EM Workflow for Beginners

EM Single Particle Analysis (SPA) Workflow

The typical workflow for SPA projects in EM follows characteristic stages, often requiring iterative optimization at specific points.

A quick way to assess initial project feasibility.
Mainly used to assess particle size, homogeneity and distribution.
Can collect small datasets to generate initial 2D classes and low-resolution volumes.

Start with concentrations 5-10x higher than negative stain for holey grids or use a transparent surface, like thin continuous carbon, graphene or graphene oxide.
Find a blotting condition with a distribution of ice thicknesses.
Screen different ice thicknesses to determine particle behavior at various thicknesses.
Try different cryo-grid foil types (carbon, gold, hole diameter, etc.).
Optimize particle concentration, so you have many particles-per-image with enough space to properly pick them.

Particle aggregation? Try a different grid foil (i.e., holey gold).
Low particle concentration and cannot concentrate further? Use a thick surface (i.e., graphene or graphene oxide) to concentrate particles.
Complex instability? Try mild crosslinking (i.e., BH3 on ice) or more advanced fixing techniques (i.e., grafix).

Load optimized grids into a 200kV microscope for automated imaging over hundreds-to-thousands of images.
Perform initial image processing to determine 2D classes and 3D reconstruction(s) from the dataset.
In many cases, a 200kV dataset will be sufficient resolution for answering the biological question.

For challenging projects with small complexes, high heterogeneity and preference for thick ice, use the optimized grids to collect a dataset on a 300kV microscope. This will help you to achieve the highest possible resolution dataset for your grid.
Grids screened on our 200kV are compatible with all typical autoloader-based systems (i.e., you can transfer the same grid from one system to another).