Griffith, O. Hayes
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Browsing Griffith, O. Hayes by Author "Habliston, D. L."
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Item Open Access Biological Photocathodes(Proceedings of the National Academy of Sciences, 1989-03) Griffith, O. H.; Habliston, D. L.; Birrell, G. B.; Skoczylas, W. P.; Hedberg, K. K.Biological surfaces emit electrons when subjected to UV light. This emission is increased greatly after exposure to cesium vapor. Increases from 2 to 3 orders of magnitude are observed, depending on the biochemicals present. Heme and chlorophyll exhibit unusually high photoemission currents, which are increased further after cesiation. Photoemission from proteins and lipids is much less but also is increased by exposure to cesium. The formation of photocathodes with cesium greatly increases the practical magnifications attainable in photoelectron microscopy of organic and biological specimens. Photoelectron micrographs taken at magnifications ;;;: x 100,000 of chlorophyll-rich thylakoid membranes and of colloidal gold-labeled cytoskeleton preparations of cultured epithelial cells demonstrate the improvement in magnification. The selectivity and stability of the photocathodes suggest the possibility of detecting chromophore binding proteins in membranes and the design of photoelectron labels for tagging specific sites on biological surfaces.Item Open Access Immunophotoelectron Microscopy: The Electron Optical Analog of Immunofluorescence Microscopy(Proceedings of the National Academy of Sciences, 1985-01) Birrell, G. B.; Habliston, D. L.; Nadakavukaren, K. K.; Griffith, O. H.The electron optical analog of immunofluo rescence microscopy combines three developments: (i) photoelectron microscopy to produce a high-resolution image of ex posed components of the cell, (jQ site-specific antibodies, and (iii) photoemissive markers coupled to the antibodies to make the distribution of sites visible. This approach, in theory, pro vides a way to extend the useful immunofluorescence micros copy technique to problems requiring much higher resolution. The resolution limit of fluorescence microscopy is limited to about 200 nm by the wavelength of the light used to form the image, whereas in photoelectron microscopy the image is formed by electrons (current resolution: 10-20 nm; theoretical limit: 5 nm or better depending on the electron optics). As a test system, cytoskeletons of CV-1 epithelial cells were pre pared under conditions that preserve microtubules, and the microtubule networks were visualized by both indirect immu nofluorescence and immunophotoelectron microscopy using colloidal gold coated with antibodies. Colloidal gold serves as a label for immunophotoelectron microscopy, providing en hanced photoemission from labeled cellular components so that they stand out against the darker background of the re maining unlabeled structures. In samples prepared for both immunofluorescence and immunophotoelectron microscopy, individual microtubules in the same cells were visualized by both techniques. The photoemissionof the colloidal gold mark ers is sufficiently high that the microtubules are easily recog nized without reference to the immunofluorescence micro graphs, indicating that this approach can be used, in combina tion with antibodies, to correlate structure and function in cell biological studies.Item Open Access Photoelectron microscopy and immunofluorescence microscopy of cytoskeletal elements in the same cells(Proceedings of the National Academy of Sciences, 1983-07) Nadakavukaren, K. K.; Chen, L. B.; Habliston, D. L.; Griffith, O. H.Pt K2 rat kangaroo epithelial cells and Rat-I fibroblasts were grown on conductive glass discs, fixed, and permeabilized, and the cytoskeletal elements actin, keratin , and vimentin were visualized by indirect immunofluorescence. After the fluorescence microscopy, the cells were postfixed and dehydrated for photoelectron microscopy. The contrast in these photoelectron micrographs is primarily topographical in origin, and the presence of fluorescent dyes at low density does not contribute significantly to the material contrast. By comparison with fluorescence micrographs obtained on the same individual cells, actincontaining stress fibers, keratin filaments, and vimentin filaments were identified in the photoelectron micrographs. The apparent volume occupied by the cytoskeletal network in the cells as judged from the photoelectron micrographs is much less than it appears to be from the fluorescence micrographs because the higher resolution of photoelectron microscopy shows the fibers closer to their true dimensions. Photoelectron microscopy is a surface technique, and the images highlight the exposed cytoskeletal structures and suppress those extending along the substrate below the nuclei. The results reported here show marked improvement in image quality of photoelectron micrographs and that this technique has the potential of contributing to higher resolution studies of cytoskeletal structures.