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

Abstract

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...

Immunophotoelectron microscopy: the electron optical analog of immunofluorescence microscopy.

“…The photoelectron label, in this case the colloidal gold with the third antibody (RAG) bound to it, does not contribute to, nor interfere with, the fluorescence image. Similarly, the rhodamine fluorochrome on the second antibody does not significantly affect the photoelectron image (15). The gold-labeled microtubules are clearly visible in Fig.…”

“…This sensitivity to topographical detail is very useful for imaging fine details but can exceed the useful range in cases of larger-scale structures, such as some nuclear regions and rounded-up cells. Topographical contrast has been used alone to visualize cytoskeletal structures by photoelectron imaging in a previous study (15). In that case, the cytoskeletal elements were indirectly identified by comparison with immunofluorescence images of the same cells.…”

“…CV-1 (African green monkey) kidney epithelial cells were grown on sterile tin oxide-coated 5-mm glass coverslips (Bellco Glass) prepared as described (15). The cells were cultured in Dulbecco's modified Eagle's medium (GIBCO) containing 10% fetal bovine serum (GIBCO), 25 mM Hepes, and 2 mM L-glutamine in a 10% C02/90% air incubator at 37°C.…”

Photoelectron Imaging in Cell Biology

“…A comparison of photoelectron and immunofluorescence images of the same cells has recently been made (22). This type of study is illustrated in Figure 6, using RAT-1 fibroblast cells prepared for the visualization of actin.…”

Multiple Labeling of Cellular Constituents by Combining Surface Reflection Interference and Fluorescence Microscopy

“…Because of the growing awareness of extensive interactions between the different components of the cytoskeleton, such as microfilaments, microtubules and intermediate filaments [2,6,9,10,12,26,27,34] it is not surprising that double immunofluorescence labeling has been extensively used in studies of cytoskeletal organization and function for visualizing the distribution of two different antigens in the same cell [28], Information about the overall organization of the cyto skeleton, however, cannot be obtained by this approach. Therefore a variety of nonspe cific techniques which can be used in con junction with fluorescence microscopy [11,17,18,19,36] such as transmission [4] and scanning [30] electron microscopy, videoenhanced contrast microscopy [1], surface re flection interference (SRI) microscopy [18,19,21], and photoelectron microscopy [17] have been developed to visualize the cyto skeleton.…”