High Voltage Electron Microscopy and Low Voltage Scanning Electron Microscopy of Human Neoplastic Cell Culture

Improved procedures were developed to correlate cell culture data with the images provided by advanced ultrastructural technologies. These procedures were compatible with the two main types of cellular behavior: adherent, spreading (melanomas, rhabdomyosarcomas) and non-adherent in suspension (leukemias). The ultrastructure and function of spreading neoplastic cells primarily depend on surface properties of the attaching substrates. Therefore, the films used for cultured cell whole-mount ultrastructural analysis must have adherence features identical to those of standard cell culture vessels. Improved procedures were developed to produce the polystyrene films of required qualities. These films allowed processing of cells for electron microscopy including chemical fixation, cryoimmobilization, and immunolabelling. Furthermore, these polystyrene films permitted observations of the same cell in the high voltage electron microscope to reveal the internal organization and in the low voltage scanning electron microscope to reveal the surface topography. Neoplastic cells in suspension may dramatically change their ultrastructure as a result of interactions with substrates or other cells. Therefore, immobilization of cellular processes must occur rapidly while cells remain in suspension. These processes were cryoimmobilized by high pressure freezing through the use of the newly designed specimen carrier. Procedures allowing high yield attachment of cryofixed neoplastic cells to amino-propyl-derived glass carriers enabled observations of cell surface topography. Furthermore, freeze-substitution and drying of freeze-fractured cells revealed their three-dimensional internal organization in the low voltage scanning electron microscope

Backscattered Electron Imaging for High Resolution Surface Scanning Electron Microscopy with a New Type YAG-Detector

Double Layer Coating for backscattered electron imaging is a coating and imaging method especially suitable for high resolution scanning electron microscopy (SEM) of large biological samples. Since the backscattered electron (BSE) signal from thin metal coating layers is quite low, field emission SEM\u27s and very sensitive BSE-detectors are required for this method. In this study an improved BSE-detector of the YAG type was used with an in-lens type field emission SEM. Two samples were investigated in order to demonstrate and to improve the potential of this new approach: (1) cryo-prepared cultured kidney cells were shadowed by electron beam evaporation with platinum-carbon (unidirectionally at a fixed angle of 45°) and then coated with an additional 10 nm carbon layer; and (2) cryo-prepared trichocyst matrixes (paracrystalline structures contained in secretory granules, the trichocysts, found in Paramecium) were coated by ion beam sputtering with about 1 nm of platinum. This sample was rotated and tumbled during coating in order to obtain as uniform a metal layer as possible and then an additional 10 nm carbon layer was evaporated over the metal. When these samples were viewed at a primary beam accelerating voltage (Vo) of 10 kV or higher, contrast was good on the unidirectionally coated cell culture samples. However, trichocyst matrixes with the thinner and more uniform coating showed very poor contrast because most of the BSE detected represented beam-specimen interactions from the bulk of the sample and not in the thin platinum layer. The situation was improved by using low Vo (4 kV). Under these conditions the penetration depth of the electrons is reduced and a greater proportion of the BSE electrons are scattered by the platinum layer. The results were compared with freeze-fracture and deep-etch transmission electron microscope studies of the trichocyst matrixes from the literature: Almost similar resolution is achieved on the biological structures but a better impression of the three dimensional arrangement of the whole trichocyst matrix is obtained with the SEM. The globular particles form disc-like structures that are connected with each other by thin fibers

Improved Methods for Preserving Macromolecular Structures and Visualizing Them by Fluorescence and Scanning Electron Microscopy

To determine the optimal procedures to preserve cytoskeletal and other macromolecular structures for microscopic studies we have evaluated the effects of various methods to extract cultured cells. In this report, we compare results using different fixatives, crosslinking reagents, and permeabilization methods on (1) the labeling of cells for fluorescence microscopy with phalloidin or antibody against tubulin; and (2) the morphological preservation of macromolecular structures for scanning electron microscopy. Maximal labeling of F-actin with phalloidin was obtained by fixing cells in 4% para formaldehyde (PFA) and labeling the unextracted cells with methanolic phalloidin, whereas maximal labeling of tubulin required prefixation with either PFA or the bi functional protein crosslinking reagent, dithiobis (succinimidylpropionate) (DSP) and extraction with ethanol or Triton in a high salt buffer. However, for both qualitative and quantitative light and electron microscopic studies of intracellular macromolecular structures, prefixation with DSP and extracting with Triton X-100 in a stabilizing buffer is the overall method of choice for both labeling and morphological studies. Although other methods provide maximal labeling or preservation of specific structures, this method provides excellent preservation of morphological structure while allowing proteins to be preserved and labeled by specific probes

