Ponceau-Fuchsin as a Routine Countebstain

A new technique for studying the morphology of the cochlea is described. The development of back-scattered electron (BSE) detectors has allowed the examination of heavy-metal stained tissues by scanning electron microscopy. Comparison with light microscopy on adjacent resin sections through whole decalcified cochleae demonstrated that the back-scattered electron technique provides equal or superior clarity and resolution throughout the light microscope range of magnification, allows identification of lysosomes, mitochondria and endoplasmic reticulum, and extends useful magnification into the range previously associated only with transmission electron microscopy. Back-scattered electron imaging enables the study of sections of the undissected cochlea at high magnifications and resolution.     

Back-scattered electron imaging of sections through the cochlea: a new technique for studying cochlear morphology

A new technique for studying the morphology of the cochlea is described. The development of back-scattered electron (BSE) detectors has allowed the examination of heavy-metal stained tissues by scanning electron microscopy. Comparison with light microscopy on adjacent resin sections through whole decalcified cochleae demonstrated that the back-scattered electron technique provides equal or superior clarity and resolution throughout the light microscope range of magnification, allows identification of lysosomes, mitochondria and endoplasmic reticulum, and extends useful magnification into the range previously associated only with transmission electron microscopy. Back-scattered electron imaging enables the study of sections of the undissected cochlea at high magnifications and resolution.     

Surface Characteristics of Gram-Negative and Gram-Positive Bacteria in an Atomic Force Microscope Image

Bacterial images can be obtained rather easily with an atomic-force microscope (AFM) in the magnification range of 5,000 to 30,000 times without any pretreatment of the specimens for such observations as chemical fixation, dehydration or staining. The bacterial shapes or the presence of flagella can be clearly recognized in these magnification ranges. In addition, we were also able to distinguish between Gramnegative and Gram-positive bacteria based on the specific wavy surface appearance of the former. AFM could thus be a useful tool for the identification of bacteria in the resolution range between electron and light microscopy.     

Localization of immunoreactive tyrosine hydroxylase in the goldfish retina with pre-embedding immunolabeling with one-nanometer colloidal gold particles and gold toning.

The goal of this study was to develop an alternative to silver intensification for visualizing small colloidal gold particles by light and electron microscopy. The isolated goldfish retina was labeled with rabbit antiserum to tyrosine hydroxylase and 1-nm colloidal gold-conjugated goat anti-rabbit IgG. The gold particles were enlarged by toning with gold chloride, followed by reduction in oxalic acid. Dopaminergic interplexiform cells were clearly visible by light microscopy and, in lightly-fixed material treated with detergent, they were labeled in their entirety. Labeling was qualitatively similar, although less extensive, in material fixed and processed for electron microscopy. The labeled processes were apparent in ultra-thin sections viewed at low magnification, but the gold-toned particles were not so large that they obscured subcellular structures. The procedure apparently had no deleterious effects on the tissue, since the ultrastructural preservation was comparable to that seen with other pre-embedding immunolabeling methods. The technique was simple, reliable and, since the gold solutions were so dilute, relatively inexpensive.     

The use of silver-enhanced 1-nm gold probes for light and electron microscopic localization of intra- and extracellular antigens in skin.

We used colloidal gold (1-nm diameter) with silver enhancement, in conjunction with a low-temperature post-embedding immunolabeling technique, to localize several antigens in normal skin at both the light and the electron microscopic level within the same tissue blocks. Normal skin subjected to cyrofixation and cryosubstitution and embedded in Lowicryl K11M was used as a substrate. Semi-thin sections (1 micron) were incubated in primary antibody (against epidermal basement membrane zone associated antigens and two keratin sub-types), biotinylated secondary antibodies, and then in 1-nm gold-conjugated streptavidin. Finally, the 1-nm gold label was enhanced using silver staining. Labeling of both basement membrane and keratin antigens was well demonstrated, and the area in the semi-thin sections showing the best structural preservation and the greatest intensity of immunolabeling was used to identify the part of the block to be used for ultra-thin sectioning. Ultra-thin sections were treated using a similar procedure to that employed for semi-thin sections. The labeling with silver-enhanced 1-nm gold probes was intense and readily visible by electron microscopy, even at low magnification. We have found this technique to have a high degree of specificity and sensitivity for labeling both intra- and extracellular antigens in skin, with the added advantage of providing the means for studies at both light microscopic and electron microscopic level.     

