In the traditional cryo-EM sample preparation workflow, blotting is the process of removing excess aqueous sample from the grid with filter paper prior to vitrification. Inherently a difficult process to control, users must take into consideration the risks that this poses to their results.
Blotting risks sample integrity
Although commonly used, its effects on sample integrity remain to relatively be poorly defined and understood. It is difficult to know how shear forces, as well as changes to buffer concentration due to evaporation, affect individual particles. To add to the uncertainty, these effects are coupled with the often-harmful influence of the air water interface (AWI), which risks alignment of orientation, as well as particle dissociation or even denaturation.
Researchers are still trying to untangle the relationship between all these forces at play, and to better understand exactly how individual samples are adversely affected by blotting.
Blotting risks image resolution
Blotting leaves behind an uneven residual fluid layer, and therefore produces an uneven ice layer following vitrification. Ice thickness is a critical factor in image resolution, and as such, it is very difficult to reliably achieve optimal grid preparation with blotting. This oftens results in suboptimal output image resolution, wasting valuable time and resource at the microscope.