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J B Pawley

Publications and source records attributed to J B Pawley.

12 recordsLinked to original sources
PubMed1992

High-resolution scanning electron microscopy.

The spatial resolution of the scanning electron microscope is limited by at least three factors: the diameter of the electron probe, the size and shape of the beam/specimen interaction volume with the solid for the mode of imaging employed and the Poisson statistics of the detected signal. Any practical consideration of the high-resolution performance of the SEM must therefore also involve a knowledge of the contrast available from the signal producing the image and the radiation sensitivity of the specimen. With state-of-the-art electron optics, resolutions of the order of 1 nm are now possible. The optimum conditions for achieving such performance with the minimum radiation damage to the specimen correspond to beam energies in the range 1-3 keV. Progress beyond this level may be restricted by the delocalization of SE production and ultimate limits to electron-optical performance.

Electrons↗
PubMed1992

High-resolution scanning electron microscopy of frozen-hydrated cells.

Cryo-fixed yeast Paramecia and sea urchin embryos were investigated with an in-lens type field-emission SEM using a cold stage. The goal was to further develop and investigate the processing of frozen samples for the low-temperature scanning electron microscope (LTSEM). Uncoated frozen-hydrated samples were imaged with the low-voltage backscattered electron signal (BSE). Resolution and contrast were sufficient to visualize cross-fractured membranes, nuclear pores and small vesicles in the cytoplasm. It is assumed that the resolution of this approach is limited by the extraction depth of the BSE which depends upon the accelerating voltage of the primary beam (V0). In this study, the lowest possible V0 was 2.6 kV because below this value the sensitivity of the BSE detector is insufficient. It is concluded that the resolution of the uncoated specimen could be improved if equipment were available for high-resolution BSE imaging at 0.5-2 kV. Higher resolution was obtained with platinum cryo-coated samples, on which intramembranous particles were easily imaged. These images even show the ring-like appearance of the hexagonally arranged intramembranous particles known from high-resolution replica studies. On fully hydrated samples at high magnification, the observation time for a particular area is limited by mass loss caused by electron irradiation. Other potential sources of artefacts are the deposition of water vapour contamination and shrinkage caused by the sublimation of ice. Imaging of partially dehydrated (partially freeze-dried) samples, e.g. high-pressure frozen Paramecium and sea urchin embryos, will probably become the main application in cell biology. In spite of possible shrinkage problems, this approach has a number of advantages compared with any other electron microscopy preparation method: no chemical fixation is necessary, eliminating this source of artefacts; due to partial removal of the water additional structures in the cytoplasm can be investigated; and finally, the mass loss due to electron beam irradiation is greatly reduced compared to fully frozen-hydrated specimens.

Animals↗
PubMed1991

Early results using high-resolution, low-voltage, low-temperature SEM.

Recent advances in the design of the scanning electron microscope (SEM) column, such as the coupling of a field-emission gun to a low-aberration immersion lens and the availability of a high-stability cryo-transfer stage, make low-temperature, low-voltage SEM (LTLVSEM) possible at very high resolution. We have used this combination to obtain results with uncoated biological specimens. The trichocyst from a Paramecium was used as a test specimen to observe the shrinkage of this structure as the temperature is raised from 170 K to room temperature following freeze-drying. High-magnification stereo images were obtained of trichocysts that had been prepared by freezing, freeze-substitution and critical-point drying and which were subsequently viewed by LTLVSEM to reduce beam damage and contamination.

Animals↗
PubMed1991

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's 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 degrees) 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.

Animals↗
PubMed1990

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.

Blood Platelets↗
PubMed1990

High-resolution immunogold localization of Giardia cyst wall antigens using field emission SEM with secondary and backscatter electron imaging.

We describe here the ultrastructural localization of Giardia cyst antigens in the filaments associated with the outer portion of intact cysts and on developing cyst wall filaments in encysting trophozoites. Post-embedding immunogold labeling of thin sections of intact Giardia cysts with polyclonal and monoclonal antibodies specific for cyst wall antigens (major protein bands of approximately 29, 75, 88, and 102 KD on Western blots) showed strong labeling of the filamentous cyst wall, whereas no labeling was seen on the membranous portion. High-resolution field emission scanning electron microscopy (FESEM) of Giardia cysts revealed that the cyst wall-specific polyclonal rabbit antisera and monoclonal mouse antibody produced gold labeling of 20-nm filaments in the cyst wall as detected with secondary electron imaging (SEI) and backscatter electron imaging (BEI) at 10 kV, despite coating of the cells with platinum by ion sputtering. FESEM studies of encysting Giardia trophozoites demonstrated that immunostaining with antibodies to cyst wall antigens produced colloidal gold labeling of developing cyst wall filaments on the cell surface; however, the intervening membrane domains were unlabeled. Substitution of normal serum for cyst wall-specific antibodies, or preabsorption of specific antibodies with Giardia cysts, eliminated immunolabeling of the filaments.