Scanning Electron Microscopy of High-Pressure-Frozen Sea Urchin Embryos

High-pressure-freezing permits direct cryo-fixation of sea urchin embryos having a defined developmental state without the formation of large ice crystals. We have investigated preparation protocols for observing high-pressure-frozen and freeze-fractured samples in the scanning electron microscope. High-pressure-freezing was superior to other freezing protocols, because the whole bulk sample was reasonably well frozen and the overall three-dimensional shape of the embryos was well preserved. The samples were either dehydrated by freeze-substitution and critical-point-drying, or imaged in the partially hydrated state, using a cold stage in the SEM. During freeze-substitution the samples were stabilized by fixatives. The disadvantage of this method was that shrinking and extraction effects, caused by the removal of the water, could not be avoided. These disadvantages were avoided when. the sample was imaged in the frozen-hydrated state using a cold-stage in the SEM. This would be the method of choice for morphometric studies. Frozen-hydrated samples, however, were very beam sensitive and many structures remained covered by the ice and were not visible. Frozen-hydrated samples were partially freeze-dried to make visible additional structures that had been covered by ice. However, this method also caused drying artifacts when too much water was removed

Accessing Nuclear Structure for Field Emission, in Lens, Scanning Electron Microscopy (FEISEM)

Scanning electron microscopy (SEM) has had a shorter time course in biology than conventional transmission electron microscopy (TEM) but has nevertheless produced a wealth of images that have significantly complemented our perception of biological structure and function from TEM information. By its nature, SEM is a surface imaging technology, and its impact at the subcellular level has been restricted by the considerably reduced resolution in conventional SEM in comparison to TEM. This restriction has been removed by the recent advent of high-brightness sources used in lensfield emission instruments (FEISEM) which have produced resolution of around 1 nanometre, which is not usually a limiting figure for biological material. This communication reviews our findings in the use of FEISEM in the imaging of nuclear surfaces, then associated structures, such as nuclear pore complexes, and the relationships of these structures with cytoplasmic and nucleoplasmic elements. High resolution SEM allows the structurally orientated cell biologist to visualise, directly and in three dimensions, subcellular structure and its modulation with a view to understanding its functional significance. Clearly, intracellular surfaces require separation from surrounding structural elements in vivo to allow surface imaging, and we review a combination of biochemical and mechanical isolation methods for nuclear surfaces

Sample Preparation Techniques for Conventional and High Resolution Scanning Electron Microscopy of the Central Nervous System. The Cerebellum as a Model

In the present paper some basic sample preparation techniques for scanning electron microscopy of nervous tissue are described. These basic preparative methods include conventional scanning electron microscopy or slicing technique, ethanol-cryofracturing technique, freeze-fracture method using either liquid nitrogen (slow freezing) or Freon 22 cooled by liquid nitrogen (fast freezing), improved freeze-fracture method with delicate specimen preparation and chromium coating, ultrasonic microdissection, and creative tearing technique. Some basic principles, advantages and limitations are critically considered. In addition, some specific applications in neurobiological research are reported. Emphasis is placed upon understanding the sources and nature of artifacts that are likely to be produced in each preparatory step. Examples are given of the results obtained with the different types of nerve tissue preparation, using the cerebellar cortex as a model of the central nervous system. According to the author\u27s experience, the slicing technique is recommended for studying cytoarchitectonic arrangement of gray centers, the ethanol-cryofracturing technique for tracing short intracortical circuits, and the freeze-fracture methods for analysis of nerve cell cytoplasmic and nuclear compartments. An attempt is made to explain results obtained in relation with nerve cell biology

Three-Dimensional Morphology and Platelet Adhesion on Pyrolytic Carbon Heart Valve Materials

Low-temperature isotropic pyrolytic carbon (LTIC) is the preferred material for mechanical heart valve prosthetics due to its durability and good thromboresistance, although thromboembolic complications remain a significant clinical problem. LTlC morphology has been previously studied using scanning (SEM) and transmission electron microscopy (TEM), and scanning tunneling microscopy (STM). However, these microscopies have limitations with imaging rough surfaces. In this study, LTIC valve leaflets from CarboMedics, Inc. and St. Jude Medical, Inc. were prepared and polished exactly as used in clinical prosthetics, and examined at magnifications up to macromolecular resolution using stereo-pair low-voltage SEM (LV -SEM). LV -SEM reveals that LTIC leaflets have a complex topography of 10 nm to 1 ~m features, with height differences of 100- 500 nm occurring over lateral distances of 10-50 nm. Compared to previous reports using conventional SEM and STM, LV -SEM shows a much rougher surface. In contrast to studies that have reported minimal platelet interaction with LTIC, very ex tensive adhesion and spreading were observed. That our observations are different from previous reports may be ex plained by the physics of SEM image formation at low and conventional (higher) accelerating voltages. Due to the low atomic density of LTIC and platelets, obscuration of small features due to specimen coatings, and since platelets closely follow LTIC\u27s three-dimensional contours, the surface sensitivity of conventional SEM is unable to provide sufficient contrast to image either the material topography or thin adherent platelets. These results suggest that the ex tent of platelet interaction on L TIC vascular prosthetics may have been previously underestimated

