Colour development of immunogold-labelled antibodies for light microscopy

We describe an improvement of the immunogold technique, which is based on the colour development of silver-intensified immunogold signals. This method (referred to as the coloured-SIG technique) was found to be as sensitive as the silver-intensified immunogold method and more sensitive (in two of the three tested systems) than immunoenzymatic procedures, such as the peroxidase/antiperoxidase technique or the avidin-biotin system. The coloured SIG-method results in either a magenta-red or a cyan-blue final reaction product. Therefore, this new improvement of the immunogold technique should be useful in double-staining methods, immunoblot methods and conventional immunostaining methods.     

Shedding light on the system: studying embryonic development with light sheet microscopy

Light sheet-based fluorescence microscopy (LSFM) is emerging as a powerful imaging technique for the life sciences. LSFM provides an exceptionally high imaging speed, high signal-to-noise ratio, low level of photo-bleaching and good optical penetration depth. This unique combination of capabilities makes light sheet-based microscopes highly suitable for live imaging applications. There is an outstanding potential in applying this technology to the quantitative study of embryonic development. Here, we provide an overview of the different basic implementations of LSFM, review recent technical advances in the field and highlight applications in the context of embryonic development. We conclude with a discussion of promising future directions.     

Cryogenic light microscopy and the development of cooling protocols for the cryopreservation of filamentous fungi

One hundred and ninety five strains of fungi were observed during freezing and thawing using a cryogenic light microscope. There was no obvious link between taxonomic position and their morphological response to freezing and thawing. The viability of seven of these strains was examined following freezing and thawing in the presence or absence of the cryoprotectants glycerol and dimethyl sulphoxide. Intracellular ice and hyphal shrinkage were not necessarily lethal events, but in many cases they affected the rate and quality of growth. Both cryoprotectants reduced shrinkage, shifted the cooling rate where intracellular ice formed in many cases, and improved the recovery of strains. The results presented aid the development of successful cryopreservation protocols.     

Colored silver-intensified gold technique for light microscopy

The development of silver-intensified immunogold-labeled antibodies for light microscopy described by Fritz et al. (4) has been investigated. Principles and chemistries used in color photographic science have been applied to immunogold enhancement. In this technique, colloidal gold acts as the catalytic center for the reduction of silver ions to metallic silver with subsequent color development in the presence of hydroquinone. Silver ions and hydroquinone are adsorbed onto the surface of colloidal gold. The reduction of silver ions to metallic silver is further catalyzed by autometallography. The colored-SIG technique offers several advantages. It has sensitivity comparable to the silver-intensified gold (SIG) method and greater sensitivity than immunoenzymatic procedures, takes approximately one hour, results in one of three color reaction products (magenta, cyan, or yellow), and produces better contrast between the reaction products and the background (Figure 1). Thus, this method should prove useful in double- and even triple-staining procedures.     

Monoclonal antibodies against calcitonin. Characterization and application in light and electron microscopy immunocytochemistry

Twenty-seven monoclonal antibodies (MAbs) to synthetic human calcitonin (CT) were characterized for their reactivities with human CT peptide fragments by dot-blot analysis on nitrocellulose paper. Most of the antibodies bound to the C-terminus and fewer to the mid-region of CT. We have studied thyroid tissue specimens from several animal species after fixation in paraformaldehyde-, glutaraldehyde- or picric acid-containing mixtures and cryostat sectioning or embedment in paraffin or plastic (Epon 812 or Lowicryl 4KM) using this panel of MAbs. The site of antigen-antibody reaction was revealed either by immunoperoxidase, immunoalkaline phosphatase or by silver-enhanced immunogold staining methods. All MAbs were able to localize CT in human, rat and mouse thyroid C cells. Nineteen MAbs recognizing synthetic salmon CT and synthetic [Asu1,7]-eel CT by dot-blot, reacted with chicken ultimobranchial body C cells. One MAb recognizing native porcine CT by dot-blot, stained C cells in hog thyroid. Immunopositivity was confined to the cytoplasm and ultrastructural immunogold labelling demonstrated that cytoplasmic secretory granules were stained. Surgical specimens from human medullary thyroid carcinoma were also analysed for the presence of CT and a variable number of positive cells was found. Furthermore, Congo red-positive areas were shown to react with the MAbs. All conventional staining and immunoabsorption controls were negative. Hence, these MAbs may be suitable for use in routine immunopathological diagnosis of CT-producing tumors and for immunocytochemical localization of the three major CT variants in different animal species.     

Calibrating excitation light fluxes for quantitative light microscopy in cell biology

Power output of light bulbs changes over time and the total energy delivered will depend on the optical beam path of the microscope, filter sets and objectives used, thus making comparison between experiments performed on different microscopes complicated. Using a thermocoupled power meter, it is possible to measure the exact amount of light applied to a specimen in fluorescence microscopy, regardless of the light source, as the light power measured can be translated into a power density at the sample. This widely used and simple tool forms the basis of a new degree of calibration precision and comparability of results among experiments and setups. Here we describe an easy-to-follow protocol that allows researchers to precisely estimate excitation intensities in the object plane, using commercially available opto-mechanical components. The total duration of this protocol for one objective and six filter cubes is 75 min including start-up time for the lamp.     

Plastic sectioning for light microscopy of Pyrenomycetes.

In preparation for light microscopy, ascocarps of Sordaria fimicola Ces. & DeNot. were embedded in Spurr's medium and sectioned at 1-1.5 mum on an ultramicrotome. Sections were floated on Giemsa staining solution at 60 C for 10-30 min, washed in distilled water, affixed to slides by drying, and mounted in immersion oil. Best preservation of the delicate sterile tissues of the centrum was obtained by fixation in 1% KMnO4 for 2.5-3 hr, followed by the Giemsa stain. This method is suggested for future studies on the morphology of perithecial ascomycetes.     

