Charge accumulation in electron cryomicroscopy.

When irradiated in a transmission electron microscope, plunge-frozen, amorphous water ice specimens accumulate a pattern of static charge that changes dynamically as the specimen is irradiated, and which can deflect the transmitted electrons and blur the resultant micrographs. Here we provide a physical description of this charge accumulation and characterise its dynamic behaviour in the context of low-dose electron cryomicroscopy (cryoEM). We observe the accumulation of positive charge in the primary irradiation area as expected from earlier work. To our surprise, we also observed a build-up of negative charge in nearby unirradiated regions of the specimen. Using a standard carbon support foil containing a pure water ice specimen, we collect a portion of this negative charge in the micrometer sized specimen holes which act as electrostatic lenses. These unusual, diverging micro-lenses are extremely sensitive charge detectors that allow us to directly measure the magnitude and dynamics of charge accumulation and neutralisation that occur during cryoEM imaging. Using these measurements, we find that the build-up of charge on the specimen saturates to a dynamic equilibrium at an electron fluence which is orders of magnitude lower than required for a typical low-dose micrograph. The measurements here will guide the development of optimal imaging conditions for biological specimens and contribute to a complete theory of information loss in electron cryomicroscopy.