How do you optimize sample preparation for cryo-EM?

2 Prepare a thin layer of vitrified sample

The next step in cryo-EM sample preparation is to prepare a thin layer of vitrified sample on a grid. Vitrification is the process of freezing your sample rapidly and without ice crystals, preserving its native structure and hydration. To achieve vitrification, you need to apply a small amount of your sample (usually a few microliters) on a grid coated with a thin film of carbon or other support material. Then, you need to blot away the excess liquid with a filter paper and plunge the grid into liquid ethane or another cryogen. This will create a thin layer of vitrified sample (usually less than 100 nm thick) on the grid, ready for cryo-EM imaging.

3 Store and transport your grids

The third step in cryo-EM sample preparation is to store and transport your grids safely and securely. Your grids are very sensitive to temperature, humidity, and contamination, so you need to protect them from any environmental changes or damage. You can store your grids in liquid nitrogen or in a freezer at -80°C or below, using special containers or boxes designed for cryo-EM grids. You can transport your grids in liquid nitrogen dewars or in dry shippers, which are insulated containers that maintain a low temperature for several days. You should always handle your grids with care and use appropriate tools and gloves to avoid touching them directly.

4 Evaluate your grid quality

The fourth step in cryo-EM sample preparation is to evaluate your grid quality before proceeding to cryo-EM imaging. You can use different methods to assess your grid quality, such as optical microscopy, scanning electron microscopy (SEM), or low-dose transmission electron microscopy (TEM). These methods can help you to check the thickness, uniformity, and distribution of your vitrified sample layer, as well as the presence of any contaminants, ice crystals, or artifacts. You can also use these methods to select the best areas of your grid for cryo-EM imaging, based on the density and morphology of your specimen.

5 Optimize your imaging conditions

The final step in cryo-EM sample preparation is to optimize your imaging conditions for cryo-EM analysis. You need to consider several factors that can affect the quality and resolution of your cryo-EM images, such as the electron dose, the magnification, the defocus, the tilt, and the alignment. You can use different software and hardware tools to control and adjust these factors, depending on the type and model of your cryo-EM instrument. You can also use different strategies to improve the signal-to-noise ratio and the contrast of your images, such as phase plates, energy filters, or image processing techniques.