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.     

A scanning electron microscopy study of micro-arterial damage and repair.

The effects of microvascular clamps on the femoral vessels of rats were studied, using the SEM. The early changes observed were (1) local fusiform dilatation of the area secondary to necrosis of the muscular wall, (2) flattening of the longitudinal ridges in the endothelial (3) loss of laminar flow, (4) endothelial sloughing, (5) platelet aggregation, and (6) leukocyte adherence and diapedesis. The repair of the endothelium occurred by an early replication of the adjacent undamaged endothelial cells -- with their subsequent migration across a platelet bed. The coverage was complete in one week, although reorientation of the neo-endothelial cells took longer. On the basis of this study and our clinical experience, we think the ideal microvascular clamp would possess the following characteristics: small size, light weight, mechanical simplicity, flat jaws (one to two mm in diameter) coated with a non-slip surface, and calibrated to produce a pressure less than 30 gm per mm2. In addition the clamp should be unaffected by blood, autoclaving, or repeated use. No such clamp is commercially available now, but we hope that one will be available in the near future.     

Relationship between membrane fluidity and capping of receptors for concanavalin A

The activities of a range of phenylalaninol-related compounds on capping of concanavalin A and induction of rounding of Chinese hamster ovary tsHl cells, as well as on the fluidity of phosphatidylcholine-cholesterol (1:1) liposomes, have been examined. These compounds include phenylalaninol, histidinol, leucinol, benzyl alcohol, benzylamine, 2-phenylethanol, 2-phenylamine, 3-phenyl-1-propanol, 3-phenyl-1-propylamine, and 3-phenylpropionic acid. The results indicate a strong correlation between the capacities of these compounds to enhance fluidity and their capacities to inhibit capping of concanavalin A. The specificity of this correlation is suggested by the finding that both types of capacities are poorly correlated with the capacities of the various compounds to induce cell rounding.     

A scanning electron microscope study of concanavalin a receptors on retinal rod cells labeled with latex microspheres

Con A-methacrylate microsphere conjugates prepared by a two-step glutaraldehyde reaction were used to label Con A-binding sites on bovine rod photoreceptor cells for visualization by scanning electron microscopy. A dense distribution of markers was observed on the surface of the rod outer segment, the inner segment, and the synaptic region. Disk membranes also appear to be heavily labeled with the Con A-microsphere conjugates. The Con A inhibitor, α-methyl mannoside, inhibited the binding of the conjugate to the surface of these visual cells.     

A scanning electron microscopy study of vascular casts from senile human brains.

A scanning electron microscope study was performed of vascular casts from 26 senile human brains. In 15 of these, three types of arterial deformities (glomerular loop formations, vascular wickerworks and bundles) were frequently encountered. They were compared with the appearance in microangiograms and histological sections.     

Development of Dictyostelium discoideum: A study by scanning electron microscopy

Stages in the development of the cellular slime mould Dictyostelium discoideum have been examined by scanning electron microscopy using specimens prepared by freeze-drying, infiltration with polyethylene glycol or freezing. Formation of the slime sheath characteristic of D. discoideum was first observed early in aggregation and by the time discrete aggregates had formed, they were completely enveloped in the slime sheath. At the first finger-like stage, the slime sheath appeared to be thicker at the base than at the tip of the aggregate but such variations in thickness of the slime sheath could not be observed in the migrating pseudoplasmodium although it has previously been suggested that this may be important in controlling cell differentiation during this migratory stage.     

Applications of field emission scanning electron microscopy to polymer membrane research

The recent development of ultra-high resolution field emission scanning electron microscopy has opened exciting new opportunities in many scientific and engineering applications at the molecular scale. It overcomes the instrumentation limitations of low resolution in SEM and uncertainty in TEM due to artifacts imposed by sample preparation.Applications of field emission scanning electron microscopy (FESEM) to polymer membrane research such as studies of surface morphology of finely porous membranes and mechanisms of membrane fouling are illustrated with examples. The advantages of the technique, especially the low voltage requirements of FESEM for surface observation, are also discussed in comparison with TEM (replica) and conventional SEM.     

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.     

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.     

A new method of preparing insects for scanning electron microscopy.

Acidified 2,2-dimethoxypropane (DMP), used as an alternative to regular fixation and dehydration methods for insects, was found to be the only successful means of preparing the sugarbeet root maggot larva, Tetanops myopaeformis (R?der) (Diptera:Otitidae), for the scanning electron microscope. No morphological changes were evident when DMP treated sugarbeet root maggot adults were compared to fresh (unfixed) adults and glutaraldehyde-osmium tetroxide fixed adults. The method has been used with success on several gropus of insects. Acidified CMP is quickly hydrolyzed by water in tissue to acetone and methanol. DMP is advantageous in that it penetrates water impermeable cuticles rapidly and saves several steps and time in the fixation and dehydration process.     

Sugarcane somatic embryogenesis: a scanning electron microscopy study

Spindles of CUBA 87-51 sugarcane were cultured in Murashige and Skoog (MS) basal medium and supplemented with different nutrients. Embryogenic and non-embryogenic callus obtained were comparatively studied by scanning electron microscopy (SEM). Samples of embryogenic callus cultured in regeneration medium (MS without 2.4 dichlorophenoxyacetic acid) were taken at different times for analyzing the sequential process. Distinctive features of two types of callus are shown by SEM: cells organized in embryos are noted in embryogenic callus; while elongated, disorganized cells can be seen in non-embryogenic callus. The characteristics of the embryos during plant regeneration are described. Sugarcane embryoid stages are: globular, globular with lateral notch and scutellum. In this process also appear shoot meristems, leaf and root primordia and finally, true leaves and roots. It is concluded that callus plant regeneration from young leaf segments of sugarcane mainly occur via somatic embryogenesis.     

