Fig. 2.

Charge build-up and neutralisation. Series of highly defocussed 300 keV images of a 30 µm square area of a holey carbon film with amorphous ice prepared by plunge freezing. (a) shows the area prior to illumination apart from the electron irradiation used to record this highly defocused, low magnification image (1.2 $\times {10}^{-4}$ e${}^{-}$/Ų) and (b)-(i) shows successive images after irradiation of the centre of the square (5 µm diameter focused beam) by 1.4 e${}^{-}$/Ų. Panel b shows the image immediately after irradiation and frames (c)–(i) are after increasing amounts of illumination of the whole region, 1.2 $\times {10}^{-4}$ e${}^{-}$/Ų per image for b–h. The final frame (i) is the last frame in the series of 300, which is after 3.6 $\times {10}^{-2}$ e${}^{-}$/Ų of cumulative dose, corresponding to 25 min. The side panel (j) shows magnified views of a surface contaminant particle, which is shown by an arrow in (a) and (i). Scale bars are 10 µm and 1 µm; high intensity is white and low intensity is black in all panels.