Quantitative light and scanning electron microscopy of ferret sperm.

Sperm were obtained via electroejaculation from Domestic ferret, (Mustela putorius furo), Siberian ferret (M. eversmanni), Black-footed ferret (M. nigripes), and a hybrid between Siberian and Domestic, called the Fitch ferret (M. sp.). Comparisons of sperm were made by four different microscopy techniques to determine whether differences exist among species. First, Nomarski differential interference microscopy could be used to distinguish domestic ferret sperm from the others on the basis of the structure of the posterior part of the acrosome. Second, both silver staining, which demonstrates argentophilic protein distribution, and scanning electron microscopy (SEM), revealed differences among the morphology of sperm for each species; variation in the unique appearance of the acrosome in ferret sperm was detected especially well by SEM. To quantify differences in morphology, five sperm head parameters were measured using image analysis; light microscopy produced significantly larger values than did SEM (all parameters and all species but Fitch), and there were significant differences owing to species for all parameters but one. Generally, our data demonstrate the value of complementary techniques to distinguish among sperm of closely related species and more specifically may help establish evolutionary relationships among the ferret species studied. In addition, they provide baseline data important for the captive breeding of the endangered Black-footed ferret.     

Probing the building blocks of eumelanins using scanning electron microscopy

Scanning electron microscopy (SEM) is used to examine the structure of natural and synthetic melanins. Eumelanin from Sepia officinalis and synthetic eumelanin are found to be structurally dissimilar. The natural sample has a significant structural order with subunits that have a lateral dimension of approximately 15 nm. The synthetic samples, on the other hand, appear to be amorphous solids. These results lend support for the existence of fundamental structural units proposed from the analyses of wide-angle X-ray diffraction measurements and previous mass-spectrometry results. These findings also provide insight into the disparate photophysical behavior of Sepia and synthetic eumelanin.     

An analysis of human sperm chromosome breakpoints.

Sperm chromosome analysis of 19 sperm donors with either normal or balanced karyotypes was carried out in order to explore the nature of sperm chromosome structural aberrations. A total of 2,389 cells (range 36-298/donor) were karyotyped after in vitro penetration of hamster eggs. The median percentage of sperm structural aberrations was 9.3% (SD +/- 4.7; range 0%-17.8%), with a total of 247 breakpoints, of which 220 could be characterized fully. Two sets of donors were studied in two different centers: center 1 (United States) and center 2 (Spain). The frequencies of nonrejoined and rejoined chromosome-type aberrations were very similar between center 1 and center 2: 83.6% and 10.0%, and 75.0% and 10.3%, respectively. Chromatid-type aberrations were more frequent in center 2 (14.7%) than in center 1 (6.4%) (P = .037). Chromosome 4 had less than the expected number of breakpoints (P < .001). A positive significant correlation was found between sperm breakpoints reported in this study and sites of balanced chromosome de novo rearrangements detected at prenatal diagnosis and reported in the literature (P = .0001).     

麦蛾茧蜂触角感器的扫描电镜观察

应用扫描电镜对麦蛾茧蜂BraconhebetorSay的触角感器进行观察。结果表明:麦蛾茧蜂的触角上存在6种感器,分别为毛形感器,板形感器,刺形感器,鳞状感器,锥形乳头状感器和嗅孔。其中毛形感器和板形感器是主要感器,数量较大,分布较广。雌雄蜂的触角感器差异不明显。     

Imaging of anisotropic cellulose suspensions using environmental scanning electron microscopy

The effect of concentration on anisotropic phase behavior of acid-hydrolyzed cellulose suspensions has been examined using conventional polarizing microscopy and the novel technique of environmental scanning electron microscopy (ESEM). Microcrystalline cellulose dispersed in water formed biphasic suspensions in a narrow concentration range, 4-12 wt % for a suspension pH of 4, where the upper and lower phases were isotropic and anisotropic (chiral nematic), respectively. It is known from previous work that within the biphasic regime total suspension concentration affects only the volume fractions of the two phases, not phase concentration or interfacial packing. As the total suspension concentration surpassed the upper critical limit (c), however, a single anisotropic phase of increasing concentration was observed. It was evident from polarizing microscopy that the chiral nematic pitch of the anisotropic phase decreased with increasing concentration, which has been attributed to a reduction in the electrostatic double layer thickness of the individual rods, thus increasing intermolecular interactions. Chiral nematic textures were also visible using ESEM. This technique has the advantage of studying individual rod orientation within the liquid crystalline phase as it permits the high resolution of electron microscopy to be applied to hydrated samples in their natural state. To our knowledge this is the first time such lyotropic systems have been observed using electron microscopy.     

