Immunophotoelectron microscopy: the electron optical analog of immunofluorescence microscopy.

Abstract

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.

Fluorescence Quenching by Colloidal Heavy Metals Nanoparticles: Implications for Correlative Fluorescence and Electron Microscopy Studies

“…Previous studies demonstrated that when fluorophores (fluorescein or rhodamine) and cAu 30 are attached to the same molecule (such as IgG or ligand), the fluorescence signal was quenched (Goodman, et al , 1991;Birrell, et al , 1985).…”

“…Most fluorophores are small, compatible with biological samples and easily conjugated to different proteins or ligands. However, resolution attainable with LM is limited (Birrell et al , 1985). Therefore, subsequent EM imaging may be beneficial to obtain higher spatial resolution for structural studies (Nisman et al , 2004;Albrecht et al , 1993).…”

Protein kinase C inhibitor H‐7 alters the actin cytoskeleton of cultured cells

“…The Oregon photoelectron microscope has been described (Griffith and Rempfer, 1987) and has been used to image a variety of biological specimens (Griffith and Birrell, 1985;Hedberg and Griffith, 1986;Habliston et al, 1986). In short, the microscope is an oil-free ultrahigh vacuum instrument utilizing liquid nitrogen temperature sorbtion prepumps and an ion pump for attaining final vacuum.…”

Challenges in Applying Photoemission Electron Microscopy to Biological Systems†

“…From a historical perspective, O. Hayes Griffith (4) was one of the pioneers of PEEM technology and applied the technique to biological samples in 1972 (4), reporting first PEEM images of rat epididymis. 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.…”