Morphology of the cyst of Giardia microti by light and electron microscopy

Cysts of Giardia microti, isolated from feces and intestinal contents of Microtus ochrogaster, were examined by light and electron microscopy. These cysts differed morphologically from cysts of other G. duodenalis morphological types in that these cysts often contained two apparently differentiated trophozoites with mature ventral discs. Cysts more closely resembling those reported for G. lamblia and G. muris were in greater abundance in preparations made from intestinal contents and were interpreted as immature cysts. "Multiple fission" cysts, reported in G. muris and G. microti by earlier workers, were not observed; however, endosymbiotic bacteria were found in the cysts of G. microti and may have been responsible for reports of multiple fission in the cysts of Giardia.     

Processing scarce biological samples for light and transmission electron microscopy

Light microscopy (LM) and transmission electron microscopy (TEM) aim at understanding the relationship structure-function. With advances in biology, isolation and purification of scarce populations of cells or subcellular structures may not lead to enough biological material, for processing for LM and TEM. A protocol for preparation of scarce biological samples is presented. It is based on pre-embedding the biological samples, suspensions or pellets, in bovine serum albumin (BSA) and bis-acrylamide (BA), cross-linked and polymerized. This preparation provides a simple and reproducible technique to process biological materials, present in limited quantities that can not be amplified, for light and transmission electron microscopy.     

Size analysis of biological membrane vesicles by gel filtration, dynamic light scattering and electron microscopy

Biological membrane vesicles are analysed in terms of size and size distribution using gel filtration on Sephacryl S-1000, electron microscopy and quasi-elastic light scattering. The agreement between the three methods is satisfactory particularly for homogeneous dispersions. Gel filtration on Sephacryl S-1000 is a quick and convenient method for the routine size analysis of membrane vesicles up to a diameter of about 250 nm.     

Visualizing aquatic bacteria by light and transmission electron microscopy

The understanding of the functional role of aquatic bacteria in microbial food webs is largely dependent on methods applied to the direct visualization and enumeration of these organisms. While the ultrastructure of aquatic bacteria is still poorly known, routine observation of aquatic bacteria by light microscopy requires staining with fluorochromes, followed by filtration and direct counting on filter surfaces. Here, we used a new strategy to visualize and enumerate aquatic bacteria by light microscopy. By spinning water samples from varied tropical ecosystems in a cytocentrifuge, we found that bacteria firmly adhere to regular slides, can be stained by fluorochoromes with no background formation and fast enumerated. Significant correlations were found between the cytocentrifugation and filter-based methods. Moreover, preparations through cytocentrifugation were more adequate for bacterial viability evaluation than filter-based preparations. Transmission electron microscopic analyses revealed a morphological diversity of bacteria with different internal and external structures, such as large variation in the cell envelope and capsule thickness, and presence or not of thylakoid membranes. Our results demonstrate that aquatic bacteria represent an ultrastructurally diverse population and open avenues for easy handling/quantification and better visualization of bacteria by light microscopy without the need of filter membranes.     

Morphology of Giardia agilis: observation by scanning electron microscopy and interference reflexion microscopy

The flagellated protozoan, Giardia agilis, was isolated from tadpole small intestine and examined by scanning electron microscopy and interference reflexion microscopy. The general morphology of the G. agilis trophozoite is similar to G. muris and G. duodenalis, but with modifications that reflect its elongated form. Interference reflexion microscopic analysis of attachment of G. agilis reveals a pattern of focal contacts by the lateral crest of the ventral disc, the ventrolateral flange, the lateral shield, and by numerous microvillus-like appendages found along the lateral border of the trophozoite. The pattern of focal contacts was observed to be dynamic; trophozoites were observed to make and break the focal contacts in a relatively short time and to glide along the surface of the substratum without breaking focal contacts.     

Viruses and the development of quantitative biological electron microscopy

The electron microscope has become an important tool for determining the structure of biological materials of all kinds. Many technical advances in specimen preparation and in sophisticated methods of image analysis, initially based on optical systems but latterly on computer processing, have contributed to the development of the subject. Viruses of various kinds have often provided a convenient and appropriate test specimen. This paper describes the major technical advances and shows how viruses have had an important role in most of the developments.     

