Electron dense granules and the role of buffers: artefacts from fixation with glutaraldehyde and osmium tetroxide

Electron dense granules may appear in tissues after glutaraldehyde prefixation and osmium tetroxide postfixation. In order to determine the conditions under which the granules are formed various vehicles in pre- and post-fixatives were tested on lymph node, thymus and heart. If granules appeared they were found in all cell types investigated, particularly in the nuclei. There was no difference in the distribution of the granules in the different compartments of these organs. The granules probably consist of complexes of glutaraldehyde, osmium and phosphate. The ultimate phosphate concentration in the tissues during the postfixation was shown to determine whether or not the artefacts appeared. Local conditions in the tissues also contributed to the appearance of the granules. It is concluded that phosphate buffers can be used in the double fixation procedure, but to avoid the granules in lymph node, thymus and heart, a concentration of 0·1 mol/l or less phosphate should be used. For brain and kidney other conditions apply.     

A simple and rapid scanning electron microscope preparative technique for delicate "gymnodinioid" dinoflagellates

Light microscopy (LM) is routinely used to investigate delicate (unarmoured and lightly armoured) "gymnodinioid" dinoflagellate species but at this level of resolution, morphological features such as apical grooves, apical pores, thin thecal plates, and scales are often difficult to observe, thereby necessitating the use of scanning electron microscopy (SEM). Good results were obtained when harvested cells were fixed with osmium tetroxide (OsO(4)) as the primary fixative, adhered with poly-L-lysine to round glass coverslips, dehydrated in an ethanol series, and dried with hexamethyldisilazane (HMDS). Poly-L-lysine has in the past effectively been used to adhere biological material such as human red blood cells, mouse leukemic cells, and marine dinoflagellates to glass coverslips. HMDS has been used to substitute critical point drying (CPD) to dry soft insect tissues, rat hepatic endothelial cells, and the cilia of rat trachea. By combining and fine-tuning these two protocols in SEM studies of delicate "gymnodinioid" dinoflagellates, it is possible to overcome cell distortion such as shrinking and collapsing that result from centrifuging, filtering, and CPD. The combination of poly-L-lysine and HMDS not only produces good results but also requires limited expertise and equipment, is inexpensive, and is less time-consuming.     

2D and 3D immunogold localization on (epoxy) ultrathin sections with and without osmium tetroxide

For nearly 50 years immunogold labeling on ultrathin sections has been successfully used for protein localization in laboratories worldwide. In theory and in practice, this method has undergone continual improvement over time. In this study, we carefully analyzed circulating protocols for postembedding labeling to find out if they are still valid under modern laboratory conditions, and in addition, we tested unconventional protocols. For this, we investigated immunolabeling of Epon‐embedded cells, immunolabeling of cells treated with osmium, and the binding behavior of differently sized gold particles. Here we show that (in contrast to widespread belief) immunolabeling of Epon‐embedded cells and of cells treated with osmium tetroxide is actually working. Furthermore, we established a “speed protocol” for immunolabeling by reducing antibody incubation times. Finally, we present our results on three‐dimensional immunogold labeling.     

A preparation method for observing intracellular structures by scanning electron microscopy

In order to observe intracellular structures by scanning electron microscopy, excess cytoplasmic matrix must be removed from the fractured surface of cells. Previously we reported an Osmium-DMSO-Osmium method devised for this purpose. This method is very effective in revealing intracellular structures, but requires osmium tetroxide for initial fixation with some consequent disadvantages. In the present study, a revised Osmium-DMSO-Osmium method is reported, in which an aldehyde mixture is used as the initial fixative instead of osmium tetroxide. As fixation is carried out by perfusion in this revised method, better preservation of fine structures is achieved than by the original method, especially in the central nervous tissue which tends to suffer from post-mortem degeneration. Moreover this method can be applied to cytochemical studies of intracellular structures with a scanning electron microscope (SEM). In this study, acid phosphatase of lysosomes is demonstrated in a coloured SEM micrograph.     

Use of microwave fixation in the preparation of cell cultures for observation with the scanning electron microscope

This paper describes a method for primary fixation of cultured cells for scanning (SEM) and transmission (TEM) electron microscopy using microwaves alone. This method of fixation takes 8 seconds and is therefore quicker and less expensive than conventional fixation techniques. The preservation of cell morphology is excellent and cultures of mammalian immune system cells and peripheral nervous tissue have been examined using this fixation.     

A simple method for overcoming some problems when observing thick reflective biological samples with a confocal scanning laser microscope

A simple device is described, which allows the range of depth of scanning to be reduced when observing thick reflecting biological samples with a confocal scanning laser microscope (CSLM). Thick histological sections of human skin and rat brain stem were mounted between two coverslips (‘sandwich’ style) and the optical tomography was performed from both sides by turning the ‘sandwich’ upside-down. The samples were impregnated using standard Golgi–Cox, ‘rapid Golgi’ or other silver methods. The ability to turn the ‘sandwich’ upside-down is particularly useful when the reflective structure inspected is deep inside the section, i.e. near the lower surface of the specimen, or when it is opaque to the laser beam or excessively reflective.     

A method to compensate for light attenuation with depth in three-dimensional DNA image cytometry using a confocal scanning laser microscope

A method to compensate for attenuation of detected light with increased depth of the collected optical section, and its application in three-dimensional (3-D) DNA image cytometry is described. The method is based on studying the stack of 2-D histograms that can be formed from each consecutive pair of sections in a stack of optical serial sections. An attenuation factor is calculated interactively and a new compensated section series is computed. Formalin-fixed paraffin-embedded rat tissue was stained with propidium iodide. Each cell nucleus is extracted by thresholding and its total intensity is calculated. The coefficient of variation (CV) of the total intensity of all cells in each stack is computed. For comparison the CV of the same cells is computed in the uncompensated stacks. This study shows a significantly lower CV for the compensated data, thus contributing to the accuracy of DNA quantification in 3-D DNA image cytometry.