RNA molecules is taking advantage of the novel single-molecule probing and imaging... Show moreNowadays, the development of experimental procedures for the determination of the secondary structure of RNA molecules is taking advantage of the novel single-molecule probing and imaging techniques. We report a method for the mapping of the secondary structure of RNA molecules spread on a flat surface by means of the atomic force microscope. Globular domains comprising groups of RNA secondary and tertiary structure elements separated by unstructured domains can be discerned in the micrographs and their position along the molecule contour can be measured directly on unstained specimens. We have analyzed the morphology of a population of single molecules of 3' fragments of the Turnip Yellow Mosaic Virus RNA shorter than 1 kb in different temperature and electrolytic conditions. We found a satisfying agreement of the shape of the imaged structures with previously available evidence. The method we have developed can be used to map also different types of RNA molecules and has the advantage of showing the distribution of the single molecule conformations within the population. (C) 2005 Wiley-Liss, Inc. Show less
This paper describes a structured approach for a standard setup of a computer program for 3D reconstruction from serial sections. Three-dimensional reconstruction as a technique increases in... Show moreThis paper describes a structured approach for a standard setup of a computer program for 3D reconstruction from serial sections. Three-dimensional reconstruction as a technique increases in importance as, along with modern immunohistochemical techniques, it is a tool in the understanding of three-dimensional development patterns. In order to apply 3D reconstruction technique in a standard laboratory setup, an attempt was made to streamline the input and the manipulation of the data such that results are obtained easily. One will find a combination of two approaches in this paper: the first is a strict ordering of the complex data, and the second is an ordering of the processes that one wishes to apply on the data (together, these two approaches constitute an information analysis); because it was observed that developmental biologists tend to work from simple lines to describe their observations, the contour model was chosen as the vehicle to build a reconstruction model from. Consequently, the data is ordered in a database that has to be manipulated to get the data out in the desired format. The most important output format is a display of the reconstructed contour stack on a graphical computer screen. Together with the other data manipulation processes, such as the input, the inspection, the revision (correction), and the reconstruction, all processes are described using the reconstruction of an 11 embryonic days (ED) rat embryo as an example. Finally, the merits of the program are illustrated with an example from the development of the human embryonic heart. (C) 1995 Wiley-Liss, Inc. Show less