Effects of bromodeoxyuridine substitution on metaphase chromosome structures examined by scanning electron microscopy

Chinese hamster chromosomes were differentially substituted with 50 M 5-bromodeoxyuridine (BrdU) to obtain chromosomes with bifilarly and unifilarly substituted (BB-TB) and unifilarly and non-substituted (TB-TT) chromatid constitutions. To avoid the effect of Giemsa staining on the ultrastructure of chromosomes, unstained preparations were exclusively used. When TB-TT chromosomes were prepared with the conventional air-drying method followed by the osmium tetroxide-thiocarbohydrazide (OsO₄-TCH) technique and examined by scanning electron microscopy (SEM), the TB-chromatid appeared somewhat more slender and showed more conspicuous spiral structures, thereby appearing more loosened compared to the TT-chromatid. At higher magnifications, however, 30 nm chromatin fibres which were seen to constitute both chromatids showed no discernible differences in dimension between the TT- and TB-chromatids. On the other hand, TB-TT chromosomes specially prepared for SEM without the process of air-drying appeared in their entirety less extended and no spiral configuration was observed even in the TB-chromatid. The TB-chromatid instead appeared rather less loosened than the TT-chromatid whereas thick fibre-like structures which in turn seemed to consist of 30 nm fibres were more easily discernible in the TT-chromatid compared to the TB. Such seemingly contradictory results obtained from the two different preparatory procedures were tentatively explained on the basis of our multiple coiling model (Taniguchi and Takayama 1986).     

Comparative atomic force and scanning electron microscopy: An investigation of structural differentiation of hepatic stellate cells

The molecular mechanism leading to the transdifferentiation of hepatic stellate cells (HSC) into myofibroblast-like cells following liver injury is not well understood. The state of cultured rat HSCs was determined using primarily fluorescence microscopy (UV), immunofluorescence (IF) (Glial fibrillary acidic protein (GFAP), Desmin, alpha-smooth muscle actin (alpha-SMA), F-actin) and immunocytochemistry (ICC) (GFAP, Desmin, alpha-SMA, Fibulin-2). Additionally, tapping-mode atomic force microscopy (TM-AFM) and field-emission scanning electron microscopy (FE-SEM) with low-resistivity indium-tin-oxide (ITO) thin-film were performed to observe the micro-morphological character of cells during HSC differentiation. Quiescent HSCs changed to the activated state were identified via UV, IF, and ICC observations. Normal rat HSCs (NHSCs) and thioacetamide-induced rat HSCs (THSCs) were demonstrated to be UV⁻, GFAP⁺, Desmin⁺, alpha-SMA⁺ and Fibulin-2⁻. After F-actin staining, lamellipodia and filopodia were found in both NHSCs and THSCs, but membrane ruffles were only seen in THSCs. The micro-structures of lamellipodia and filopodia in both NHSCs and THSCs were confirmed using FE-SEM and TM-AFM with ITO; in contrast, the micro-projection was not found. Moreover, “aerial root” structures were observed for the first time in the filopodia of THSCs using TM-AFM. These results reveal that HSC transdifferentiation to a myofibroblastic-like cell (activated HSC) from thioacetamide-induced rat HSC induces extensive changes in the cytoskeleton.     

Production of injection replicas of microvessels for scanning electron microscopy using resin "PN-8"]

A method for obtaining injection replications of microvessels for scanning electron microscopy using nonsaturated polyether resin PH-8 is described. Possible applications of the method in question to study microcirculatory bed are discussed. With resine PH-8 it is possible to obtain complete and detailed replications which give information on three-dimensional organization both of the microcirculatory bed and of the vascular microrelief peculiarities.     

Improved preservation of rat epididymal sperm for high-resolution low-voltage scanning electron microscopy (HR-LVSEM)

Various fixation protocols were used in an attempt to improve preservation of rat epididymal sperm for high-resolution low-voltage scanning electron microscopy (HR-LVSEM). Wash solutions and fixatives of different composition and osmolarity were tested. Paraformaldehyde and glutaraldehyde concentrations were varied between 0.5% and 3%. Ruthenium red was tested as an additive in both primary fixation and postfixation, or in postfixation alone. HR-LVSEM revealed various degrees of ruffing, folding, blebbing, and peeling off of the plasma membrane, as well as holes of different sizes. The plasma membrane overlying the acrosome and the connecting piece proved to be particularly sensitive to varying fixation conditions. Consistent topographical differences were revealed among the different domains over the sperm head. Most of the differences were considered to be artifacts. Their consistency, however, suggests that structural and biochemical differences exist either within the membrane or in the structures subjacent to the membrane. Primary fixation turned out to be less critical than postfixation. Preservation of a smooth plasma membrane without holes could only be achieved when primary fixation in low aldehyde concentrations, with or without ruthenium red, was followed by postfixation with OsO₄ and 1,000 ppm ruthenium red. Examination of thin sections of the same material confirmed that even a considerable number of small holes are difficult to detect in transmission electron microscopy. These results show that with the recent increase in resolution of LVSEM there is need for further effort to improve sample processing. © 1993 Wiley-Liss, Inc.     

Microwave processing for scanning electron microscopy

The normal processing of biological samples for Scanning Electron Microscopy, includes treatment with aldehyde (1 to 2 hours), postfixation with Osmium (1 hour), followed by dehydration in a ascending grade of ethanol (30 a 100%), 10 to 15 minutes in each step, and finally drying. This procedure takes at least 8 hours. In this work, samples of mosquitoes (Aedes), protozoa (Tritrichomonas muris), bacteria (Clostridium oceanicum), murine liver, and small intestine were processed in the same manner in a domestic microwave oven for two minutes at 20% of its maximum power. The complete procedure from the initial fixation to dehydration in 100% ethanol was reduced to one hour with good preservation of the ultrastructural details of the specimens.