A post-embedding avidin-biotin peroxidase system to demonstrate the light and electron microscopic localization of lectin binding sites in rat kidney tubules

Summary A post-embedding method for the light and electron microscopic demonstration of lectin binding sites in rat kidney tubules is described. The use of biotinylated lectins, followed by treatment with avidin peroxidase and the DAB—H₂O₂ sequence, produced intense staining of acrylic sections at the electron microscope level: brush borders and associated structures, cytoplasmic granules, basal infoldings and basement membrane—plasmalemmal interfaces of proximal tubules bound erythrophytohaemagglutinin, while distal tubules were mainly unstained. At the light microscope level, epoxy resin sections showed a similar staining pattern after etching, as did acrylic resin sections after intensification of the final reaction product. The binding of wheatgerm agglutinin to cytoplasmic granules and brush border structures in the proximal tubules was abolished, at both the light and electron microscope levels, by the competing sugar tri-N—acetylchitotriose. Epoxy resin ultrathin sections required etching before staining was achieved in the electron microscope, and results were far inferior to those obtained with acrylic resin. This method allows rapid and inexpensive screening of large numbers of lectins, if required, at both the light and electron microscope levels, using reagents that are stable for long periods of time.     

The use of organic dyes in electron microscopy

The extinctions produced by several organic dyes in the light microscope and the electron microscope are compared. The two have the same order of magnitude. The influence of the methacrylate embedding medium is not great enough to hamper visibility in the electron microscope. It is pointed out that a wide variety of organic stains should be useful for electron microscopic studies.     

Pollen morphology of the alangiaceae

This paper presents a pollen-morphological study of Alangium, a genus mainly restricted to the tropics of the Old World, of which 18 of the 19 known species were studied. The pollen grains, studied with the use of a light microscope, a transmission electron microscope and a scanning electron microscope, were classified into 15 pollen types, to which a key is given. Comparison of fossil and recent grains produced some information about both the evolutionary trends in the characters of the pollen grains and the phytogeography of Alangium.     

FIB/SEM tomography with TEM-like resolution for 3D imaging of high-pressure frozen cells

Focused ion beam/scanning electron microscopy (FIB/SEM) tomography is a novel powerful approach for three-dimensional (3D) imaging of biological samples. Thereby, a sample is repeatedly milled with the focused ion beam (FIB) and each newly produced block face is imaged with the scanning electron microscope (SEM). This process can be repeated ad libitum in arbitrarily small increments allowing 3D analysis of relatively large volumes such as eukaryotic cells. High-pressure freezing and freeze substitution, on the other hand, are the gold standards for electron microscopic preparation of whole cells. In this work, we combined these methods and substantially improved resolution by using the secondary electron signal for image formation. With this imaging mode, contrast is formed in a very small, well-defined area close to the newly produced surface. By using this approach, small features, so far only visible in transmission electron microscope (TEM) (e.g., the two leaflets of the membrane bi-layer, clathrin coats and cytoskeletal elements), can be resolved directly in the FIB/SEM in the 3D context of whole cells.     

Biomimetic zinc oxide replica with structural color using butterfly (Ideopsis similis) wings as templates

Nano-structured colorful zinc oxide (ZnO) replicas were produced using the wings of the Ideopsis similis butterfly as templates. The ZnO replicas we obtained exhibit iridescence, which was clearly observed under an optical microscope (OM). Field emission scanning electron microscope analysis shows that all the microstructure details are maintained faithfully in the ZnO replica. A computer model was established to simulate the diffraction spectral results, which agreed well with the OM images.     

Patterns of blood-vessel invasion by mammary tumor cells

Rat mammary tumor TMT-081 was employed as a model for blood vessel invasion because its mode of metastasis resembles that of human tumors. The invasive mechanism was studied with two methods of serial transplantation: transfer of enzymatically dispersed solid tumors, and transfer of buffy coat containing circulating tumor cells. The latter method produced greater invasion of blood vessels, including larger veins and occasionally arteries, perhaps by obviating damage to tumor cells during enzyme treatment. The course of migration was traced by three-dimensional examination in the high voltage electron microscope, as well as the light microscope. Two broad patterns were found for the course of invasion of small and large vessels respectively.     

