Measurement of ice thickness on vitreous ice embedded cryo-EM grids: investigation of optimizing condition for visualizing macromolecules

Background: Cryo-electron microscopy is an excellent method for the structural analysis of biological materials. Advantage of its use over conventional electron microscopy techniques is the preservation of the sample in a near-native, hydrated state. To achieve the analysis with greatly improved structural details, optimization of various parameters involved in sample vitrification is required. Most considerable parameter is the thickness of ice: thick and thin layers are ideally in favor for larger and smaller target objects. Findings: We measured the thickness of vitreous ice from different types of widely used holey carbon grids using cryo-EM and electron energy loss spectroscopy. It showed that Quantifoil grids are suitable for the structural analysis of large biological macromolecules (>100 nm in size), whereas the use of lacey and C-flat grids are ideal for smaller particles. Conclusions: This report provides informative details that may help increasing chances of obtaining optimal vitreous ice for various biological objects with different sizes, hence facilitate the successful application of cryo-electron microscopy.