Liquid Phase Biological Electron Microscopy: Many Published Results and Claimed Benefits Are Fantasy, Not Fact.

While the idea of imaging biological molecules by electron microscopy in the liquid phase might seem to be quite attractive, fundamental problems inherent in this approach preclude success at high resolution. One attractive goal, for example, is to image macromolecular machines in action, but radiation-inactivation of enzymatic function severely limits the electron exposures that can be used. Furthermore, although nanometer resolution or better has been claimed in some papers for macromolecular complexes said to be freely suspended in the liquid phase, Brownian motion must limit the achievable resolution to dimensions that are much larger than the macromolecules themselves. While Brownian motion can be avoided by adsorption of particles to a substrate or a thin window, there still is a risk that air drying occurs. We have analyzed publicly-available EM images of GroEL that were putatively obtained in the liquid phase, and show that these particles clearly had been dried. While the original authors argued that the contrast reversal seen in their images must have been due to liquid water scattering 120 keV electrons much more strongly than either vitreous ice or proteins, we show that the particles were likely negatively stained, presumably by remaining buffer salts.