![]() ![]() ![]() Micrographs are obtained by conventional transmission electron microscopy (CTEM) at low fluences of 20–100 e −/Å 2 and imaged at micrometer-defocuses to enhance low-resolution contrast of the macromolecules. Thus far, complete three-dimensional (3D) single-particle cryo-EM structures of biological macromolecules at close-to-atomic resolution, have not been determined by images of the STEM technique.Įlectron cryomicroscopy (cryo-EM) has become a very successful structural biology technique that commonly produces near-atomic resolution 3D structures of ice-embedded biological macromolecules. More recently, micrographs of rotavirus and HIV-1 virus-like particles were imaged using ptychography reporting the principal applicability to biological specimens albeit at low resolution 27. Furthermore, cryo-STEM was applied to single-particle specimens of Fe or Zn-loaded ferritin to precisely visualize and locate metals within the protein cage 26. Although low in resolution, it was shown that image contrast can be obtained even from micrometer-thick samples mainly by inelastically scattered electrons 25. More recently, STEM tomography has been applied to thick vitrified cells 23, 24. One of the early STEM applications to freeze-dried biological samples was the molecular mass determination by annular dark field (ADF) scattering, as the number of atoms is directly related to the scattering intensity 22. For these dose-sensitive low-contrast specimens, iDPC–STEM enables direct interpretation of the image without the need of defocusing and a subsequent contrast-transfer function (CTF) correction of the images 21. Recently, large biological sections, including thick ones (~500 nm), have also been imaged 20. By using iDPC–STEM with a low-dose exposure of as little as 40 e −/Å 2, a resolution of 1.8 Å was obtained successfully from a single micrograph for such materials 19. Other investigated materials are known as metal-organic frameworks that can only be imaged using electron doses smaller than 50 e −/Å 2 before damaging the structure 18. One of these samples included an individual aromatic hydrocarbon molecule trapped within a porous framework structure 17. Moreover, iDPC–STEM was demonstrated to successfully image different crystalline as well as amorphous materials including beam-sensitive ones such as zeolites 14, 15, 16. In the same manner, specimens such as metal hydrides were successfully visualized at subatomic resolution, including heavy elements alongside light elements such as hydrogen 13. Among these STEM imaging modalities is integrated differential phase contrast–STEM (iDPC–STEM) 7, 8, which has been routinely applied to a variety of specimens such as GaN, NdGaO 3-La 0.67Sr 0.33MnO 3, Ni–YSZ interfaces and Bi 2Sr 2CaCu 2O 8+δ superconductors 9, 10, 11, 12. ![]() 4, 5), the latter becoming the method of choice for obtaining the highest possible spatial resolutions 6. STEM methods and a derivate known as ptychography were shown to image dose-resistant specimens reaching resolutions better than 0.5 Å (refs. Scanning transmission electron microscopy (STEM) is a well-established methodology in characterizing materials at micro, nano and atomic scales 1, 2, 3. These data show that STEM imaging in general, and in particular the iDPC–STEM approach, can be applied to vitrified single-particle specimens to determine near-atomic resolution cryo-EM structures of biological macromolecules. The micrographs show complete contrast transfer to high resolution and enable the cryo-EM structure determination for KLH at 6.5 Å resolution, as well as for TMV at 3.5 Å resolution using single-particle reconstruction methods, which share identical features with maps obtained by CTEM of a previously acquired same-sized TMV data set. Here, we apply scanning transmission electron microscopy (STEM) using the integrated differential phase contrast mode also known as iDPC–STEM to two cryo-EM test specimens, keyhole limpet hemocyanin (KLH) and tobacco mosaic virus (TMV). In electron cryomicroscopy (cryo-EM), molecular images of vitrified biological samples are obtained by conventional transmission microscopy (CTEM) using large underfocuses and subsequently computationally combined into a high-resolution three-dimensional structure. ![]()
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