Scanning electron microscopy最新文献

Implantation of bits of marrow in ectopic sites is followed by reorganization of tissue and the formation of a hemopoietic nodule surrounded by a shell of bone. This regenerative process is reminiscent of marrow ontogeny and the model can serve to study marrow ontogeny in a relatively short period of time. Early events during this regeneration were studied by scanning (SEM) and transmission electron microscopy (TEM). Within 24 hours the implant elicited an angiogenic reaction and new vessels penetrated the implant. Intense circulation, thus established, divested the implant from hemopoietic cells, leaving the stroma behind. Stromal cells proliferated and the impetus for this proliferation appeared to result from an impulse caused by the presence of bony fragments outside and within the stromal cells. Previous studies of this model have not appreciated the presence of non-viable bone in the implant, although the fact that non-viable bone can trigger osteogenesis and new bone marrow formation is well-known. This experimental model lends itself to the study of the interrelationship of hemopoietic cells and their supporting stroma as well as the interrelationship of bone and hemopoiesis.

This paper is intended as an introduction to the field of proton microprobe analysis with special emphasis on applications in biological sciences. It is mainly intended for users of electron microscopes equipped with microprobes or other analytical equipment. The basic principles of Particle Induced X-ray Emission analysis are discussed as well as the instrumental requirements for the proton microprobe. The analytical characteristics including quantification procedures are compared with those of the electron microprobe and a review is given of various analytical applications of the proton microprobe within biology and medicine.

To investigate the microstructure of in situ (eutopic) and autotransplanted (ectopic) splenic remnants, adult Sprague-Dawley rats were studied 60 days after subtotal (approximately 80%) splenectomy, total splenectomy followed by single or multiple remnant intraperitoneal autotransplantation, or sham operation. Total nucleated cell counts were determined in excised splenic remnants, and immunohistochemical staining using monoclonal antibodies to rat B- and T-cell antigens was performed in serial tissue sections. Immunoarchitecture of eutopic remnants was indistinguishable from that of intact spleens and total nucleated cell counts remained proportional to weight. In contrast, ectopic remnants showed sparsity and abnormal mixing of B and T lymphocyte subpopulations with widespread loss of follicles and periarteriolar lymphoid sheaths in addition to lower density and marked reduction of total nucleated cells. These findings provide immunohistologic evidence that preservation of intact vasculature is critical to splenic architecture, which may account in part for the demonstrable functional inferiority of ectopic remnants.

Cystic fibrosis (CF) is a generally lethal, congenital, genetic disease of unknown etiology. It is likely that a defective regulation of ion and water transport in exocrine glands and possibly also in other epithelial cells has a central role in the pathogenesis of this disease. Calcium has been implicated in the basic defect underlying CF because of findings of abnormally high calcium levels in some secreted fluids and some cells of CF patients. Using X-ray microanalysis, we have demonstrated elevated calcium concentrations in cultured fibroblasts and in goblet cells of the bronchial epithelium of CF patients. A factor produced by CF fibroblasts in culture can increase the calcium concentration in healthy cells, although this may be an indirect effect. In animal models for CF, such as the chronically reserpinized rat and the chronically isoproterenol-treated rat, abnormally high calcium levels in the acinar cells of the submandibular gland could be demonstrated, similar to the situation in CF patients. In the acinar cells of the parotid gland in these animal models, the calcium levels are, however, abnormally low. This suggests that the changes in cell calcium content are secondary to other changes, possibly changes in the secretory proteins. A study of the effect of the serum calcium level and of the calciotropic hormone calcitonin suggested that neither of these factors could be directly linked with CF. It is concluded that several lines of evidence point to a secondary rather than a primary role for calcium in the pathogenesis of CF.

We studied two aspects of the human gastric mucosa: the surface morphology of mucous cells, as viewed by scanning electron microscopy (SEM); the glycosidic components of intracellular mucins, characterized by means of lectins. The latter were conjugated with fluorescein isothiocyanate and with colloidal gold-silver for the visualization of the reaction products in light microscopy (LM) and in SEM (backscattered mode) respectively. The surface morphology of mucous cells appears to be correlated to the secretory state. In gastric ulcers we found a prevalence of non-secreting cells. A decrease in glycosidic receptors for fucose-binding lectin and galactose-(1-3)-N-acetyl-galactosamine-binding lectin was also observed. This suggests the presence of an impaired mucus secretion which may play a role in the pathogenesis of gastric ulcer. Spiral bacteria, supposed to be aetiologically related to peptic ulcer and gastritis, were easily detected by SEM. Intestinal metaplasia defined "complete" in LM showed surface morphology and glycosidic components different from those of true intestinal mucosa. This implies the necessity of taking into account also these parameters when classifying this lesion. The same applies to polyps. Our data indicate that correlative SEM may contribute further information on the pathogenesis and pathology of gastric diseases.