A removable polar embedding medium for light microscopy

A new plastic embedding medium for light microscopy is described. The monomer mixture consists of equal proportions by volume of acrylonitrile, dimethyl acrylamide and methyl methacrylate, and may be polymerized by exposure to ultraviolet light in the presence of benzoin methyl ether as catalyst. Dithiothreitol may also be added to the monomer mix to limit the degree of polymerization. The resulting polymer is soluble in dimethyl formamide.     

Bismuth staining for light and electron microscopy.

Bismuth salts on aldehyde fixed tissue give a highly selective pattern of staining suitable for light and electron microscopy. Structures stained include the nucleolus, ribosomes, inter- and perichromatin granules, the Golgi complex beads and the outer face of the tubule doublets of mouse sperm, certain neurosecretory vesicles believed to contain biogenic amines, some junctions (some central synapses, neuromuscular junctions, tight junctions), specialized membranes such as the post acrosomal dense lamina of mouse sperm and the inner alveolar membrane of Paramecium, and a variety of structures associated with the cytoplasmic face of membranes, such as plasma membrane plaques, cleavage furrows, the leading edge of the spreading acrosome and sperm annuli.Staining is not reduced by nucleases and spot tests show no reaction between nucleic acids and bismuth under conditions similar to those used to stain tissues. However, spot tests do show strong binding of bismuth by basic proteins and by some phosphorylated molecules.It is hypothesized that bismuth reacts with cell components in two ways, distinguishable by their glutaraldehyde sensitivity. For example, staining of the nucleolus and ribosomes is blocked by glutaraldehyde but the inter- and perichromatin granules and the GC beads are unaffected. Spot tests show that basic proteins (histones, protamines, polylysine and polyargenine) and other molecules with free amino groups (5HT, tryptamine, dopamine) bind bismuth strongly, a reaction that is blocked to varying degrees by glutaraldehyde. We presume that most bismuth staining of tissues is due to reaction with amine groups and is glutaraldehyde sensitive and some may be due to guanidine groups which are less sensitive to fixation by glutaraldehyde. Organic phosphates may be the cause of the glutaraldehyde insensitive staining since ATP and some other phosphates bind bismuth in a reaction that is not blocked by glutaraldehyde.     

Mapping myosin light chains by immunoelectron microscopy. Use of anti- fluorescyl antibodies as structural probes

The two classes of light chains in vertebrate fast muscle myosin have been selectively labeled with the thiol specific reagent 5- (iodoacetamido) fluorescein to determine their location in the myosin head. The alkali light chains (A1 and A2) were labeled at a single cysteine residue near the COOH terminus, whereas the regulatory light chain (LC2) was reacted at either cysteine 125 or 154. The two cysteines of LC2 appear to be near each other in the tertiary structure as evidenced by the ease of formation of an intramolecular disulfide bond. Besides having favorable spectral properties, fluorescein is a potent haptenic immunogen for raising high affinity antibodies. When anti-fluorescyl antibodies were added to the fluorescein-labeled light chains, the fluorescence was quenched by greater than 90%, thereby providing a simple method for determining an association constant. The interaction with antibody was the same for light chains exchanged into myosin as for free light chains. Complexes of antibody bound to light chain could be visualized in the electron microscope by rotary shadowing with platinum. By this approach we have shown that the COOH- terminal regions of the two classes of light chains are widely separated in myosin: the cysteine residues of LC2 lie close to the head/rod junction, whereas the single cysteine of A1 or A2 is located approximately 90 A distal to the junction. These sites correspond to the positions of the NH2 termini of the light chains mapped in earlier studies (Winkelmann, D. A., and S. Lowey. 1986. J. Mol. Biol. 188:595- 612; Tokunaga, M., M. Suzuki, K. Saeki, and T. Wakabayashi. 1987b. J. Mol. Biol. 194:245-255). We conclude that the two classes of light chains do not lie in a simple colinear arrangement, but instead have a more complex organization in distinct regions of the myosin head.     

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 development of porcine zona pellucida using monoclonal antibodies: II. Electron microscopy

Following our previous study on the immunohistochemistry of porcine zonae pellucidae (ZP), we undertook the present study to localize the components of the ZP with immunoelectron microscopy, using three types of anti-porcine-ZP monoclonal antibodies (Mabs), named STA-1, STA-2, and STA-3. Some organelles of the oocyte were seen to react with STA-2 and STA-3 prior to ZP formation. As soon as a follicle began to mature, STA-2 and STA-3 reacted with the perinuclear space and the endoplasmic reticular membrane of the oocyte. The follicle first reacted with STA-1 at the secondary follicle stage. At this stage, the positive reaction involved the follicular cell layer as well as the oocyte and ZP. Positive reaction was scattered within and limited to the interfollicular cell space and was never found in the cytoplasm of follicular cells. At the antral follicle stage, the oocyte was surrounded by a thick, electron-dense ZP. A strong reaction was observed in the outer layer, but no significant reaction occurred in the inner layer. The convex and ragged outer margin of the ZP was characterized by the strongest reaction.     

Scanning electron and light microscopy of appressorium development in Piptocephalis unispora (Mucorales)

Appressorium development in the mycoparasite Piptocephalis unispora was studied by means of scanning electron microscopy using the techniques of critical point drying, sputter coating and light microscopy. The germ tube which contacts both the young host hypha or a germinating spore swells at the tip to form an appressorium closely adpressed to the surface of the host. Lateral proliferation of hyphae may occur from the mature appressorium. Factors affecting the sites of appressorium development are suggested and their significance discussed.