An automatic focusing and astigmatism correction method for high resolution electron microscopy

When based on the image formation theory of an object studied in high resolution electron microscopes, the amount of focusing associated with the astigmatism was derived from measurements of the phase transfer function of the computer generated diffractograms, together with a line spectrum integration using the one-dimensional Fourier transform of the specimen. It was successfully found that an automatic focusing and astigmatism correction system with higher accuracy can be achieved by an on-line computer system.     

Adaptation of a non-radioactive in situ hybridization method to electron microscopy: detection of tenascin mRNAs in mouse cerebellum with digoxigenin-labelled probes and gold-labelled antibodies

In this study we describe a method for the detection of mRNAs at the ultrastructural level using a non-radioactive in situ hybridization method based on digoxigenin-labelled cRNA probes and gold-labelled digoxigenin-specific antibodies. We applied this protocol to an analysis of the expression of the extracellular matrix protein tenascin in the developing cerebellar cortex of the mouse. To gain an impression of the sensitivity attainable with digoxigenin-labelled probes, we first established at the light microscopic level that the hybridization signal obtained with the non-radioactive probe is as sensitive as that obtained with a ³⁵S-labelled probe. The non-radioactive hybridization protocol was then combined with electron microscopic post-embedding and immunogold detection techniques. Tenascinspecific, digoxigenin-labelled cRNA probes were hybridized to ultrathin sections of Lowicryl K4M-embedded tissue and the probe/target mRNA hybrids were detected using gold-labelled antibodies to digoxigenin. In agreement with the observations from in situ hybridization at the light microscopic level, specific labelling was observed in Golgi epithelial cells in the region of the Purkinje cell layer and cells in the internal granular layer, which could be identified as astrocytes by ultrastructural criteria. Labelling was detectable in association with free ribosomes and ribosomes of the rough endoplasmic reticulum. In addition, focal hybridization signals were occasionally found in the nucleus. No signal was observed in Golgi epithelial cells or astrocytes using sense or in any other cerebellar cell type using either sense or anti-sense probes. The described in situ hybridization technique uses ultrastructural criteria to associate the presence of a given mRNA species with a particular cell type. Additionally, it provides information about the target mRNA's subcellular distribution, thus offering the possibility to study intracellular transport of particular mRNAs.     

Polystyrene embedding: a new method for light and electron microscopy.

Polystyrene embedments of histological specimens can be obtained with a solution of 1:14 polystyrene-toluene, 5% benzyl alcohol and 1% dibutyl phthalate, allowing the solvent to evaporate in polyethylene containers for 2-3 days at 58 C. The resulting blocks are easily cut into truncated pyramids, each containing a piece of tissue, which are then glued to a Plexiglas support. Drying is completed at 80 C for 20 hr. The pyramids can then be sectioned to produce thick sections with a steel knife or to produce semi- or ultrathin sections with a glass knife. A 10% paraldehyde solution is used to mount the light microscopy sections on a slide heated on a hot plate to 80 C; these can be treated with the same techniques used with paraffin sections. The results are of high quality. Semithin sections of tissues fixed for electron microscopy can be stained directly after mounting, or by a wider range of stains once the polystyrene has been removed by organic solvents. In electron microscopy, the ultrathin sections obtained with the usual techniques are highly electron beam-resistant and given acceptable results.     

Fluorescence in situ hybridization method for co-localization of mRNA and GEP

Co-localization studies using green fluorescent protein (GFP) and fluorescence immunohistochemistry have become commonplace. However, co-localization studies using GFP and mRNA in situ hybridization are rare, in large part because typical in situ hybridization reaction conditions often lead to the loss of GFP fluorescence. Here, we describe a new fluorescence mRNA in situ hybridization protocol using cRNA riboprobes that leaves GFP fluorescence intact. This protocol is based on a urea-based hybridization buffer and the Tyramide Signal Amplification system. This protocol should provide researchers engaged in the use of GFP with a solid starting point for adapting their own in situ hybridization protocols.     

