Real-Time Measurement of the Liquid Amount in Cryo-Electron Microscopy Grids Using Laser Diffraction of Regular 2-D Holes of the Grids

Mol Cells. 2020 Mar 31;43(3):298-303. doi: 10.14348/molcells.2020.2238.


Cryo-electron microscopy (cryo-EM) is now the first choice to determine the high-resolution structures of huge protein complexes. Grids with two-dimensional arrays of holes covered with a carbon film are typically used in cryo-EM. Although semi-automatic plungers are available, notable trial-and-error is still required to obtain a suitable grid specimen. Herein, we introduce a new method to obtain thin ice specimens using real-time measurement of the liquid amounts in cryo-EM grids. The grids for cryo-EM strongly diffracted laser light, and the diffraction intensity of each spot was measurable in real-time. The measured diffraction patterns represented the states of the liquid in the holes due to the curvature of the liquid around them. Using the diffraction patterns, the optimal time point for freezing the grids for cryo-EM was obtained in real-time. This development will help researchers rapidly determine highresolution protein structures using the limited resource of cryo-EM instrument access.

Keywords: cryo electron microscopy; grid preparation; laser diffraction; real-time measurement; vitreous ice.

MeSH terms

  • Cryoelectron Microscopy / methods*
  • Lasers / standards*