Topographic imaging of chromium-coated frozen-hydrated cell and macromolecular complexes by in-lens field emission scanning electron microscopy

An in-lens Schottky field emission scanning electron microscope (SEM) combined with a transmission electron microscope (TEM)-type cold-stage and a chromium (Cr) sputter-coating system was developed to rapidly prepare and cryo-image biological specimens to attain accurate nanometer-level structural information. High-resolution topographic images at high primary magnification (greater than or equal to 200,000x) were digitally recorded with very short dwell times and without beam damage. Plunge freezing in ethane, followed by fracturing, Cr coating, and in-lens cryo-high-resolution scanning electron microscope (HRSEM) imaging directly revealed macromolecular features of yeast cells, platelets, and cell-free elastin analogues. The "vitreous" nature of bulk water in its solid state appeared featureless in cryo-HRSEM images, suggesting that if ice crystals were present they would be less than or equal to 2-3 nm (the approximate instrument resolution on cryo-specimens). Compared to technically difficult and indirect freeze-fracture TEM replicas, cryo-HRSEM samples are fully hydrated, unfixed, non-cryoprotected specimens immersed in featureless ice. The time necessary to cryo-immobilize the specimen and record the image is <3 hr. The hexagonal arrays of intramembrane particles on the protoplasmic face of yeast cells and differences in surface morphology between thrombin-stimulated and quiescent platelets were assessed. A clear interface line between collapsed elastin fibril lacework and vitreous lakes was commonly observed. These experiments demonstrate the feasibility of this technique to rapidly evaluate macromolecular features in cryofixed cells and cell-free systems.