Morphology of the second- and third-instar larvae of Dermatobia hominis by scanning electron microscopy

Larvae of Dermatobia hominis 10–27 days old were collected from experimentally infected rats and their morphology was studied by scanning electron microscopy. The moult from the second to third instar occurs at 18 days, with emergence from the host at 30 days post-infection. The second-instar larvae bear on the pseudocephalon, antennae (coeloconic sensilla), and coeloconic and basicoconic sensilla on the maxillary sensory complex. The thoracic segments bear small backwardly-directed spines anteriorly and ventral trichoid and campaniform sensilla. The first four abdominal segments have small and large backwardly-directed spines that are absent on segments five and six. The seventh and eighth abdominal segments have medium-sized forwardly-directed spines. Abdominal segments are encircled by campaniform sensilla. The terminal end of the eighth abdominal segment bears the anus, prominent anal lobes and two spiracular openings on each spiracular plate. Spiracular plates show a radial sun ray pattern. The rear abdomen also bears an ecdysal aperture, several pores and eight coeloconic sensilla. Although there are slight morphological differences, the spines (predominantly flat and thorn-like) and sensilla (campaniform and coeloconic) of the third-instar larvae show a similar arrangement to that of second-instar larvae. Thoracic trichoid sensilla are not seen in third-instar larvae. A perispiracular gland aperture is situated above each posterior spiracular opening. These morphological features are compared with those of other cuterebrid larvae.     

Visualization of identified GFP-expressing cells by light and electron microscopy.

We have developed a procedure for visualizing GFP expression in fixed tissue after embedding in LR White. We find that GFP fluorescence survives fixation in 4% paraformaldehyde/0.1% glutaraldehyde and can be visualized directly by fluorescence microscopy in unstained, 1 microm sections of LR White-embedded material. The antigenicity of the GFP is retained in these preparations, so that GFP localization can be visualized in the electron microscope after immunogold labeling with anti-GFP antibodies. The ultrastructural morphology of tissue fixed and embedded by this protocol is of quality sufficient for subcellular localization of GFP. Thus, expression of GFP constructs can be visualized in living tissue and the same cells relocated in semithin sections. Furthermore, semithin sections can be used to locate GFP-expressing cells for examination by immunoelectron microscopy of the same material after thin sectioning.     

Postembedding immunocytochemical localization of paramyxovirus antigens by light and electron microscopy

A postembedding method is described to localize antigens specific for various paramyxoviruses in sections of cells and tissues that have been fixed and embedded in epoxy resins for conventional electron microscopy. Viral antigens were localized in CV-1 cell cultures infected with simian virus 5 (SV5), brains of suckling hamsters inoculated with either neuroadapted mumps virus or hamster-adapted measles virus, and brains of adult mice infected with Sendai (parainfluenza I) virus. Both 1-micrometer-thick and thin (gold) tissue sections were etched with alcoholic sodium hydroxide-solution and then treated following either the unlabeled antibody peroxidase-antiperoxidase or the biotinylated protein A:avidin peroxidase procedure. Primary reagents included immunoglobulin isolated from hyperimmune rabbit sera with specificity to the major viral components of SV5 or SV5 hemagglutinin-neuraminidase, to whole mumps virus or mumps virus nucleocapsids, and to whole Sendai virus. Crude rabbit anti-Sendai virus antiserum and whole human subacute sclerosing panencephalitis (SSPE) sera were used in parallel. The results indicate that tissues processed for conventional evaluation by electron microscopy may be suitable, within limits, for postembedding immunocytochemical staining of paramyxovirus antigens.     

Errors in measurement of section areas and perimeters of microvessels from their profiles in sections for light and electron microscopy]

The error of measuring the section areas and perimeters of microvessels was estimated, which is due to a deviation of the section plane from the direction perpendicular to the longitudinal axis of microvessels. The estimates of the microvessel nonperpendicular sectioning error correspond to the case of three-dimensional isotropic distribution of microvessels without their own eccentricity. Only those microvessel profiles are selected for morphometry that meet the condition theta min < or = b/c < or = 1.00, where b is the minor and c is the major radii of microvessel profile on a section, and theta min is the tolerance for profile nonperpendicularity.     