两种脱矿体系制备人工龋损的病理学比较

目的评价两种不同脱矿体系在人牙釉质与牛牙釉质形成人工龋样损害的情况,确定模拟早期龋实验研究的脱矿系统。方法两种脱矿体系：部分饱和酸缓冲溶液和酸性凝胶溶液,分别在人牙釉质与牛牙釉质制备人工早期釉质龋损,采用偏振光显微镜和扫描电子显微镜观察实验人工龋的组织病理学变化。结果部分饱和的酸缓冲体系制备的人工龋损都具有完整的表层和表层下脱矿,牛牙釉质除了脱矿深度大于人牙釉质外,其它病理学特点都类似于人牙釉质,与早期自然龋损相似。而凝胶液脱矿体系形成的人工龋损不管是人牙还是牛牙都没有明显的表层结构,实验牙表面多出现被腐蚀或溶解。结论部分饱和的酸缓冲液脱矿系统形成的人工龋损病理学变化更接近于自然龋;与人牙具有相似化学组成的牛牙很适合代替人牙来检测脱矿和再矿化的效果。     

Hemagglutinins and fimbriae of Providencia spp.

A series of 25 strains of Providencia spp. produced at least five different patterns of hemagglutination as judged by results from hemagglutination tests in the presence and absence of D-mannose and at least six distinct types of fimbriae as seen from observations with the electron microscope.     

Phagocytic inability of Kurloff cells in the lung and spleen of the guinea pig

Summary Adult female guinea pigs received subcutaneous implants of diethylstilbestrol-cholestrol pellets which produced splenomegaly and increased numbers of splenic Kurloff cells. Latex spheres subsequently injected intravenously were not phagocytized by Kurloff cells within the lungs and spleen as examined with the electron microscope. This is considered as evidence that Kurloff cells are probably not phagocytic. The origin of these cells is discussed.     

Surface ultrastructure of trypsin-banded chromosomes

The surface ultrastructure of trypsin-banded chromosomes has been examined by electron microscopy. Trypsin pretreatment removed cellular debris and produced banding patterns recognizable with the electron microscope as ridges in platinum-carbon replicas. The ridges observed in replicas of trypsin-treated chromosomes corresponds to Giemsa-stained bands observed by light microscopy. The bands appeared as an area of tightly compacted fibrils on the surface of the chromosomes. Prolonged treatment in trypsin resulted in collapsed chromosomes and loss of ridges in the replicas. Interchromosomal fibers were also noted and in some preparations appeared to be localized to banded regions.     

Characterization and localization of the naturally occurring cross-links in vaccinia virus DNA

The vaccinia virus genome is a single, linear, duplex DNA molecule whose complementary strands are naturally cross-linked. The molecular weight has been determined by contour length measurements from electron micrographs to be 122 ± 2.2 × 10⁶. Denaturation mapping techniques indicate that the nucleotide sequence arrangement of the DNA is unique. Two forms of cross-linked vaccinia DNA were observed in alkaline sucrose gradients. The relative S-values of the two cross-linked species were appropriate for a single-stranded circle and a linear single strand, each with a molecular weight twice that expected for an intact, linear, complementary strand of vaccinia DNA. The fraction of sheared vaccinia DNA able to “snap back” after denaturation suggested a minimum of two crosslinks per molecule. Full-length single-stranded circles were observed in the electron microscope after denaturation of vaccinia DNA. Partial denaturation produced single-stranded loops at the ends of all full-length molecules. Exposure of native vaccinia DNA to a single strand-specific endonuclease isolated from vaccinia virions caused disruption of the cross-links, as assayed by alkaline sedimentation, and produced free single-strand ends when partially denatured DNA was observed in the electron microscope. We conclude that vaccinia DNA contains two cross-links, one at or near (within 50 nucleotides) each end in a region of single-stranded DNA. Two models for the cross-links are presented.     

