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

Abstract

Abstract 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.