Charging has long been known to reduce spatial resolution in cryo-EM, since non-uniform electrostatic fields in irradiated vitreous ice cause distortions and image shifts. Charging shifts the back focal plane, misaligning the electron beam and thus complicating imaging with a phase plate. In single-particle cryo-EM, this problem is alleviated by irradiating the support film simultaneously with the specimen, or by using a conductive continuous support layer. In cryo-FIB-milled cellular lamellae, charging has been much less well characterized. A mitigation strategy that has been proposed is to apply a post-milling sputter coating of platinum. However, the conditions required to suppress charging have not been characterized. Additionally, platinum’s high density and large scattering cross section cause significant signal loss, which has limited its adoption. The extent of charging in lamellae, and its consequences for cryo-EM imaging both with and without a phase plate, has remained largely unquantified. Here, we systematically characterize charge accumulation during cryo-EM imaging of lamellae and show that it occurs irrespective of the ion species used for milling. Positive charging produces millimeter-scale shifts of the back focal plane, making stable beam alignment with a laser phase plate impractical. We demonstrate that depositing a thin (∼5 nm) amorphous carbon layer after milling reduces charging by more than 10-fold without detectable loss of signal, improving image quality. Crucially, a thin layer of carbon enabled laser phase plate imaging of lamellae while maintaining a consistent phase shift. By making laser phase plate imaging of cellular lamellae practicable, this approach will substantially improve image contrast in cellular cryo-EM and opens the door to structural studies of smaller macromolecular complexes in their native cellular context than are currently accessible.
Dickerson et al. (Sun,) studied this question.