Photoelectron microscopy of cell surfaces

Dam, Rudy J.; Nadakavukaren, Karen K.; Griffith, O. Hayes

doi:10.1111/j.1365-2818.1977.tb00061.x

Cited by 22 publications

(13 citation statements)

References 17 publications

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“…Griffith and his group realized the capabilities of PEEM as the electron-optics analog of fluorescence microscopy and used colloidal gold and silver enhanced colloidal gold particles to selectively label portions of a biologic with a higher spatial resolution (5)(6)(7). PEEM images for cells (8)(9)(10), viruses (11,12) and DNA (11,(13)(14)(15), among others (16)(17)(18), were reported using both labeling and nonlabeling techniques. In addition to the experimental evidence, reviews were written establishing PEEM as a powerful tool for biological imaging (19)(20)(21).…”

Section: Introductionmentioning

confidence: 99%

“…These three images, along with the multitude of others that Griffith and coworkers have produced, exemplify the strides his group accomplished in pioneering biologically focused PEEM. †This Photochemistry and Photobiology, 2009, 85: [8][9][10][11][12][13][14][15][16][17][18][19][20] Despite the capabilities demonstrated by Griffith and others, the technique did not flourish in the biological sciences and applications of PEEM to biological systems have lagged behind complementary electron imaging techniques, such as scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Instead, as an ultra high vacuum (UHV) instrument suited for relatively flat samples, PEEM found prominence in the fields of surface and materials sciences (2,22).…”

Section: Introductionmentioning

confidence: 99%

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Challenges in Applying Photoemission Electron Microscopy to Biological Systems^†

Peles

Simon²

2009

Photochem & Photobiology

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Photoemission electron microscopy (PEEM) is a unique surface-sensitive instrument capable of providing real-time images with high spatial resolution. While similar to the more common electron microscopies, scanning electron microscopy and transmission electron microscopy, the imaging technology relies on the photogeneration of electrons emitted from a sample through light excitation. This imaging technique has found prominence in surface and materials sciences, being well suited for imaging flat surfaces, and changes that occur to that surface as various parameters are changed (e.g. temperature, exposure to reactive gases). Biologically focused PEEM received significant attention in the 1970s, but was not aggressively advanced since that pioneering work. PEEM is capable of providing important insights into biological systems that extend beyond simple imaging. In this article, we identify and establish important issues that affect the acquisition and analysis of biological samples with PEEM. We will briefly review the biological impact and importance of PEEM with respect to our work. The article also concludes with a discussion of some of the current challenges that must be addressed to enable PEEM to achieve its maximum potential with biological samples.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Challenges in Applying Photoemission Electron Microscopy to Biological Systems^†

Peles

Simon²

2009

Photochem & Photobiology

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“…After the fluorescence microscopy, the lens immersion oil was carefully cleaned from the back of the coverslip, and the coverslip was transferred to a vial containing the glutaraldehyde fixative described above and stored at 40C. The fixed samples, were dehydrated through a graded series of aqueous ethanol mixtures to 100% ethanol, followed by ethanol/amyl acetate (1:1) and 100% amyl acetate, and then dried under a stream of warm air, as in previous photoelectron microscopy studies on intact cells (1,2,5).…”

Section: Methodsmentioning

confidence: 99%

“…Photoelectron microscopy (photoemission electron microscopy or PEM) has recently been introduced into the study of whole cells (1,2) although the origins of this technique are old, predating both transmission electron microscopy and scanning electron microscopy (for review, see ref. 3 for physics and refs.…”

mentioning

confidence: 99%

Photoelectron microscopy and immunofluorescence microscopy of cytoskeletal elements in the same cells.

