(color online) Air–water interface effects during specimen cryo-vitrification. (a) The bio-macromolecule specimen exhibits homogenous distribution and good shape in negative electron microscopy (left), but is prone to degradation and is difficult to observe in cryo-electron microscopy (right). Scale bar, 50 nm. (b) Diagram showing the physical process during specimen cryo-vitrification. Bio-macromolecules in their native state (A_V) are colored pink, those absorbed in the air–water interface (A_S) are colored orange, and those denatured (A_D) are in yellow. The thickness (t) of the solution layer after blotting is between 30 nm and 100 nm.