Receptor-mediated endocytosis of low density lipoproteins in aortic endothelial cells

Lipoprotein binding and metabolism in actively-dividing (subconfluent) and quiescent (postconfluent) bovine aortic endothelial cells (ECs) were qualitatively investigated by fluorescence microscopy using dioctadecylindocarbocyanine-labelled lipoproteins and by indirect immunofluorescence microscopy. LDL and acetylated-LDL (AcLDL) were seen bound to the surfaces of subconfluent ECs (at 4°C or at 37°C), as a random distribution of punctate foci. ECs therefore closely resembled fibroblasts in the distribution of LDL receptors on their surfaces. No binding of LDL was seen on postconfluent EC surfaces by either direct or indirect fluorescence microscopy. The patterns of AcLDL binding on postconfluent ECs resembled those on subconfluent ECs. Intracellular LDL and AcLDL occurred as perinuclear accumulations of large fluorescent disc-shaped profiles in subconfluent ECs. These accumulations were shown to arise from surface-bound material by pulse-chase experiments. Intracellular LDL was absent in the majority of postconfluent ECs, while AcLDL accumulation was massive. "Wounding" of cultures allowed simultaneous assessment of lipoprotein metabolism in quiescent and actively-dividing areas of the same culture. Quantitative assessments of the above-mentioned phenomena were made using ¹²⁵I-labelled lipoproteins. Receptor-mediated binding of LDL decreased five to ten-fold as the cultures modulated from subconfluent to postconfluent morphology. No receptor-bound LDL was detected in postconfluent ECs. Conversely, the amount of AcLDL bound increased at least fivefold during EC growth in parallel cultures. The amounts of lipoproteins endocytosed and metabolised were generally related proportionately to the amounts bound in each case. The distribution of LDL receptors on cultured cells was also investigated at the ultrastructural level using colloidal gold-conjugated LDL as a probe, and similarly labelled antibodies as probes. Whole-mounted cells with receptor probes bound to them were examined directly in the transmission electron microscope. The topographical distribution of LDL receptors has not been investigated by these techniques before. A novel method of preparing cytochemically-labelled, whole-mounted cells from styrene culture dishes was developed and used in this study. LDL Receptors expressed on the surfaces of human skin fibroblasts served to standardise these colloidal gold techniques and fortuitously led to new information on receptor distribution. Normal (FGo) and LDL receptor-negative mutant fibroblasts (GM 2000) acted as positive and negative controls respectively. Normal fibroblast LDL receptors were grouped into clusters consistent in size with coated pits (200 - 500 nm in diameter). A novel finding was the presence of a diffuse population of receptors scattered randomly amongst the clustered receptors. Another mutant fibroblast, GM 2408A, known to have an aberrant LDL receptor distribution, was also examined. Its receptors were shown to be dispersed singly, and in occasional groups of two and three, at random over the cell surfaces. No clusters were detected. The receptor-negative GM 2000 bound virtually no probes. While not as sensitive as the colloidal gold-conjugated LDL probe, an antireceptor monoclonal antibody (IgG-C7), localised by indirect immunogold labelling, gave similar results when applied to the above cells. This was taken as strong corroborative evidence that the LDL receptor distributions as determined by colloidal gold-conjugated LDL were correct. It is suggested that the dispersed population of receptors on normal fibroblasts may represent newly-emerged recycling receptors which have yet to cluster in coated pits. A further new finding reported here is the existence of the same two patterns of LD L receptors, dispersed and clustered, on the surface of subconfluent ECs. It was noted, from the study of whole-mounted and thin-sectioned cells, that the receptors were preferentially arranged in rings following the circumference of coated pit areas on the cell surface. Often these rings associated in groups or even coalesced into compound clusters. The significance of these groupings is not yet understood. In sharp contrast to the situation on subconfluent ECs, no LDL receptors (probed with the extremely sensitive colloidal-gold conjugated LDL) could be detected at the EM level on the surface of postconfluent ECs. Active cells in wounded postconfluent monolayers expressed abundant receptors detected at the EM level. It is concluded that postconfluent quiescent bovine aortic ECs in vitro metabolise virtually no LDL via the LDL-receptor pathway due to a vanishingly low number of LDL receptors. This contrasts with the ability of postconfluent cells to metabolise relatively large amounts of AcLDL via a receptor-mediated mechanism. The significance of these conclusions is discussed with respect to the interaction of plasma lipoproteins with the endothelium in vivo

The Electron Microscopy of Tissue Sections With Special Reference to the Structure of Spinal Ganglia

Abstract Not Provided

Electron Microscope Studies of Bacteriophages

Abstract Not Provided