Pitfalls of immunogold labeling: analysis by light microscopy, transmission electron microscopy, and photoelectron microscopy

The immunogold method is widely used to localize, identify, and distinguish cellular antigens. There are, however, some pitfalls that can lead to nonspecific binding, particularly in cytoskeletal studies with gold probes prepared from small gold particles. We present a list of suggestions for minimizing nonspecific binding, with particular attention to two problems identified in this study. First, we find that the method used to prepare the colloidal gold particles affects the degree of nonspecific binding. Second, the standard BSA-stabilized small gold probes evidently possess exposed regions that bind to the proteins of cytoskeletal preparations. This was investigated in whole-mount cytoskeletal preparations of cultured cells by use of light microscopy, transmission electron microscopy, and photoelectron microscopy of silver-enhanced specimens. Gold probes were made from approximately 5-nm particles generated by reduction of HAuCl4 with three different reducing agents: white phosphorus, sodium borohydride, and citrate-tannic acid. All three preparations stabilized in the conventional way showed significant levels of nonspecific binding, which was highest with citrate-tannic acid. This problem was largely solved with all three types of probes by including fish gelatin in the probe buffer, by substituting fish gelatin for the BSA stabilizer used to prepare the probes, or by pre-adsorption methods. Application of these techniques resulted in clear immunogold labeling patterns with minimal nonspecific background.     

X-ray microanalysis of biological specimens by high voltage electron microscopy

For the purpose of analyzing and imaging chemical components of cells and tissues at the electron microscopic level, 3 fundamental methods are available, chemical, physical and biological. Among the physical methods, two methods qualifying and quantifying the elements in the structural components are very often employed. The first method is radioautography which can demonstrate the localization of radiolabeled compounds which were incorporated into cells and tissues after the administration of radiolabeled compounds. The second method is X-ray microanalysis which can qualitatively analyze and quantify the total amounts of elements present in cells and tissues. We have developed the two methodologies in combination with intermediate high or high voltage transmission electron microscopy (200–400 kV) and applied them to various kinds of organic and inorganic compounds present in biological materials. As for the first method, radioautography, I had already contributed a chapter to PHC (37/2). To the contrary, this review deals with another method, X-ray microanalysis, using semi-thin sections and intermediate high voltage electron microscopy developed in our laboratory.

X-ray microanalysis is a useful method to qualify and quantify basic elements in biological specimens. We first quantified the end-products of histochemical reactions such as Ag in radioautographs, Ce in phosphatase reaction and Au in colloidal gold immunostaining using semithin sections and quantified the reaction products observing by intermediate high voltage transmission electron microscopy at accelerating voltages from 100 to 400 kV. The P/B ratios of all the end products Ag, Ce and Au increased with the increase of the accelerating voltages from 100 to 400 kV. Then we analyzed various trace elements such as Zn, Ca, S and Cl which originally existed in cytoplasmic matrix or cell organelles of various cells, or such elements as Al which was absorbed into cells and tissues after oral administration, using both conventional chemical fixation and cryo-fixation followed by cryo-sectioning and freeze-drying, or freeze-substitution and dry-sectioning, or freeze-drying and dry-sectioning producing semithin sections similarly to radioautography. As the results, some trace elements which originally existed in cytoplasmic matrix or cell organelles of various cells in different organs such as Zn, Ca, S and Cl, were effectively detected. Zn was demonstrated in Paneth cell granules of mouse intestines and its P/B ratios showed a peak at 300 kV. Ca was found in human ligaments and rat mast cells with a maximum of P/B ratios at 350 kV. S and Cl were detected in mouse colonic goblet cells with maxima of P/B ratios at 300 kV. On the other hand, some elements which were absorbed by experimental administration into various cells and tissues in various organs, such as Al in lysosomes of hepatocytes and uriniferous tubule cells in mice was detected with a maximum of P/B ratios at 300 kV.

From the results, it was shown that X-ray microanalysis using semi-thin sections observed by intermediate high voltage transmission electron microscopy at 300–400 kV was very useful resulting in high P/B ratios for quantifying some trace elements in biological specimens. These methodologies should be utilized in microanalysis of various compounds and elements in various cells and tissues in various organs.