Figure 1
From: Ice thickness monitoring for cryo-EM grids by interferometry imaging
Experimental setup for interferometric microscopy at cryogenic temperature. (a) Optical setup. The insert shows an exemplary EM grid that will be coated with a vitrified ice layer containing the protein complex. (b) Image of the custom-made cryo-stage filled with liquid nitrogen, also showing the tweezer holding the grid coated with sample (arrow 1), the aluminium heat sink supporting the grid (arrow 2), the grid-box transfer mount (arrow 3, magnification from another view in the top right corner), and a grid-box (arrow 4). (c) Picture of the aluminium heat sink on which grids are placed for imaging. The insert shows a magnification showing three grids, the middle one being illuminated by the three-wavelength light through the microscope objective. (d) Top: schematic diagrams of grid hole cross-sections of (1) ideal ice thickness where the particles tightly fit, (2) region with thicker ice regions in which particle density is too high for optimal cryo-EM imaging, and (3) too thick ice with the presence of water droplets not suitable for the measurements. Bottom: typical interferometric image of a grid coated with an ice layer containing the sample. The interference colour patterns reveal the presence of the characteristic regions described on top (three different cases indicated by the arrows). Scale bar 50 µm. (e) Corresponding low-magnification EM image of the same grid region. The EM user usually estimates ice thickness based on the brightness of the images inside the holes of the grids since a thicker ice layer transmits less electrons than a thinner film. The comparison with the interferometric in (d) confirms the presence of different ice layer thicknesses (top in d), as identified by the coloured interference patterns.