Fluorescence in situ hybridization: a new method for determining primary sex ratio in ants

The haplodiploid sex determining system in Hymenoptera, whereby males develop from haploid eggs and females from diploid eggs, allows females to control the primary sex ratio (the proportion of each sex at oviposition) in response to ecological and/or genetic conditions. Surprisingly, primary sex ratio adjustment by queens in eusocial Hymenoptera has been poorly studied, because of difficulties in sexing the eggs laid. Here, we show that fluorescence in situ hybridization (FISH) can be used to accurately determine the sex (haploid or diploid) of eggs, and hence the primary sex ratio, in ants. We first isolated the homologue coding sequences of the abdominal-A gene from 10 species of 8 subfamilies of Formicidae. Our data show that the nucleotide sequence of this gene is highly conserved among the different subfamilies. Second, we used a sequence of 4.5 kbp from this gene as a DNA probe for primary sex ratio determination by FISH. Our results show that this DNA probe hybridizes successfully with its complementary DNA sequence in all ant species tested, and allows reliable determination of the sex of eggs. Our proposed method should greatly facilitate empirical tests of primary sex ratio in ants.     

Identification of chlamydia and (or) chlamydia-like microorganisms by light microscopy confirmed by electron microscopy and fluorescent in situ hybridization

Using light microscopy, we have shown that chlamydia and/or chlamydia-like microorganisms are registered in 20-25% of the healthy part of human population, whereas in patients of the same age with gynecological problems these were found in 40-50%. Commonly, the infection was slightly manifested (less than 5% of cells are infected). These results were confirmed in four months but only in heavily infected patients. The light microscope data are confirmed by observations with electron microscopy, and by FISH hybridization of the total chlamydial DNA on cytological preparations with chlamydial inclusions. In some cases, microcolonies revealed by FISH hybridization occupied the majority of the cytoplasm volume. Occasionally, the DNA material was found on the nuclear surface. It seems likely that in heavily infected cells chlamydia are able to penetrate into the perinucular space.     

HSV hepatitis in the mouse: a light and electron microscopic study with immunohistology and in situ hybridization

In order to characterize better the morphology and immune response in acute necrotizing HSV infection, murine HSV hepatitis was examined. BALB/c mice were inoculated intraperitoneally with 10(6) plaque-forming units (PFU) of HSV-1 (Lenette) and HSV-2 (D316). In both groups half the animals were pretreated with silica particles to block macrophage function. Up to 6 days after infection four mice from each group were sacrificed at daily intervals and the livers were examined by light and electron microscopy, immunohistology, in situ hybridization, combined immunohistology/in situ hybridization and titration of viral PFU. HSV-2 infected mice developed severe necrotizing hepatitis with persistence of HSV in the liver tissue until the end of the study. HSV-1 infected mice rapidly eliminated the virus and revealed only small necrotic foci. Early phase alterations and necrotic phase lesions were distinguished and characterized and morphologic evidence of a direct cytopathic effect of HSV was detected. A specific immune reaction in late stages appeared to be mediated by T4-positive T-lymphocytes. In situ hybridization and immunohistochemistry showed a close correlation with virus titration and were valuable in characterizing early phases and in the assessment of prognosis and differential diagnosis.     

Thin graphite support films for high resolution electron microscopy

A method is described for preparing graphite films down to about 2nm in thickness. First, a suspension of small flakes in ethylene dichloride is prepared by repeated cleavage of a graphite crystal. This is then used to prepare a relatively thin film of graphite which is applied to grids previously coated with a holey carbon film. Fragments of tungsten oxide crystals are placed on the grid which is then exposed in the electron microscope to an electron beam of high intensity. As the result of an apparent reaction between the tungsten oxide and the carbon, the graphite film is etched, thereby producing a film which is both much thinner and cleaner than the original. The thickness of films prepared in this way has been estimated and their characteristics described. A brief account is given of the use of such a film using ferritin as a test object.     

Non-radioactive in situ hybridization. A comparison of several immunocytochemical detection systems using reflection-contrast and electron microscopy

A number of immunocytochemical detection systems for determining the chromosomal localization of specific nucleic acid sequences by non-radioactive in situ hybridization have been compared. The procedures were: 1. the peroxidase/diaminobenzidine (PO/DAB) combination, either or not gold/silver intensificated; 2. alkaline phosphatase marking using the nitro-blue tetrazolium plus bromochloro-indolyl phosphate substrate combination (AP/NBT + BCIP); and 3. immunogold with or without silver enhancement. The procedures were first tested and optimized in dot blot experiments and then applied to in situ hybridization. As hybridization probes, both a middle-repetitive and a unique sequence (modified with 2-acetylaminofluorene (AAF] were used. The advantages and disadvantages of the various methods for reflection contrast (RC) or transmission electron microscopic (TEM) visualization of hybrids are discussed.     

