The crater defect in boar spermatozoa: A correlative study with transmission electron microscopy,scanning electron microscopy,and light microscopy

Nuclear vacuoles resembling the “crater defect” described in bull spermatozoa were observed in 14 boars. Both the incidence of the defect and semen quality were monitored with phase contrast microscopy over a three-month period. The percentages of cratered spermatozoa varied widely both among boars and in ejaculates from the same boar taken on different days. The presence of cratered spermatozoa at a level of 5% or more appeared to be associated with low semen quality. The defect was studied with scanning and transmission electron microscopy and was found to consist of nuclear invaginations, about 0.5 μm in diameter, containing some scanty amorphous electron-dense material. In boars showing a high incidence of spermatozoa with crater defects, abnormalities of the acrosome and perforatorium were common.     

Chromosomes and karyotype analysis of a liver fluke, Opisthorchis viverrini, by scanning electron microscopy

Opisthorchis viverrini, a human liver fluke, has been categorized as the carcinogenic organism according to the strong association with carcinogenesis of cholangiocarcinoma (CCA). The infection of this food-borne parasite is a major impact on the health of humans, especially CCA patients in the northeast of Thailand. Taxonomy, morphology, epidemiology and molecular study of O. viverrini have been publicized increasingly but the precise karyotypic study is still incomplete. In this study, the chromosomes of O. viverrini were prepared from the testes of adult worms retrieved from metacercariae infected-hamsters. The chromosomes of O. viverrini were identified in haploid (n=6) meiotic metaphase and in diploid (2n=12) mitotic metaphase by light microscopy. The chromosome number, length and nomenclature of each chromosome were determined by scanning electron microscopy. The six chromosomes consist of one large-sized metacentric, one medium-sized metacentric, two small-sized metacentric, one small-sized submetacentric and one small-sized acrocentric chromosomes with the lengths of 2.84±0.03, 2.12±0.10, 1.71±0.13, 1.44±0.04, 1.23±0.03 and 0.84±0.13 μm, respectively. This is the first karyotype analysis of O. viverrini with defined complete nomenclature.     

Environmental scanning electron microscopy offers real-time morphological analysis of liposomes and niosomes

Liposomes have been imaged using a plethora of techniques. However, few of these methods offer the ability to study these systems in their natural hydrated state without the requirement of drying, staining, and fixation of the vesicles. However, the ability to image a liposome in its hydrated state is the ideal scenario for visualization of these dynamic lipid structures and environmental scanning electron microscopy (ESEM), with its ability to image wet systems without prior sample preparation, offers potential advantages to the above methods. In our studies, we have used ESEM to not only investigate the morphology of liposomes and niosomes but also to dynamically follow the changes in structure of lipid films and liposome suspensions as water condenses on to or evaporates from the sample. In particular, changes in liposome morphology were studied using ESEM in real time to investigate the resistance of liposomes to coalescence during dehydration thereby providing an alternative assay of liposome formulation and stability. Based on this protocol, we have also studied niosome-based systems and cationic liposome/DNA complexes.     

Optimizing parameters for correlative immunogold localization by video-enhanced light microscopy, high-voltage transmission electron microscopy, and field emission scanning electron microscopy

Correlative video-enhanced light microscopy, high-voltage transmission electron microscopy, and low-voltage high resolution scanning electron microscopy were used to examine the binding of colloidal gold-labeled fibrinogen to platelet surfaces. Optimal conditions for the detection of large (18 nm) and small (3 nm) gold particles are described.     

Complementary visualization of mitotic barley chromatin by field-emission scanning electron microscopy and scanning force microscopy

The surface structure of mitotic barley chromatin was studied by field-emission scanning electron microscopy (FESEM) and scanning force microscopy (SFM). Different stages of the cell cycle were accessible after a cell suspension was dropped onto a glass surface, chemical fixed, and critically point dried. Imaging was carried out with metal-coated specimen or uncoated specimen (only for SFM). The spatial contour of the chromatin could be resolved by SFM correlating to FESEM data. The experimentally determined volume of the residue chromatin during mitosis was within the range of 65-85 microm(3). A comparison with the theoretically calculated volume indicated a contribution of about 40% of internal cavities. Decondensation of chromosomes by proteinase K led to a drastic decrease in the chromosome volume, and a 3-D netlike architecture of the residue nucleoprotein material, similar to that in the intact chromosome, was obvious. Incubation of metaphase chromosomes in citrate buffer permitted access to different levels of chromatin packing. We imaged intact chromosomes in liquid by SFM without any intermediate drying step. A granular surface was obvious but with an appreciably lower resolution. Under similar imaging conditions proteinase K-treated chromosomes exhibited low topographic contrast but were susceptible to plastic deformations.     

Transmission electron microscopy of tissue prepared for scanning electron microscopy by ethanol-cryofracturing.

Tissue processed for scanning electron microscopy by ethanol-cryofracturing combined with critical point drying was embedded and sectioned for transmission electron microscopy. Study of specimens cut in a plane passing through the fracture edge indicated that preservation of cellular fine structure of fractured cells was excellent. Even at the most peripheral edge of the fracture there was no evidence that movement of cytoplasmic components occurred to distort the original structural organization of fractured cells. Lack of cytoplasmic detail in ethanol-cryofractographs has been due more to the nature of the fracturing of the tissue and to the obscuring effects of the metal coating than to structural deformation at the fracture edge or to limitations in resolving power of the scanning electron microscope used.     