A structural basis for R- and T-banding: a scanning electron microscopy study

The structure of reverse (R)-banded and telomeric (T)-banded chromosomes was studied by examination of the same chromosomes first in the light microscope (LM) followed by the scanning electron microscope (SEM). This procedure demonstrated a structural basis to both the R- and T-banding techniques. A direct correlation was shown between the LM staining patterns and the structural patterns observed in the SEM. In the R-banded chromosomes the positively stained R-bands, viewed by LM, corresponded to highly fibrous three-dimensional regions in the SEM. The negatively stained R-interbands corresponded to flatter regions from which material appeared to have been extracted. These structural observations strongly support the suggestion that chromosomal material is preferentially lost from the R-interbands with aggregation of fibres in the R-bands. T-banded chromosomes showed a similar structure to the R-banded chromosomes. The positively stained T-bands located at the telomeres corresponded to regions of highly aggregated fibres. The remainder of the chromosome, corresponding to the negatively stained area, had a flattened and extracted appearance. These similarities in morphology between the T- and R-banded chromosomes support the view that T-bands result from a progressive breakdown of the R-banded chromosome structure.     

Fixation of platelets for scanning and transmission electron microscopy.

A double fixation method of preparing platelet suspensions for both scanning and transmission electron microscopy is outlined. Prefixation in 0.1% glutaraldehyde allows for immediate preservation of morphologic characteristics induced by experimental procedures, but does not completely destroy platelet surface stickiness. Preservation of surface stickiness allows subsequent production of a platelet pellet for processing for transmission electron microscopy. This pelleting cannot be achieved when higher initial concentrations of glutaraldehyde are used for prefixation. Prefixation in 0.1% glutaraldehyde is also an appropriate initial step for preservation of platelets in suspension for scanning electron microscopy.     

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.     

The rat pituitary cleft: A correlated study by scanning and transmission electron microscopy

Summary SEM reveals that the inner surface of the pituitary cleft is lined by a continuous layer of marginal cells possessing microvillous and ciliated apical surfaces. The ciliated cells are more numerous on the posterior side (toward the pars intermedia) than on the anterior side of the cleft (toward the pars distalis). In contrast small infoldings (crypts) were occasionally noted only on the marginal layer covering the distal part of the hypophysis. In some areas of the cleft the surface features of the marginal cells are rather similar to the epithelial cells populating the upper parts of the respiratory tract in their topography and distribution. In other regions they also show striking similarities with the ependymal cells (tanycytes) lining the lateral recesses of the 3rd ventricle and the infundibular process with which the pituitary cleft has a very close topographical relationship.The parenchymal cells of the pars distalis are closely related to the flattened marginal cells of the cleft. The intercellular spaces of the pars distalis form a three-dimensional labyrinthic series of cavities continuous with the submarginal spaces of the cleft. Further SEM and TEM results demonstrate that the majority of the microvillous marginal cells lining both sides of the cleft possess surface features such as bulbous protrusions, laminar evaginations and large cytoplasmatic vacuoles, which are very likely the expression of an active transport of fluids.On the basis of these results it is concluded that the fluid-like material (colloid) present in the pituitary cleft is mainly derived from the fluids contained in the lacunar spaces of the pars distalis. Thus, marginal cells by absorbing fluids from the cleft by active endocytosis, may transport to the pars intermedia material (or hormones) produced in the distal part of the gland and vice versa.The cilia present on many marginal cells, based on their 9+2 tubular pattern, possess a kynetic role. This is very similar to that shown by the ciliated cells of the ependyma lining the brain ventricles. The occurrence of ciliated cells within the pituitary parenchyma (mainly in the follicles) suggests that they probably arise from the ciliated cells populating the marginal layer of the cleft and with which the parenchyma cells are closely related.     

Assembly of chromatin fibers into metaphase chromosomes analyzed by transmission electron microscopy and scanning electron microscopy.

The higher-order assembly of the approximately 30 nm chromatin fibers into the characteristic morphology of HeLa mitotic chromosomes was investigated by electron microscopy. Transmission electron microscopy (TEM) of serial sections was applied to view the distribution of the DNA-histone-nonhistone fibers through the chromatid arms. Scanning electron microscopy (SEM) provided a complementary technique allowing the surface arrangement of the fibers to be observed. The approach with both procedures was to swell the chromosomes slightly, without extracting proteins, so that the densely-packed chromatin fibers were separated. The degree of expansion of the chromosomes was controlled by adjusting the concentration of divalent cations (Mg2+). With TEM, individual fibers could be resolved by decreasing the Mg2+ concentration to 1.0-1.5 mM. The predominant mode of fiber organization was seen to be radial for both longitudinal and transverse sections. Using SEM, surface protuberances with an average diameter of 69 nm became visible after the Mg2+ concentration was reduced to 1.5 mM. The knobby surface appearance was a variable feature, because the average diameter decreased when the divalent cation concentration was further reduced. The surface projections appear to represent the peripheral tips of radial chromatin loops. These TEM and SEM observations support a "radial loop" model for the organization of the chromatin fibers in metaphase chromosomes.