Although there are many light microscopic methods for imaging live cells, they do not have the resolution of electron microscopy. Significant efforts have been put into the development of scanning electron microscopes (SEM) which are capable of imaging wet samples, thus eliminating many of the artefacts associated with traditional sample preparation. This has yet to be achieved in life science applications however adaptations to SEMs for the observation of partially hydrated materials have been developed. Technological improvements to achieve this goal include the development of variable pressure columns and electron detectors that are efficient when imaging samples in a vapour environment (environmental SEM, ESEM). Using this approach, imaging of cells and tissues at acceptable resolution and obtaining adequate contrast is questionable [1].A new technology has been developed allowing cells grown on an electron lucent partition membrane to be stained using either one of a number of protocols including conventional electron microscopy fixation and staining, cytochemistry and immunogold immunocytochemistry. The cells are then sealed in a capsule that contains a liquid imaging media that dissipates electrons and provides some thermal insulation. This capsule maintains a pressure of one atmosphere and the cells can be observed using any SEM equipped with a backscatter electron (BE) detector. Thus unlike ESEM and similar techniques, the sample is completely isolated from the vacuum within the microscope. The images themselves are dependent upon a number of factors including accelerating voltage, probe current and backscatter electron (BE) detection efficiency. This wet SEM technique uses BEs the majority of which originate from the portions of the cells that are adhered to the partition membrane of the chamber. Although an electron scanning reflectance mode is used, the image is generated from material within the cells that yield significant atomic contrast. Subcellular organelles can be distinguished based on differences in local concentration of lipids and salts within biological samples and a wide variety of stains and labels can be used to enhance contrast [2].In a series of preliminary experiments, both pseudomonas aeruginosa bacteria and an astrocytoma derived cell line (U343MG) were grown on the partition membrane of the capsules. The cells were fixed with glutaraldehyde, stained with uranyl acetate and sealed in the capsules immersed in imaging buffer. They were then imaged in a field emission SEM (FESEM) using a conventional solid state BE detector. Bacterial flagella were easily distinguished as well as structures that looked like pili in the pseudomonas samples. In the capsules that contained the astrocytoma derived cells, cell filipodia, cytoskeleton and nuclei were easily distinguished. Some of the astrocytoma cells grown on the partition membrane were fixed in paraformaldehyde and immunogold labelled with 30nm colloidal gold bound to an antibody against CD44 and sealed in the capsules immersed in imaging...
The microscopic surface potential distributions were measured from the onset energies of secondary electron spectra using a scanning Auger electron microscope. The spatial resolution is several tens of nanometers and a sensitivity of the surface potential measurement is 0.05 eV. We demonstrated the calibration of the instrument for local surface potential analysis based on the onset energy measurement of the secondary electron spectrum. Several applications of this technique for Ni polycrystal grains of different orientations, and a potential profile along the p–n junction of a light-emitting diode were demonstrated.
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