The myth of high-resolution liquid phase biological electron microscopy

Cryo-electron microscopy (cryo-EM) has transformed structural biology over the past 12 years, with it now being routine rather than exceptional to reach a near-atomic level of resolution for proteins and macromolecular complexes. Samples are immobilized by vitrification and this sample can be maintained at liquid nitrogen temperatures in the vacuum of the electron microscope with negligible sublimation. Due to the low electron doses needed to avoid radiation damage, averaging over tens of thousands to hundreds of thousands of particle images is used to achieve a high signal-to-noise ratio. An alternative approach has been proposed where samples are at room temperature in the liquid state, maintained in the vacuum of the electron microscope by thin film enclosures that are relatively transparent to electrons while preventing evaporation of the liquid. A paper has argued that using this liquid-phase approach, higher resolution (3.2 Å) can be achieved than using cryo-EM (3.4 Å) when imaging and reconstructing adeno-associated virus particles. I show here that these assertions are untrue, and that basic principles in mathematics and physics would need to be violated to achieve the stated resolution in the liquid state. Thus, high resolution liquid phase EM of macromolecules remains science fiction.