Animals↗
PubMed1978

A chamber attached to the SEM for fracturing and coating frozen biological samples.

A chamber for introducing, fracturing and coating frozen biological samples has been developed as an attachment to the sepcimen chamber of a scanning electron microscope. Together with a eucentric-tilt cold-stage, this chamber constitutes a complete system for viewing fractured biological surfaces of the type normally only seen by replica techniques. An air-lock on the chamber accepts a transfer module to allow insertion of the frozen sample without frost build-up. Fracturing is carried out with a precisely adjustable cooled knife under a 10--100X binocular microscope. The sample can tilt and rotate while being coated with carbon or metals evaporated from rechargeable sources introduced through the air-lock. Cooling in the chamber is provided by a cylindrical copper tank filled with liquid nitrogen. The chamber has its own LN2 trapped high vacuum system. After preparation the sample can be placed directly into the SEM through an isolation valve. The cold-stage utilizes a Joule-Thomson refrigerator. The sample can be kept below 103 K at all times though there are provisions for heating it in the fracturing and cold-stage positions. A system of controls, sensors and interlocks simplifies the operation of the system.

Animals↗
PubMed1977

Using simultaneous three colour X-ray mapping and digital-scan-stop for rapid elemental characterization of coal combustion by-products.

A system is described for rapid, simultaneous three colour elemental mapping with a scanning electron microscope (SEM) and an energy dispersive X-ray analyser. The technique, which use a SEM scanning at TV rate, minimizes the disadvantages of long scan times such as inefficient use of linear amplifer and observable dead-time shadowing. The system also employs a digital scan-stop assembly utilizing a light pen to rapidly and reproducibly direct the beam to an object of choice for spot-mode analysis. Application of the system to analysis of fine particulates with emphasis on fly ash derived from coal-fired electric power plants is discussed. Chemical heterogeneity of fine particles in standard reference material fly ash and in phagocytized fly ash within pulmonary macrophages is demonstrated. This system combines the morphologic capability of the SEM with X-ray multielement mapping to provide a needed tool for particulate source identification.

Air Pollutants↗
PubMed1975

Osteoblasts and collagen orientation.

Bone was removed from the calvaria of anaesthetized 70 g rats or freshly killed young monkeys and the fibrous periosteum dissected off the inner, formative surface under 0.15 M cacodylate buffer. The bone and undisturbed osteoblasts were fixed in 3% glutaraldehyde in the same buffer for 24 to 48 hours, critical point dried and coated with evaporated carbon and gold for scanning electron microscopy (SEM). Fields of osteoblasts were photographed and chosen cells dissected off the osteoid using a tungsten needle. The control of the dissection was made possible by the use of a system of real-time stereo tv-speed SEM. The fields were rephotographed and the orientations of the osteoblasts were compared with that of the underlying collagen fibres. 62% of all osteoblasts lay with their long axes within 15 degrees of the collagen fiber orientation below and 80% within 30 degrees. Montages of large areas of osteoblasts were also made, and then compared with ones of the same area after the cells had been stripped off on adhesive tape. In general, the orientation of the collagen tended to be the same as the cell that formed it. Collagen fibers below cells at the periphery of a domain sometimes had the orientation of the cells in the adjacent patch. It is not possible to determine whether the cells controlled the orientation of the collagen, or vice versa, from this experiment, but other SEM evidence suggests that the collagen orientation in hard tissue matrices depends on the freedom of cells to move with respect to matrix surface.

Animals↗
PubMed1975

A robust micromanipulator for the scanning electron microscope.

A simple device for holding and moving mechanical tools in the region of a sample being viewed in the scanning electron microscope is described. The unit has a 20:1 mechanical reduction and when fitted with a tungsten carbide dental chisel, it is sufficiently rigid to cut biological hard tissues. Alternately, when fitted with an electro-etched tungsten needle, it can be used, in conjunction with specimen stage controls, to remove individual cells from the surface of soft tissues. Examples of these applications are illustrated.

Animals↗