Recent technological advancements have led to significant gains in higher quality structures and cryo-EM becoming the go-to method for structural biologists. Sample preservation in vitrified ice (vitrification) is widely acknowledged as the first step in the cryogenic electron microscopy (Cryo-EM) workflow. However, before sample preparation, researchers must consider the sample quality and determine its suitability for high-resolution structure determination.
A robust understanding of these critical considerations and the correct instrumentation is vital before beginning any CryoEM project to ensure its subsequent success.
Read our blog series on getting started in sample preparation for single particle cryo-EM
Certainly, sample preparation is a recognized and as yet unresolved bottleneck in the conventional cryo-EM workflow. A standard cryo-EM workflow involves an iterative process of freezing many grids of varying quality and then screening for successful specimens using a cryogenic electron microscope. This process requires the manual handling of small, fragile grids under cryogenic conditions. The quality of resulting specimen grids is often user-dependent leading to poor consistency. Furthermore, substantial researcher experience with cryo-EM is necessary to determine ‘good’ or 'bad' sample quality as outcomes tend to be inconsistent and varied.
The main obstacle to routine structure determination of single particles by high-resolution cryo-EM remains protein adsorption to the air-water interface. It is best practice to avoid air bubbles while handling bulk protein solutions due to denaturation at the air-water interface (AWI).
In the context of thin-film formation for sample vitrification, the increased AWI surface area leads to undesirable effects such as limited particle orientation and distribution, degradation, and aggregation.