By Utkur M. Mirsaidov, Haimei Zheng, Yosune Casana and Paul Matsudaira
Biophysical Journal, 2012 Volume 102 Issue 4, Pages L15-L17
We demonstrate an in situ transmission electron microscopy technique for imaging proteins in liquid water at room temperature. Liquid samples are loaded into a microfabricated environmental cell that isolates the sample from the vacuum with thin silicon nitride windows. We show that electron micrographs of acrosomal bundles in water are similar to bundles imaged in ice, and we determined the resolution to be at least 2.7 nm at doses of ∼35 e/Å2. The resolution was limited by the thickness of the window and radiation damage. Surprisingly, we observed a smaller fall-off in the intensity of reflections in room-temperature water than in 98 K ice. Thus, our technique extends imaging of unstained and unlabeled macromolecular assemblies in water from the resolution of the light microscope to the nanometer resolution of the electron microscope. Our results suggest that real-time imaging of protein dynamics is conceptually feasible.Imaging proteins in a liquid cell. (A) The liquid cell is assembled and a protein solution is loaded through two large reservoirs that are then sealed by a gasket. The protein solution is drawn by capillary force into the liquid cell and forms a thin film between two 10-nm-thick Si3N4 membranes. (B) Low-magnification image of 80-nm-diameter acrosomal bundles in liquid. (C) An acrosomal bundle in h0l orientation. The unit cell is boxed. (D) Fourier transform pattern from a portion of the image of the acrosomal bundle in C. The meridional reflection at 2.7 nm is circled. Red box: Unit cell reconstructed from reflections with S/N ratio (E) IQ plot of the Fourier transform and the resolution shells at 5 and 2.7 nm. (F) MAP-assembled microtubules show rod-shaped structures (enhanced contrast), whereas the protofilament substructure is not resolved.
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