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.
Highlights
Cryo-electron microscopy is an excellent method for the structural analysis of biological materials
In comparison to stain-embedded samples of which the resolution is limited to the size of heavy metal salts, cryoelectron microscopy (cryo-EM) can achieve structure determination of macromolecular assemblies at near-atomic resolution because the image formation directly results from electron scattering of biological specimen (Gonen et al 2005)
In order to entrap biological specimen in vitreous ice that is free of supporting material, EM grid coated with a thin carbon film with perforated holes, namely “holey carbon grid”, is used
Summary
Cryo-electron microscopy is an excellent method for the structural analysis of biological materials. In comparison to stain-embedded samples of which the resolution is limited to the size of heavy metal salts, cryo-EM can achieve structure determination of macromolecular assemblies at near-atomic resolution because the image formation directly results from electron scattering of biological specimen (Gonen et al 2005). In order to entrap biological specimen in vitreous ice that is free of supporting material, EM grid coated with a thin carbon film with perforated holes, namely “holey carbon grid”, is used.
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