Electron dense markers of a size suitable for transmission electron microscopy and scanning electron microscopy have been prepared with gold granules labeled with a monolayer of specific macromolecules. The optimum conditions for preparing the markers have been ascertained. The method is simple, rapid and seems to be general since gold granules have been labeled with polysaccharides and proteins. As homogeneous populations of gold granules having different sizes can be prepared, the method is also suitable for double marking experiments. The gold technique is illustrated by the localization of polysaccharides and glycoproteins on yeast cell walls and erythrocyte membranes by transmission electron microscopy and on yeast cells and intact erythrocytes by scanning electron microscopy. Good spatial resolution of the marker was achieved in all cases. The method is also suitable for marking thin sections. Spectrophotometric measurements were used to determine the number of gold granules adsorbed per cell.

Colloidal gold is orange to red, displays electron-opaque properties and is capable of strong emission of secondary electrons. As gold particles can be produced in different sizes and can be labelled with macromolecules which keep their specific properties, gold markers have found ases in light and fluorescent microscopy, and many applications in scanning and transmission electron microscopy.In biology, the most sophisticated analysis performcd in vitro will never suppress the urge of an investigator to visualize the exact location of a cell component.From the time the Dutch naturalist Antonie van Leeuwenhoek (1632-1723) made his first discoveries with a simple instrument, microscopy has indeed proved to be a powerful tooi for understanding the microcosm. Thanks to patient efforts and to an amazing skill in using lenses, van Leeuwenhoek was able to obtain results which, at the time, were thought to be truly marvellous. His experiments became so popular that it was fashionable in the upper class society of Delft to Eind distraction, satisfaction and even joy in observing nature through his primitive microscopes. These inner feelings are still strongly feit by modern researchers using much more powerful instruments.Clearly, van Leeuwenhoek was a self-taught microscopist, but his observations led to many discoveries of great importante. He was the first to observe protozoa, spermatozoids and red blood cells, and the first representation of a bacterium is to be found in one of his drawings of 1682.The development of optical microscopes has continued since that time, but the limits imposed by the wavelengths of visible light prompted the search for high resolution techniques. The first transmission electron microscopes having better resolution than light microscopes were built in 1932. Thirty years later, the scanning electron microscope was introduccd. Scanning electron microscopy (SEM) gives a three-dimensional quality to specimen images. Normally, the instrument is operated by scanning, or sweeping, a very narrow beam of electrons back and forth across a metal-coated specimen, revealing its surface features rather than its internal structure.The possibility of localizing specific tissue components attracted the early microscopists, but it was Raspail (1794-1878), a French botanist, pharmacist, microscopist and politician, who first used the microscope to study the chemical reactions of tissue materials. He is considered to be the founder of histochemistry and some of the reactions which he discovered are still applied today. Over the years, a large number of techniques has been developed to identify, locate and quantify specific tissues and cell components. Among the methods having a narrow specificity, the use of fluorescent antibodies was introduced in 1941 by Coons et al. (1). A great advance was then achieved at the ultrastructural level with the development of enzyme cytochemistry (2, 3, 4) and the application of particulate markers, such as ferritin, conjugated to antibodies (5, 6). Ferritin is a large molec...

The gold method was further developed for fluorescent microscopy. Gold granules (12 nm in size) were labelled with rhodamine conjugates of Concanavalin A and avidin. The fluorescent markers were used to mark cell wall mannan on the yeast Saccharomyces cerevisiae either by the one-step, or by the two-step method via a biotinyl derivative of ConA. By fluorescence or transmission electron microscopy, the two-step method was found to achieve a higher density of marking.

A rapid method has been developed to visualize cell surface receptors in the SEM. Thus mannan at the surface of Candida utilis cells was localized by stabilized colloidal gold granules coated with either anti-mannan antibodies or Con A.

Mannan was located on thin sections of Saccharomyces cerevisiae and Candida utilis with the homologous anti-mannan antibodies or with Concanavalin A, both labelled with gold granules. Fully synthesized mannan was found in the cell walls, on the plasmalemma and within the cytoplasm sometimes associated with vesicles and vacuoles. Chitin or its oligomers were located with wheat germ agglutinin in the bud scars but also in the cell wall and the cytoplasm near the plasmalemma. Both mannan and chitin or its oligomers were found in the forming septum and are synthesized within the cytoplasm. The gold method was also suitable for marking mannan and chitin simultaneously.