Rp. Apkarian et al., Topographic imaging of chromium-coated frozen-hydrated cell and macromolecular complexes by in-lens field emission scanning electron microscopy, MICROS MICR, 5(3), 1999, pp. 197-207
An in-lens Schottky field emission scanning electron microscope (SEM) combi
ned with a transmission electron microscope (TEM)-type cold-stage and a chr
omium (Cr) sputter-coating system was developed to rapidly prepare and cryo
-image biological specimens to attain accurate nanometer-level structural i
nformation. High-resolution topographic images at high primary magnificatio
n (greater than or equal to 200,000x) were digitally recorded with very sho
rt dwell times and without beam damage. Plunge freezing in ethane, followed
by fracturing, Cr coating, and in-lens cryo-high-resolution scanning elect
ron microscope (HRSEM) imaging directly revealed macromolecular features of
yeast cells, platelets, and cell-free elastin analogues. The "vitreous" na
ture of bulk water in its solid state appeared featureless in cryo-HRSEM im
ages, 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 repli
cas, cryo-HRSEM samples are fully hydrated, unfixed, non-cryoprotected spec
imens immersed in featureless ice. The time necessary to cryo-immobilize th
e specimen and record the image is <3 hr. The hexagonal arrays of intramemb
rane particles on the protoplasmic face of yeast cells and differences in s
urface 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.