Use of confocal laser scanning microscopy on soil thin-sections for improved characterization of microbial growth in unconsolidated soils and aquifer materials

Confocal laser scanning microscopy (CLSM) was utilized to examine samples from an aquifer microcosm that was used to investigate microbially mediated losses in hydraulic conductivity. Samples were fixed, dehydrated and dried to prepare the biological material in a fashion similar to that used previously for viewing under the scanning electron microscope. Then, samples were prepared as thin-sections by employing soil micromorphological techniques. Serial images generated by the CLSM technique were visualized using computer three-dimensional rendering software. Results from the CLSM technique were compared with simple fluorescence microscopy of thin-sections and scanning electron microscopy (SEM) of samples from the microcosm. Computer visualization of serial sections with the CLSM technique provided images on a submicron scale in three dimensions. SEM has a much higher resolution, on a nanometer scale, but the results are not three dimensional. Artifacts associated with thin-section preparation are minimal for natural porous media composed mostly of sand, such as aquifer materials. Also, CLSM images are affected minimally by changes to biological material due to sample preparation, whereas artifacts associated with SEM images are very prominent, due to the higher magnification and resolution. CLSM of thin-sections and SEM are very powerful methods for viewing microbial growth in natural porous media, but CLSM is preferable because it allows three-dimensional visualization and measurements of cells and aggregates with few artifacts.     

Use of anti-horseradish peroxidase antibody-gold complex in the ABC technique.

We report a modification of the avidin-biotin-peroxidase complex (ABC) technique for the light and electron microscopic detection of antigens in tissue sections. An immunological approach was used instead of the DAB reaction to reveal ABC bound to antigen-antibody complexes. Affinity-purified polyclonal antibodies against horseradish peroxidase were complexed to particles of colloidal gold and applied for reaction with the horseradish peroxidase molecules of the ABC. For light microscopic immunolabeling, the signal produced by the anti-horseradish peroxidase antibody-gold complex required silver intensification. The ABC immunogold reaction as compared with the standard ABC technique, in particular with silver intensification of the DAB reaction product, provided superior resolution in paraffin sections. Furthermore, section pre-treatment to block endogenous peroxidase activity could be omitted and no potentially hazardous substrate was used. The ABC immunogold reaction was successfully applied for electron microscopic immunolabeling on Lowicryl K4M thin sections. We propose that the ABC immunogold reaction is a useful alternative to the standard ABC technique and can be equally well applied to light and electron microscopy.     

A novel thin section preparation and staining protocol to increase contrast and resolution of cell details for light microscopy

ABSTRACT

We developed a novel sectioning and staining method to make high contrast, high resolution sections of plant tissue for light microscopy. Specimens of teosinte (Zea mays L., ssp. mexicana) root tips were fixed and embedded in Technovit 7100? plastic resin. Thin sections, 1?2.5 μm, were cut and mounted on glass slides. The sections were either treated with RNase or not, then stained with 0.1% toluidine blue O and observed through ∞/0 objective lenses. For light microscopy, the enzyme staining procedure increased resolution and contrast. High magnification ∞/0 objective lenses produced high quality images for digital photography without using a coverslip or immersion oil. Our slide preparation and microscopic analysis were less labor intensive and more rapid than previous methods and enabled rapid and precise alignment of serial transverse sections for both tracking cell lineages and tissue measurements.     

Routine use of backscattered electron imaging to visualize cytochemical and autoradiographic reactions in semi-thin plastic sections.

The scanning electron microscope (SEM) was used to examine cytochemical and autoradiographic reactions in 2-microns semi-thin sections of tissues conventionally fixed and embedded in various resins. The sections were examined using both the secondary and backscatter modes of the SEM at magnifications within the range attainable with the light microscope. Both modes allowed the imaging of phosphatase reaction product using cerium and lead capture, lectin-gold, and immunogold labeling, with and without silver enhancement, and autoradiography. Backscattered electron imaging (BEI), however, provided images with more contrast and structural details. This approach allows examination of large sections, with more contrast and resolution than the light microscope, and visualization of reactions not visible with this instrument. The improved imaging and the simple and conventional preparation of specimens indicate that BEI can be used routinely to examine tissue organization, cell structure, and the content of the various cell compartments with a resolution approaching that of transmission electron microscopy.     

Minimally invasive method for murine brain fixation

Complete brain fixation can be achieved with transthoracic cardiac infusion without thoracotomy. Light and electron microscopy tissue sections reveal preservation of cytoplasmic and nuclear structure at all magnification levels. Punched samples were obtained from the fixed tissue specimens in precisely localized areas for study using electron microscopy. This perfusion fixation technique provides both faster tissue harvesting capability and higher quality tissue preservation, without the artifacts of brain swelling and ventricular dilation observed in direct cardiac perfusion. Acute, discrete change in brain tissue can be studied.     