On the spinning apparatus and the structure of the capture threads ofDeinopis subrufus (Araneae,Deinopidae)

Summary The spinnerets and spigots of two adult femaleDeinopis subrufus and one adult male were studied with the scanning electron microscope. The organization of the spinning apparatus corresponds very closely to that of Uloboridae. The capture threads produced by the male while subadult were studied with the light microscope and with the SEM. This study includes the questions of the glandular origin and the functions of the components of these threads. A hypothesis for howD. subrufus constructs capture threads is proposed.     

ELECTRON MICROSCOPE STUDIES ON ENZYME ACTIVITY AND THE ISOLATION OF THIOHYDANTOIN-INDUCED MYELIN FIGURES IN RAT LIVER

Characteristic cytoplasmic inclusions (myelin figures), consisting of concentric multilaminar paired membranes surrounding one or more lipid bodies, were produced in rat liver parenchymal cells by incorporating high doses of an anticonvulsant agent (Bax 422Z) into the animals' diet. Enzymatic reaction product (presumably lead phosphate) was found around the central fat of these myelin figures in liver which had been fixed in glutaraldehyde, incubated in Wachstein and Meisel's medium containing adenosine triphosphate or inosine tri- or diphosphate, postosmicated, embedded in epoxy resin, and examined in the electron microscope. In an attempt to isolate myelin figures, fresh liver from medicated rats was homogenized and differentially centrifuged. Thin sections of osmium tetroxide-fixed, Epon-embedded pellets from each fraction were examined with the electron microscope. The concentric membranous whorls, which are probably derived from cisternae of the endoplasmic reticulum, broke up as the cells were disrupted and became inextricably mixed with the microsomal fraction. However, when liver previously fixed in formalin for 24 hours was homogenized, the myelin figures remained intact.     

Fungal adhesion in aquatic hyphomycetes

The attachment of propagules of aquatic fungi often takes place under turbulent conditions, i.e. spores of marine lignicolous fungi and conidia of freshwater Hyphomycetes. It has been demonstrated that attachment in Lemonniera aquatica is effected by three arms of the conidium making contact with the surface and forming appressoria. In this study the mechanisms of attachment of conidia of 10 aquatic Hyphomycetes to a variety of natural and artificial substrata were followed at the light microscope and scanning electron microscope levels.Five species produced appressoria, while in the remaining species attachment and subsequent retention to the substratum was effected solely by the production of mucilage. Examination of selected appressorium-forming species in the transmission electron microscope showed that conidium attachment involved the production of mucilage in the regions of initial contact and only then were appressoria formed. The ecological significance of appresoiria to the aquatic Hyphomycetes is discussed.     

Characterization of chromosome replication during S-phase with bromodeoxyuridine labelling in Chinese hamster ovary and HeLa cells

The same C-banded human polymorphic chromosomes were observed in the light microscope (LM) and then in the scanning electron microscope (SEM) to investigate the structural changes produced by the C-banding technique. C-banded regions, which stained positively in LM, were highly condensed with tightly packed chromatin fibres, resembling non-banded chromosomes. In striking contrast, adjacent non-C-banded regions were represented by loosely arranged fibres, resembling G-banded chromosomes. The significance of these observations in relation to current theories on the effects of C-banding on chromosome structure is discussed.     

Energy filtered electron tomography of ice-embedded actin and vesicles.

Semiautomatic single-axis tilt electron tomography has been used to visualize the three-dimensional organization of actin filaments in "phantom cells," i.e. lipid vesicles. The instrumentation consisted of a 120-kV electron microscope equipped with a postcolumn energy filter, which was used in the zero-loss imaging mode. Apart from changing the tilt angle, all steps required for automated tomography, such as recentering the image area, refocusing, and centering the energy-selecting slit, were performed by external computer control. This setup permitted imaging of ice-embedded samples up to a thickness of 800 nm with improved image contrast compared with that produced by tomography with a conventional electron microscope. In spite of the missing-wedge effect that is especially obvious in the study of membrane-filament interaction, single-axis tilt tomography was found to be an appropriate (in fact the only available) method for this kind of investigation. In contrast to random actin networks found in actin gels, actin filaments in and on vesicles with a bending radius of less than approximately 2 microns tend to be arranged in single layers of parallel filaments and often induce an elongated shape of the vesicles. Actin filaments located on the outside usually associate with the vesicle membrane.     