Nadakavukaren¹,

Chen²,

Habliston³

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

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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.Photoelectron microscopy (photoemission electron microscopy or PEM) has recently been introduced into the study of whole cells (1, 2) although the origins of this technique are old, predating both transmission electron microscopy and scanning electron microscopy (for review, see ref. 3 for physics and refs. 4 and 5 for biological applications). Photoelectron microscopy differs significantly from the established techniques of transmission and scanning electron microscopy even though the image is formed by electrons. The photoelectron microscope can be considered to be the electron optics analogue of the fluorescence microscope. UV light from a short arc lamp is focused on the specimen as in fluorescence microscopy but, instead of imaging the emitted fluorescent light with a light optics system, emitted electrons are accelerated and imaged with an electron lens system. Photoelectron microscopy has several advantages, including high sensitivity to topographic detail (3, 6), a new source of contrast based on the photoelectric effect (7-9), and an unusually short depth of information (10). The increase in image quality during the development of the photoelectron microscope over the past few years has been substantial. Although the basic mechanisms by which the photoelectron microscopy image arises are understood, the interpretation of photoelectron microscopy images of biological specimens is the focus of current research. Here we report the comparison of photoelectron micrographs with fluorescence micrographs of the same cells that have been labeled by indirect immunofluorescence techniques with antibodies specific fo...

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“…Immunophotoelectron microscopy was first proposed in 1972 (4). Three developments were needed to bring this idea to fruition: the development of a high-resolution ultra-high vacuum photoelectron microscope with image intensification (5), theory and experiments on the photoelectric behavior of biological macromolecules (6)(7)(8)(9), and a search for suitable photoemissive markers (10)(11)(12). While this work was progressing two important developments, the production of monoclonal antibodies (13) and the introduction of colloidal gold as a marker in electron microscopy (14), have extended the capabilities of immunological mapping approaches, including immunophotoelectron microscopy.…”

Section: Introductionmentioning

confidence: 99%

Immunophotoelectron microscopy: the electron optical analog of immunofluorescence microscopy.

Birrell¹,

Habliston²,

Nadakavukaren³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

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The electron optical analog of immunofluorescence microscopy combines three developments: (i) photo-electron microscopy to produce a high-resolution image of exposed components of the cell, (ii) site-specific antibodies, and (iii) photoemissive markers coupled to the antibodies to make the distribution of sites visible. This approach, in theory, provides a way to extend the useful immunofluorescence microscopy 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 prepared under conditions that preserve microtubules, and the microtubule networks were visualized by both indirect immunofluorescence and immunophotoelectron microscopy using colloidal gold coated with antibodies. Colloidal gold serves as a label for immunophotoelectron microscopy, providing enhanced photoemission from labeled cellular components so that they stand out against the darker background of the remaining unlabeled structures. In samples prepared for both immunofluorescence and immunophotoelectron microscopy, individual microtubules in the same cells were visualized by both techniques. The photoemission of the colloidal gold markers is sufficiently high that the microtubules are easily recognized without reference to the immunofluorescence micrographs, indicating that this approach can be used, in combination with antibodies, to correlate structure and function in cell biological studies.

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Photoelectron microscopy of cell surfaces

Cited by 22 publications

References 17 publications

Challenges in Applying Photoemission Electron Microscopy to Biological Systems^†

Challenges in Applying Photoemission Electron Microscopy to Biological Systems^†

Photoelectron microscopy and immunofluorescence microscopy of cytoskeletal elements in the same cells.

Immunophotoelectron microscopy: the electron optical analog of immunofluorescence microscopy.

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Photoelectron microscopy of cell surfaces

Cited by 22 publications

References 17 publications

Challenges in Applying Photoemission Electron Microscopy to Biological Systems†

Challenges in Applying Photoemission Electron Microscopy to Biological Systems†

Photoelectron microscopy and immunofluorescence microscopy of cytoskeletal elements in the same cells.

Immunophotoelectron microscopy: the electron optical analog of immunofluorescence microscopy.

Contact Info

Product

Resources

About

Challenges in Applying Photoemission Electron Microscopy to Biological Systems^†

Challenges in Applying Photoemission Electron Microscopy to Biological Systems^†