Sample Preparation for In Situ Cryotomography of Mammalian Cells

The scope of this research is to develop a methodology for sample preparation that is both accessible and reliably reproducible. By achieving this, the power of direct cellular cryo-electron tomography can be used to answer a wide variety of biological questions. The challenges of grid preparation are extensive.

Cells do not readily attach to grids, and manipulation can result in damage to the carbon layer, resulting in the loss of imageable grid areas. Additionally, the prevalence of thin cellular protrusions is reduced without attention to proper sample preparation conditions. Current protocols typically require specialized equipment to solve the major bottlenecks of seeding, such as micro patterning and 3D printed holders for cell attachment and manipulation, respectively.

This protocol provides solutions with greater accessibility, while still maintaining flexibility for these specialized tools to be used for downstream applications.