Imaging of plant microtubules with high resolution scanning electron microscopy

Summary High resolution scanning electron microscopy was used to obtain images of cortical microtubules and associated structures in onion root tips. Specimens were prepared using a modified quick-freeze deep-etch technique utilising cytosolic extraction with saponin and conductive staining with osmium.Abbreviations DMSO dimethylsulfoxide - HRSEM high resolution scanning electron microscope/microscopy - MTSB microtubule stabilising buffer - TEM transmission electron microscope/microscopy     

Human amelogenesis: high resolution electron microscopy of nanometer-sized particles

Growth of inorganic crystals of enamel is described as a two-stage process with growth of ribbon-like crystals in length and width, followed by their development in thickness. In early stages of crystal growth during human amelogenesis nanometer-sized particles with a mean diameter of 1.1 nm were described between ribbon-like crystals. These small particles had a crystalline structure but their lattice parameters did not seem to be directly related to those of calcium phosphates. The nanometer-sized particles appear to correspond to initial stages of apatite crystal growth. Their localization close to ribbon-like crystals and their progressive increase in size and number may indicate that they represent a precursor phase for these crystals. Nucleation areas at both extremities, of elongated ribbon-like crystals could be involved in the two-directional growth of ribbons and/or in nanometer-sized particle nucleation.     

A new method of the immunohistochemical detection of cellular antigens for light and electron microscopy

A new immunohistochemical method for light and electron microscopy of tissue- and cell-specific antigens by using ferric colloid-labeled antibody is presented. The antibodies labeled with the cationic cacodylate ferric colloid are stable and bind specifically to the target antigens to show clearly the site of antigens in tissue sections and on free cells by Prussian blue reaction for light microscopy and by the specific figure of electron opaque ferric colloid particles for electron microscopy. The staining procedure is very simple and it gives clear picture. So the method will be of beneficial for general laboratory use in immunohistochemical researches.     

A new method of the immunohistochemical detection of cellular antigens for light and electron microscopy

Summary A new immunohistochemical method for light and electron microscopy of tissue- and cell-specific antigens by using ferric colloid-labeled antibody is presented. The antibodies labeled with the cationic cacodylate ferric colloid are stable and bind specifically to the target antigens to show clearly the site of antigens in tissue sections and on free cells by Prussian blue reaction for light microscopy and by the specific figure of electron opaque ferric colloid particles for electron microscopy. The staining procedure is very simple and it gives clear picture. So the method will be of beneficial for general laboratory use in immuno-histochemical researches.     

High resolution scanning electron microscopy of isolated and in situ cytoskeletal elements

Evidence is presented that cytoskeletal structures (actin filaments, intermediate filaments, and microtubules) can be resolved by scanning electron microscopy after osmium impregnation of biological material, using thiocarbohydrazide as a ligand, followed by critical-point drying. These different classes of filaments or tubules can be identified both as purified protein polymers and as structured organelles within cryofractured or detergent-extracted cells.     

A simple method of staining juxtaglomerular granules in the rat kidney for high resolution light microscopy

A simple method for the demonstration of juxtaglomerular granules in Epon embedded semithin (0.5-1 micrometer) sections has been developed as follows: sections are prepared as for routine electron microscopy except that before dehydration, the tissues are immersed in 0.5% uranyl acetate in Veronal acetate buffer (pH 5.0) overnight at room temperature. After sectioning on an ultramicrotome, the semithin sections are briefly stained with toluidine blue-pyronin Y. After staining, the section is rinsed in running tap water and then air dried. Under a light microscope with a 40 X or a 100 X objective, the juxtaglomerular granules appear as deep purple particles and are thus easily separated from the bluish cytoplasm of the juxtaglomerular cells. Cellular organelles in other cells of the kidney were also clearly stained and their fine structure distinguishable.     

Calbindin-D immunolocalization in developing chick thyroid: a light and electron microscopic study

Antiserum to calbindin-D, a 28 KD vitamin D-dependent calcium binding protein, was used to localize the protein immunocytochemically in developing chick thyroid by both light and electron microscopy. The protein first appeared in future follicular cells of developing thyroid tissue from 8-day-old embryos. The number of calbindin-D-containing cells increased rapidly to a near-plateau level at day 10; this concentration was sustained until day 15, and then declined to an undetectable level just before hatching. The protein was distributed throughout organelle-free areas of the follicular cell cytoplasm and extended into the nucleus; it was not present in the follicular colloid. Comparison of the time course of changes in calbindin-D content with known differentiative changes taking place in follicular cells suggests that the protein may function in some yet to be determined mechanism related to normal development of the thyroid.