Preparation and properties of pollen sporoplasts

Summary Methods for the removal of exine from mature, ungerminatedLilium longiflorum pollen and release of intact gametophytes (sporoplasts) have been developed. These methods rely on the low temperature solvolytic activity of 4-methylmorpholine N-oxide (MMNO), which allows partial or complete detachment of exine from intine during subsequent washing procedures. These methods are: aqueous MMNO combined with cyclohexylamine (method I), aqueous MMNO at alkaline pH (method II), and aqueous MMNO containing a high Ca²⁺ concentration with added cellulysin and macerase (method III). Sporoplasts produced by methods I and II are most frequently completely separated from exine and, as shown by histochemical tests, enveloped by the intine layer. Selected enzyme activities in method II sporoplasts are measurable but, as indicated by other tests, considerable damage to the plasma membrane accompanies this treatment. Sporoplasts produced by melhod III largely remain attached to their ruptured exine layer and retain substantial biological competence in terms of extractable enzyme activities, membrane integrity, and respiration.Abbreviations MMNO 4-methylmorpholine N-oxide - SEM scanning electron microscope - TEM transmission electron microscope     

Scanning and transmission electron microscopy of human ejaculate spermatozoa with special reference to their abnormal forms

Summary Spermatozoa from fertile and infertile human ejaculates were observed under the scanning electron microscope. A parallel study of sections was performed by transmission electron microscope.The normal head shows under the scanning electron microscope vesicular elevations in the region of the acrosome and a smooth and rigid appearance corresponding to the postnuclear cap whose occurrence is confirmed under the transmission electron microscope. Immediately anterior to this cap a shallow furrow transverses the head. Duplicated, unusually large or small and deformed heads are found under the scanning electron microscope. Most of these abnormal heads show no surface structure suggesting an acrosome.The neck and middle piece are occasionally, though frequently in abnormal spermatozoa, covered by a cytoplasmic droplet. Otherwise, the mitochondrial sheath is recognized under the scanning electron microscope as a beaded thickening in the middle piece. The lack of mitochondria is manifested by a smooth middle piece thinner than the principal portion. Transmission electron microscopy of sections reveals various types of anomalies in the number of cores, core filaments and mitochondria embedded in the cytoplasmic droplets.Abnormalities in the principal portion of the tail such as duplication, unusual thickness and length are shown under the scanning electron microscope.The investigation indicates that scanning electron microscopy is suited for the clinical as well as cytological examination of human ejaculate spermatozoa.     

The classic Golgi apparatus and vacuoles

The dense vacuoles, considered to be the classic Golgi apparatus in the root meristem ofFagopyrum, were studied by the following methods: 1. Impregnation methods for the demonstration of the Golgi apparatus, 2. cytochemical methods, 3. electron microscopic methods in the light microscope and 4. the electron microscope. A comparison was made with the classic Golgi apparatus in animal cells in the light and electron microscope. Dense vacuoles inFagopyrum and also evidently in other plants, were taken for the classic Golgi apparatus on account of their morphological similarity to the Golgi apparatus in animal cells on impregnation with silver and osmium and their staining preperties with lipoid methods. Dense vacuoles differ from the classic Golgi apparatus in other chemical properties, such as content of phenol substances, etc. No formations were found in animal cells which were similar to dense vacuoles on investigating by electron microscopy. In the electron microscope dense vacuoles have the appearance of derivatives of the normal light vacuoles known in plant cells. They therefore belong to vacuome of plant cell and cannot be analogous to the classic Golgi apparatus in animal cells. Thus the use of the term Golgi apparatus for dense vacuoles is not well founded. A comparison was made of fixation and impregnation used in the light microscope with fixation in the electron microscope. After fixation with permanganate, dense vacuoles have the same shape as after impregnation. After fixation with permanganate, they stain an intense black in the same way as after impregnation with silver and osmium. The form of the vacuoles is dependent on the fixation used. The comparison was made in the light microscope.