High resolution scanning electron microscopy of plasmodesmata

Symplastic transport occurs between neighbouring plant cells through functionally and structurally dynamic channels called plasmodesmata (PD). Relatively little is known about the composition of PD or the mechanisms that facilitate molecular transport into neighbouring cells. While transmission electron microscopy (TEM) provides 2-dimensional information about the structural components of PD, 3-dimensional information is difficult to extract from ultrathin sections. This study has exploited high-resolution scanning electron microscopy (HRSEM) to reveal the 3-dimensional morphology of PD in the cell walls of algae, ferns and higher plants. Varied patterns of PD were observed in the walls, ranging from uniformly distributed individual PD to discrete clusters. Occasionally the thick walls of the giant alga Chara were fractured, revealing the surface morphology of PD within. External structures such as spokes, spirals and mesh were observed surrounding the PD. Enzymatic digestions of cell wall components indicate that cellulose or pectin either compose or stabilise the extracellular spokes. Occasionally, the PD were fractured open and desmotubule-like structures and other particles were observed in their central regions. Our observations add weight to the argument that Chara PD contain desmotubules and are morphologically similar to higher plant PD.     

Image analysis of Artemia salina ribosomes by scanning transmission electron microscopy.

A dedicated scanning transmission electron microscope (STEM) at Brookhaven National Laboratory was used to visualize unstained freeze-dried ribosomal particles under conditions which considerably reduce the specimen distortion inherent in the heavy metal staining and air-drying preparative steps used in routine transmission electron microscopy (TEM). From high-resolution STEM images it is possible to determine molecular mass and the mass distribution within individual ribosomal particles and perform statistical evaluation of the data. Analysis of digitized STEM images of Artemia salina ribosomes provided evidence that a standard preparation of these eukaryotic ribosomes consists of a population of heterogenous particles. Because of the integrity of rRNAs established by agarose gel electrophoresis, variations in the composition and structure of the 80S monosomes and the large (60S) and small (40S) ribosomal subunits, as monitored by their mass, were attributed to the loss of ribosomal proteins, from the large subunits in particular. These results are relevant not only to the degree of ribosomal biological activity, but should also be taken into consideration for particle selection in the reconstruction of the "native" eukaryotic ribosome 3-D model.     

Rapid phenotypic analysis of uncoated Drosophila samples with low-vacuum scanning electron microscopy

Research projects featuring repetitive phenotypic analysis of insects, such as taxonomic studies, quantitative genetics, and mutant screens, could be greatly facilitated by a simpler approach to scanning electron microscopy (SEM). Here, we have applied low-vacuum SEM to wild type and mutant Drosophila and demonstrate that high quality ultrastructure data can be obtained quickly using minimal preparation. Adult flies, frozen live for storage, were mounted on aluminum stubs with carbon cement and directly imaged, with no chemical treatment or sputter coating. The key imaging parameters were identified and optimized, including chamber pressure, beam size, accelerating voltage, working distance and beam exposure. Different optimal conditions were found for eyes, wings, and bristles; in particular, surface features of bristles were obscured at higher accelerating voltages. The chief difficulties were charging, beam damage, and sample movement. We conclude that our optimized protocol is well suited to large-scale ultrastructural phenotypic analysis in insects.     

A combined light and scanning electron microscopy study

The uppermost Eocene Florissant Formation, Rocky Mountains, Colorado, has yielded numerous insect, vertebrate and plant fossils. Three previous comprehensive palynological studies investigated sections of lacustrine deposits of the Florissant Formation and documented the response of plant communities to volcanic eruptive phases but overall found little change in plant composition throughout the investigated sections. These studies reported up to 150 pollen and spore phenotypes. In the present paper, we used a taxonomic approach to the investigation of dispersed pollen and spores of the Florissant Formation. Sediment samples from the shale units containing macrofossils were investigated using light microscopy (LM) and scanning electron microscopy (SEM). The general picture of the palynoflora is in agreement with previous studies. However, the combined LM and SEM investigation provides important complementary information to previous LM studies. While a fairly large amount of previous pollen determinations could be confirmed, the purported taxonomic affinities of several pollen phenotypes need to be revised. For example, pollen referred to as Podocarpus or Podocarpidites sp. belongs to the Pinaceae Cathaya, Malus/Pyrus actually belongs to Dryadoideae, pollen of the form genus Boehlensipollis referred to as Proteaceae/Sapindaceae/Elaeagnaceae or Cardiospermum belongs to Sapindaceae but not to Cardiospermum, and pollen of Persicarioipollis sp. B with previously assumed affinities to Polygonaceae actually belongs to Thymelaeaceae. Pandaniidites and one type of Malvacipollis cannot be linked with Pandanaceae and Malvaceae. A few taxa are new records for Florissant (Ebenaceae: Diospyros; Mernispermaceae; Trochodendraceae: Tetracentron). In general, SEM investigations complement the LM palynological studies and improve the identification of dispersed pollen and spores and enable integration of data from dispersed fossil pollen into a wide range of comparative morphological, taxonomic, evolutionary, biogeographic and phylogenetic studies.