Measuring Membrane Potentials with Cryo-EM?

Abstract

In materials science it is well known that electrostatic potentials produce phase-shifts of electron waves which are detectable in phase-contrast electron microscopy. The question arises, are membrane potentials preserved during rapid freezing and cryo-EM imaging of liposomes? Imaged with 200 keV electrons, a 200 mV membrane potential in a 30 nm liposome is expected to produce a phase shift of about 50 mrad, resulting in approx. 10% change in image intensity. (The maximum phase shift due to the liposome membrane alone is about 200 mrad.) Calculations showed that phase-plate imaging would be necessary to detect the signal. We used a JEOL JEM2200FS microscope equipped with a Zernike phase plate having a cut-on frequency of approximately 1/100 nm. Liposomes with or without added valinomycin were adhered to a carbon film and then washed a buffer to create a 350:1 K+ gradient, either inwardly or outwardly directed. In liposomes in the 20-30 nm size range we observed a contrast difference relative to the no-valinomycin controls as expected from positive and negative membrane potentials ∼100 mV in size. Thus the diffusion potential set up by a K+ gradient appears to remain throughout the freezing and imaging process.