Advantages of intermediate X-ray energies in Zernike phase contrast X-ray microscopy

Understanding the hierarchical organizations of molecules and organelles within the interior of large eukaryotic cells is a challenge of fundamental interest in cell biology. Light microscopy is a powerful tool for observations of the dynamics of live cells, its resolution attainable is limited and insufficient. While electron microscopy can produce images with astonishing resolution and clarity of ultra-thin (< 1 μm thick) sections of biological specimens, many questions involve the three-dimensional organization of a cell or the interconnectivity of cells. X-ray microscopy offers superior imaging resolution compared to light microscopy, and unique capability of nondestructive three-dimensional imaging of hydrated unstained biological cells, complementary to existing light and electron microscopy.     

Cryo-electron microscopy reveals macromolecular organization within biological liquid cyrstals seen in the polarizing microscope

A method is described for examining water dispersible biopolymers in the frozen, hydrated state by electron microscopy using the filamentous bacterial viruses Pf1 and fd as examples. The technique reveals liquid-crystalline textures that correlate well with polarizing microscopy of magnetically oriented specimens. At higher magnification the packing of the virus particle is revealed to a spatial resolution of better than 30 Å, thus linking directly with data from X-ray diffraction and optical microscopy. Electron diffraction confirms that the structure is preserved to high resolution (4 Å). The technique permits a detailed understanding of the processes involved in the orientation of these samples in a strong magnetic field and clarifies the long-range bi-axial properties of some fibres as seen by X-ray diffraction and optical microscopy.     

Cell Biology and Algal Taxonomy and Evolution Absolute Orientations of Basal Bodies in Green Algae Evaluated by Light Microscopy

Using high magnification Nomarski interference microscopy a series of optical sections has been obtained through flagellated cells of several green algae in an attempt to establish the absolute orientation of their basal bodies. Using this technique we have confirmed that in Spermatozopsis similis basal bodies are displaced into the 1/7 o'clock position, whereas in gametes of Enteromorpha linza, and zoospores of Trebouxia erici basal bodies are displaced into the 11/5 o'clock position. In addition we report for the first time that in zoospores of Microthamnion kuetzingianum basal bodies are also displaced into the 11/5 o'clock position. Basal body absolute orientations can thus be determined by light microscopy and do not require serial section electron microscopy. The method should be useful for class-level recognition of unknown green algae at the light microscope level.     

The Use of ToF-SIMS for Analysis of Bioorganic Samples

The technique for the preparation of biological tissue sections developed for Electron Probe Microanalysis has been adapted for ToF-SIMS analysis of mouse GV stage oocytes. GV-oocyte sample preparation involves the following steps: plunge freezing, freeze drying, impregnation in an embedding medium, and section cutting. Molecule-specific images of the distribution of molecules in a single oocyte have been obtained with the described technique and ToF-SIMS analysis. The ToF-SIMS analysis data show that the efficient lateral image resolution is approximately 1 μm. Hence, ToF-SIMS enables us to study the distribution of chemical substances in relation to the morphological data obtained by scanning electron microscopy or conventional light microscopy.     

Structural analysis of the mitotic cycle in pre-gastrula Xenopus embryos

The long-known phenomenon of karyomere (chromosome vesicle) formation at early telophase of the nuclear cycle during early embryogenesis of a wide range of organisms including amphibians (Rubaschkin 1905; for review, see Richards 1917) was investigated in the early cleavage cycles of Xenopus laevis embryos before the mid blastula transition. Embryos were fixed and Epon embedded at successive time intervals and consecutive thick (3 m) and ultrathin sections cut. Using conventional light microscopy at low magnification as well as phase and/or interference contrast video microscopy at high magnification, a substantial amount of information could be obtained from the analysis of optical sections in thick-sectioned material. In addition, details of the ultrastructural organization could be analysed from corresponding ultrathin sections by electron microscopy. The light microscopic analysis of serial thick sections allowed precise determination of the arrangement and sizes of telophase karyomere structures during the embryonic nuclear division cycle. It was found that small, widely spaced 1st order karyomeres fuse to larger (2nd order) karyomeres which then progressively exhibit lateral fusion of neighbouring karyomeres. The final coalescence of adjacent karyomeres marks the onset of the reorganization of the typical interphase nuclear structure. The data are discussed with regard to the occurrence of karyomeres during the embryonic nuclear cycle of arthropods, dipteran insects, and echinoderms as well as recent progress in the use of Xenopus egg extracts for in vitro assembly of nuclear structures around protein-free DNA.     

