Hollow Cone Electron Imaging for Single Particle 3D Reconstruction of Proteins.
The main bottlenecks for high-resolution biological imaging in electron microscopy are radiation sensitivity and low contrast. The phase contrast at low spatial frequencies can be enhanced by using a large defocus but this strongly reduces the resolution. Recently, phase plates have been developed to enhance the contrast at small defocus but electrical charging remains a problem. Single particle cryo-electron microscopy is mostly used to minimize the radiation damage and to enhance the resolution of the 3D reconstructions but it requires averaging images of a massive number of individual particles. Here we present a new route to achieve the same goals by hollow cone dark field imaging using thermal diffuse scattered electrons giving about a 4 times contrast increase as compared to bright field imaging. We demonstrate the 3D reconstruction of a stained GroEL particle can yield about 13.5 Å resolution but using a strongly reduced number of images.
Department of Engineering and System Science, Tsing-Hua University, HsinChu 300, Taiwan.
Chun-Ying
CY
Tsai
Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 115, Taiwan.
Yuan-Chih
YC
Chang
Department of Physics/ EMAT, University of Antwerp, Groenenborgerloaan 171, B2020 Antwerp, Belgium.
Ivan
I
Lobato
Department of Physics/ EMAT, University of Antwerp, Groenenborgerloaan 171, B2020 Antwerp, Belgium.
Dirk
D
Van Dyck
Department of Engineering and System Science, Tsing-Hua University, HsinChu 300, Taiwan.
Fu-Rong
FR
Chen
D000465:Algorithms; D020285:Cryoelectron Microscopy / Q000295:instrumentation / Q000379:methods*; D007091:Image Processing, Computer-Assisted; D021621:Imaging, Three-Dimensional; D011506:Proteins / Q000737:chemistry*
D011506:Proteins