Scanning electron microscopy (SEM) permits a three-dimensional study of the surface morphology of the organ of Corti that is very useful in evaluating the condition of the apical end of the hair cells and the stereocilia. However, some laboratories have experienced problems with curling of the basilar membrane during critical point drying of cochlear specimens prepared for SEM evaluation using the Murakami or osmium thiocarbohydrazide-procedures. This curling of the basilar membrane can obstruct the view of the reticular lamina and the ciliary ends of the hair cells. We have used a dissection method, referred to as the anchor technique, to overcome basilar membrane curling. This technique removes all the structures above the reticular lamina but leaves the basilar membrane attached to the spiral ligament and the lateral bone to which the spiral ligament is anchored. Individual cochlear turns are dissected in this manner and mounted on the same examination stub for SEM evaluation. Maintenance of the lateral attachment of the basilar membrane requires additional dissection time but eliminates the problem of curling during critical point drying. An additional benefit is that mounting the individual turns on the same examination stub facilitates evaluation and photomicroscopy of the surface morphology. The anchor technique has been used successfully on the guinea pig and should be appropriate for most mammalian cochleas.

This article reviews the applications of SEM methods to human bone pathologies referring to studies made at UCL. We consider the methods which may be most suitable; these prove to be not "routine" in the context of most bio-medical applications of SEM. Valuable information can be obtained from a bone sample if its edges are ground flat, before making either a matrix surface preparation by washing away all the cells or a mineralizing front preparation, by also dissolving the osteoid-for which hydrogen peroxide is recommended to produce a robust specimen. BSE contrast from a cut block surface can be used to measure bone phase volume. SE contrasts from natural surfaces (trabeculae, canals and lacunae) can be used to study forming, resting and resorbing surfaces both qualitatively and quantitatively (except in the case of histological osteomalacia, where the existence of osteoid will go undetected and reversal lines will be difficult to distinguish from recently resorbed surfaces). We also recommend the use of PMMA embedded bone blocks, which can be used as obtained from the pathologist, but are better embedded by a more rigorous procedure. BSE image analysis can be used to quantitate bone density fractions opening up a completely new investigative method for the future. Osteoid can be measured automatically using CL if the bone sample is block stained with brilliant sulphaflavine before embedding or if a scintillant is added to the embeddant. We give examples of observations made from a number of bone diseases: vitamin D resistant rickets, osteogenesis imperfecta; osteomalacia; osteoporosis; hyperparathyroidism; fluorosis; Paget's disease; tumour metastasis to bone.

The doctor of today must adopt the 'cellular way of thinking' in the evaluation of diseases. This ultrastructural outlook provides him with much indispensable information that also serves a practical purpose. A diseased cell organelle is at the basis of every clinical sign and any attempt of therapy must be aimed at that specific point of lesion. We intend, in the light of a long experience, to propose to clinicians a new way of thinking in which a precise correlation between symptoms and submicroscopic changes of the cell is considered. Many different examples amply justify this proposal. Electron microscopy can contribute by enabling identification of structural subcellular modifications suitable for the finest differential diagnosis, more and more complete understanding of pathogenic pathways of various diseases, the establishment of guidelines for precise pharmacological interventions at the molecular level.

Nerve terminals often contain morphologically distinct populations of large (75-110 nm) and small (45-55 nm) vesicles. The small vesicles are speculated to account for release of transmitter quanta as they accumulate at presynaptic membranes. Large vesicles can co-store neuropeptides and classical transmitters but their function in neurotransmission has been disputed because they do not appear to accumulate at chemical synapses. However, there is now evidence that the large vesicles play a role in neurotransmission or its modulation even though they may not be eminently involved in synaptic release. Thus, exocytosis occurs along the synapse-lacking membranes of peripheral noradrenergic varicosities. Large vesicles may continue to function in peptide release even after the classical transmitter has been depleted as demonstrated in the pig vas deferens. Three days of reserpine administration causes a parallel loss of noradrenaline and small vesicle contents but does not decrease enkephalin-like immunoreactivity or large vesicle electron density. In the central nervous system of the rat, where substance P and enkephalin have been localized to large vesicles, exocytosis occurs from several types of terminals. The large vesicles appear preferentially to release their contents at morphologically non-specialized sites even when characteristic synapses are present. Thus different mechanisms of transmitter and neuropeptide release may coexist. The nonsynaptic discharge may allow substances to diffuse over a wider distance whereas release into a synaptic cleft could restrict receptor interaction.

This report documents phorbol ester (TPA)-induced changes in cell morphology, and in vitro growth patterns in 9 patients with hairy cell leukemia (HCL), 21 with B-type CLL and non-Hodgkin's lymphoma in leukemic phase (NHL), and 10 with acute non lymphoblastic leukemia (ANLL). TPA caused cells from HCL to adhere strongly and produce elongated cytoplasmic extensions. Many of these cells had an appearance resembling fibroblasts, while others showed marked surface ruffling and spreading containing increased dense bodies, and phagolysosomal vacuoles as seen by transmission electron microscopy. This HCL in vitro growth pattern after TPA exposure differed from that seen in B-CLL and NHL cells, which only adhered moderately after 72 hours and readily detached in clumps. CLL and NHL-cells did not show ultrastructural features of macrophages but had either plasmacytic or HCL features. It is suggested that these different growth patterns may aid in distinguishing HCL from other B-cell neoplasias. The expression of surface markers, tartrate resistant acid phosphatase (TRAP) and Ig secretion were studied in some B-CLL, NHL and HCL cells after exposure to different concentrations of TPA for up to 6 days. Results showed that the documented changes were frequently both dose and time dependent and the most striking HCL-features were encountered after 6 days incubation with higher concentrations of TPA. However, individual variation from case to case was noted. Nevertheless, it seems that TPA induces neoplastic B-cells to mature into secreting plasmacytoid lymphocytes, and cells with features of HCL with variable expression of surface markers and TRAP.