Lignin distribution in wood cell walls determined by TEM and backscattered SEM techniques

The lignin distribution in cell walls of spruce and beech wood was determined by high-voltage transmission-electron-microscopy (TEM) in sections stained with potassium permanganate as well as by field-emission-scanning-electron-microscopy (FE-SEM) combined with a back-scattered electron detector on mercurized specimens. The latter is a new technique based on the mercurization of lignin and the concomitant visualization of mercury by back-scattered electron microscopy (BSE). Due to this combination it was possible to obtain a visualized overview of the lignin distribution across the different layers of the cell wall. To our knowledge, this combined method was used the first time to analyse the lignin distribution in cell walls. In agreement with previous work the highest lignin levels were found in the compound middle lamella and the cell corners. Back-scattered FE-SEM allows the lignin distribution in the pit membrane of bordered pits as well as in the various cell wall layers to be shown. In addition, by using TEM as well as SEM we observed that lignin closely follows the cellulose microfibril orientation in the secondary cell wall. From these observations, we conclude that the polymerisation of monolignols is affected by the arrangement of the polysaccharides which constitute the cell wall.     

Focused ion beam (FIB) combined with high resolution scanning electron microscopy: A promising tool for 3D analysis of chromosome architecture

Focused ion beam (FIB) milling in combination with field emission scanning electron microscopy (FESEM) was applied to investigations of metaphase barley chromosomes, providing new insight into the chromatin packaging in the chromosome interior and 3D distribution of histone variants in the centromeric region. Whole mount chromosomes were sectioned with FIB with thicknesses in the range of 7–20 nm, resulting in up to 2000 sections, which allow high resolution three-dimensional reconstruction. For the first time, it could be shown that the chromosome interior is characterized by a network of interconnected cavities, with openings to the chromosome surface. In combination with immunogold labeling, the centromere-correlated distribution of histone variants (phosphorylated histone H3, CENH3) could be investigated with FIB in three dimensions. Limitations of classical SEM analysis of whole mount chromosomes with back-scattered electrons requiring higher accelerating voltages, e.g. faint and blurred interior signals, could be overcome with FIB milling: from within the chromosome even very small labels in the range of 10 nm could be precisely visualized. This allowed direct quantification of marker molecules in a three-dimensional context. Distribution of DNA in the chromosome interior could be directly analyzed after staining with a DNA-specific platinorganic compound Platinum Blue. Higher resolution visualization of DNA distribution could be performed by preparation of FIB lamellae with the in situ lift-out technique followed by investigation in dark field with a scanning transmission electron detector (STEM) at 30 kV.     

Light and electron microscopic demonstration of sialic acid residues with the lectin from Limax flavus: a cytochemical affinity technique with the use of fetuin-gold complexes

The development of a cytochemical affinity technique for the demonstration of sialic acid residues by light and electron microscopy is reported. The lectin from the slug Limax flavus, with its narrow specificity for N-acetyl- and N-glycolylneuraminic acid, was applied to tissue sections. Subsequently fetuin-gold complexes were used to visualize the tissue-bound lectin. Different cytochemical controls, including sugar inhibition tests, neuraminidase digestion, the use of fetuin-gold complexes alone, or acid hydrolysis of sections, proved the specificity of the technique. Postembedding staining was performed on frozen, paraffin, or semithin resin sections for light microscopy and on thin sections from low temperature Lowicryl K4M-embedded material for electron microscopy. The distribution of sialic acid residues in rat pancreas, liver, and colonic mucosa was investigated.     

生物三维电子显微学进入全新高速发展时期

生物三维电子显微学主要由三个部分组成——电子晶体学、单颗粒技术和电子断层成像术,其结构解析对象的尺度范围介于x射线晶体学与光学显微镜之间,适合从蛋白质分子结构到细胞和组织结构的解析。以冷冻电镜技术与三维重构技术为基础的低温电子显微学代表了生物电子显微学的前沿。低温单颗粒技术对于高度对称的病毒颗粒的解析最近已达到3．8A分辨率,正在成为解析分子量很大的蛋白质复合体高分辨结构的有效技术手段。低温电子断层成像技术目前对于真核细胞样品的结构解析已达到约40A的分辨率,在今后5年有望达到20A。这样,把x射线晶体学、NMR以及电镜三维重构获得的蛋白质分子及复合体的高分辨率的结构,锚定到较低分辨率的电子断层成像图像中,从而在细胞水平上获得高精确的蛋白质空间定位和原子分辨率的蛋白质相互作用的结构信息。这将成为把分子水平的结构研究与细胞水平的生命活动衔接起来的可行途径。