Photoelectron imaging of cells: photoconductivity extends the range of applicability

Abstract

Photoelectron imaging is a sensitive surface technique in which photons are used to excite electron emission. This novel method has been applied successfully in studies of relatively flat cultured cells, viruses, and protein-DNA complexes. However, rounded-up cell types such as tumor cells frequently are more difficult to image. By comparing photoelectron images of uncoated and metal-coated MCF-7 human breast carcinoma cells, it is shown that the problem is specimen charging rather than a fundamental limitation of the electron imaging process. This is confirmed by emission current measurements on uncoated monolayers of MCF-7 carcinoma cells and flatter, normal Wi-38 fibroblasts. We report here that sample charging in photoelectron microscopy can be eliminated in most specimens by simultaneous use of two light sources--the standard UV excitation source (e.g., 254 nm) and a longer wavelength light source (e.g., 325 nm). The reduction in sample charging results largely from enhanced photoconduction in the bulk sample and greatly extends the range of cells that can be examined by photoelectron imaging. The contributions of photoconductivity, the electric field of the imaging system, and the short escape depths of the photoelectrons combine to make photoelectron imaging a uniquely sensitive technique for the study of biological surfaces.

Challenges in Applying Photoemission Electron Microscopy to Biological Systems†

“…The depth of information is the distance between the surface and a point at which information from the sample contributes to the image at a specified resolution (46). To generate photoelectrons, light that penetrates the sample is absorbed, induces photoionization and the photoionized electrons are transported to the surface where they then escape from the sample to the vacuum (8). The depth‐dependent yield of photogenerated electrons is not known.…”

“…Reprinted with permission from Habliston et al . (8). (c) PEEM image of recA‐DNA revealing spatial resolution.…”

“…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).…”

Applications of Free‐Electron Lasers in the Biological and Material Sciences¶

“…PEEM imaging was applied to biological samples as early as 1972 (121). Since then, viruses and DNA (122), eukaryotic cells (123), cultured cancer cells (124) and cytoskeletons (125) have been studied. Because PEEM is a UHV technique, the samples must be fixed, and little if any water remains.…”

Investigations of the Air-Water Interface: A Structural Analysis of Metallic Surface Films and Aquatic Surface Films by Comparative Microscopy

“…The productive collaboration resulted in several important works, including Griffith and Rempfer (1985); Griffith, 1987;Birrell, Habliston and Griffith, 1994;and Habliston, Hedberg, Birrell, et al, (1995), all key articles in photoemission electron microscopy.…”