The Effect of Electron Beam Irradiation in Environmental Scanning Transmission Electron Microscopy of Whole Cells in Liquid.

Whole cells can be studied in their native liquid environment using electron microscopy, and unique information about the locations and stoichiometry of individual membrane proteins can be obtained from many cells thus taking cell heterogeneity into account. Of key importance for the further development of this microscopy technology is knowledge about the effect of electron beam radiation on the samples under investigation. We used environmental scanning electron microscopy (ESEM) with scanning transmission electron microscopy (STEM) detection to examine the effect of radiation for whole fixed COS7 fibroblasts in liquid. The main observation was the localization of nanoparticle labels attached to epidermal growth factor receptors (EGFRs). It was found that the relative distances between the labels remained mostly unchanged (<1.5%) for electron doses ranging from the undamaged native state at 10 e-/Å2 toward 103 e-/Å2. This dose range was sufficient to determine the EGFR locations with nanometer resolution and to distinguish between monomers and dimers. Various different forms of radiation damage became visible at higher doses, including severe dislocation, and the dissolution of labels.