Fig. 10. Field emission scanning and transmission EM of untransfected PtK2 cells. Untransfected PtK2 cells were fixed and immunogold labelled (with 8 nm gold particles) for K8, a natural component of the endogenous keratin filament network, as described in Materials and Methods. (A) Scanning EM image to show the degree of cytoskeleton preservation; image acquired using the SE detector (secondary electrons), at 9000x magnification and 10 mm working distance. Bar, 1 µm. (B,C,D) The keratin filament cytoskeleton at high magnification. All images were taken at 130,000x magnification and a working distance of 10.1 mm. Image in (B) was acquired using the SE detector to show maximum detail of the keratin filament surface structure. Image in (C) was acquired with the YAGBSE detector for backscattered electrons, to distinguish gold particles of the immunogold labelling; gold particles appear as white dots on the filaments from the high-energy backscattered electrons, but the image of the filament surface is poor. Both SE and YAGBSE images are combined in (D), where a keratin particle attached to the side of a keratin filament can be clearly seen (white arrow). Bar, 100 nm. (E) Transmission EM image of cell after the same fixation as in (A-D), also labelled with anti-K8 monoclonal antibody CAM 5.2. Immunogold labelling was performed before resin embedding and labelling is seen (black dots) along the keratin filaments. Keratin particles are seen adhering to the filaments (black arrows). Note that microtubules (*) are unlabelled. Bar, 100 nm.