The use of chromic potassium sulphate in bone electron microscopy

The ultrastructure of endochondral bone was studied using an aqueous solution of chromic potassium sulphate as the decalcifying agent. 0.5 mm thick sections of rat tibiae were fixed in buffered glutaraldehyde, immersed in an aqueous solution of 1% chromic potassium sulphate pH 3.4, dehydrated and embedded in Poly Bed 812 without exposure to osmium tetroxide. In unstained sections we observed clusters of crystal like structures throughout the osteoid and calcifying cartilage matrix as well as solitary needle shaped structures in association with collagen fibrils. Stained sections revealed nuclei, endoplasmic reticulum, membrane limited dense granules, mitochondrial particles and other cell components typical of bone cells. It appeared that the chromic potassium sulphate method preserves the relationship between hard and soft tissues well, gives fine cytological detail and produces images of intracellular and extracellular deposits identical to untreated crystallites. It is concluded that the chromic potassium sulphate method is indicated for ultrastructural studies of bone.     

The use of markers for correlative light electron microscopy

Bioimaging: the visualisation, localisation and tracking of movement of specific molecules in cells using microscopy has become an increasing field of interest within life science research. For this, the availability of fluorescent and electron-dense markers for light and electron microscopy, respectively, is an essential tool to attach to the molecules of interest. In recent years, there has been an increasing effort to combine light and electron microscopy in a single experiment. Such correlative light electron microscopy (CLEM) experiments thus rely on using markers that are both fluorescent and electron dense. Unfortunately, there are very few markers that possess both these properties. Markers for light microscopy such as green fluorescent protein are generally not directly visible in the electron microscopy and vice versa for gold particles. Hence, there has been an intensive search for markers that are directly visible both in the light microscope and in the electron microscope. Here we discuss some of the strategies and pitfalls that are associated with the use of CLEM markers, which might serve as a “warning” that new probes should be extensively tested before use. We focus on the use of CLEM markers for the study of intracellular transport and specifically endocytosis.     

The use of pronase for electron microscopy of protein-complexed DNA

A method is described which demonstrates the possibility of visualizing protein-complexed DNA by using pronase for both deproteinization and spreading of DNA for electron microscopy. The results show that proteolytic digestion is complete even under conditions of short formaldehyde fixation and pronase is an excellent substitute for cytochromec for spreading of DNA. The pronase method is successfully applied to virions, native and partially denatured chromatin. The procedure is highly advantageous because it does not require preliminary isolation of DNA, can be applied to microamounts of material and permits the visualization of partial denaturation of DNA in chromatin.     

Analysis of fossil bone organic matrix by transmission electron microscopy

A transmission electron microscope (TEM) study was initiated on samples of geological ages ranging from Devonian to Jurassic to analyse the ultrastructure of the organic matrix in fossil bones that have preserved a histological structure after demineralisation. All samples show a network of variably well-preserved fibrils. Within the sampling, the best results were obtained in two specimens: the scales of the Devonian sarcopterygian tetrapodomorph Eustenopteron foordi, and the humerus of Jurassic dinosaur Lappentosaurus madagascariensis. Despite an extended time difference between both specimens, their fossil bone is composed of a plywood-like structure in which the fibrils are very closely packed. These observations support the hypothesis that dense initial packing of collagen fibrils favours the preservation of the fossil bone.     

Selective use of electron microscopy in fine needle aspiration cytology

The utility of electron microscopy (EM) applied to fine needle aspiration (FNA) biopsy specimens was analyzed in order to determine the role and the diagnostic contribution of the EM examination. A rapid stain (Diff-Quik) was used to obtain a preliminary diagnostic impression and to assure the adequacy of the EM specimen for problematic cases. Our experience suggests that EM is being relied upon with greater frequency in the study of FNA specimens because it is an accurate and cost-effective diagnostic procedure. The use of a rapid interpretation (Diff-Quik stain) enhances the quality of the EM specimen and, as in surgical pathology, the EM examination increases the accuracy and specificity of the FNA biopsy diagnosis.     

The use of dyes in modern biomedicine

The discovery of the aniline dyes in the 19th century and contemporary investigation of their use as biological stains by scientists such as Koch and Ehrlich led to the idea of selectivity and formed the basis of modern chemotherapy; several of these dyes remain in pharmacopoeias. While the development of therapeutics has tended to avoid colored compounds due to unwanted coloration, the modern application of photosensitizing dyes, both in the fields of cancer therapy and anti-infection, depends on this phenomenon. In addition, the fluorescence of some anticancer photosensitizers allows their use as tumor localizing agents, which is particularly useful in precancerous conditions. It is also fitting that dyes employed in Ehrlich's original studies, such as the phenothiazinium dye, methylene blue, are now in clinical use for disinfecting donated blood products.     

The use of dyes in protein purification.

The role of the matrix, ligand and linking mechanism in affinity chromatography is discussed, special emphasis being placed on the use of dyestuff molecules as ligands. Current knowledge of dye-protein interactions is outlined and problems arising from the use of conventional textile dyes as ligands are considered. Work on the synthesis of novel dye-like molecules designed specifically for affinity chromatography is reviewed. This is seen as leading to the development of improved affinity systems capable of advancing the utility of affinity chromatography in protein purification.     

The use of dyes in modern biomedicine.

The discovery of the aniline dyes in the 19th century and contemporary investigation of their use as biological stains by scientists such as Koch and Ehrlich led to the idea of selectivity and formed the basis of modern chemotherapy; several of these dyes remain in pharmacopoeias. While the development of therapeutics has tended to avoid colored compounds due to unwanted coloration, the modern application of photosensitizing dyes, both in the fields of cancer therapy and anti-infection, depends on this phenomenon. In addition, the fluorescence of some anticancer photosensitizers allows their use as tumor localizing agents, which is particularly useful in precancerous conditions. It is also fitting that dyes employed in Ehrlich's original studies, such as the phenothiazinium dye, methylene blue, are now in clinical use for disinfecting donated blood products.     

The electron microscopy of vaccinia-diseased tissues

Summary Many hundreds of electron micrographs have been made on a corresponding number of sections through chicken embryo tissues diseased with vaccine virus. The fully developed elementary bodies of this virus are easily recognized in such sections. Search has been made for evidence of proliferation in chorioallantoic membranes infected for various lengths of time. This has given evidence bearing on several hypotheses that can be offered concerning the growth of the virus particles; but it does not select in satisfactory fashion between them.