@article{3723c37a513d439ab88e28f97b4dcc20,
title = "Scanning Transmission Electron Microscopy in a Scanning Electron Microscope for the High-Throughput Imaging of Biological Assemblies",
abstract = "Electron microscopy of soft and biological materials, or {"}soft electron microscopy{"}, is essential to the characterization of macromolecules. Soft microscopy is governed by enhancing contrast while maintaining low electron doses, and sample preparation and imaging methodologies are driven by the length scale of features of interest. While cryo-electron microscopy offers the highest resolution, larger structures can be characterized efficiently and with high contrast using low-voltage electron microscopy by performing scanning transmission electron microscopy in a scanning electron microscope (STEM-in-SEM). Here, STEM-in-SEM is demonstrated for a four-lobed protein assembly where the arrangement of the proteins in the construct must be examined. STEM image simulations show the theoretical contrast enhancement at SEM-level voltages for unstained structures, and experimental images with multiple STEM modes exhibit the resolution possible for negative-stained proteins. This technique can be extended to complex protein assemblies, larger structures such as cell sections, and hybrid materials, making STEM-in-SEM a valuable high-throughput imaging method.",
author = "Parker, {Kelly A.} and Stephanie Ribet and Kimmel, {Blaise R.} and {Dos Reis}, Roberto and Milan Mrksich and Dravid, {Vinayak P.}",
note = "Funding Information: K.A.P. and B.R.K. acknowledge the National Science Foundation Graduate Research Fellowship under Grant DGE-1842165, the ARO MURI W911NF-18-1-0200, and the Ryan Fellowship. K.A.P. acknowledges the Air Force Center of Excellence for Advanced Bioprogrammable Nanomaterials Grant AFRL FA8650-15-2-5518. S.M.R. acknowledges funding from 3M and the Ryan Fellowship. Research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health under Award Number U54CA199091. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This work used facilities of the Integrated Molecular Structure Education and Research Center, which received financial support from the State of Illinois. This work made use of the BioCryo and Keck-II facilities of Northwestern University{\textquoteright}s NU ANCE Center, which has received support from the Soft and Hybrid Nanotechnology Experimental (SHyNE) Resource (NSF ECCS-1542205); the MRSEC program (NSF DMR-1720139) at the Materials Research Center; the International Institute for Nanotechnology (IIN); and the State of Illinois, through the IIN. This research was supported in part through the computational resources and staff contributions provided for the Quest high performance computing facility at Northwestern University which is jointly supported by the Office of the Provost, the Office for Research, and Northwestern University Information Technology. The authors would like to thank Dr. Benjamin Myers, Dr. Paul Smeets and Tirzah Abbott for their help and advice during image collection and Zhaoyi Gu, Yasmine Zubi, and Prof. Jared Lewis for the use of proteins for imaging. Publisher Copyright: {\textcopyright} 2022 American Chemical Society.",
year = "2022",
month = aug,
day = "8",
doi = "10.1021/acs.biomac.2c00323",
language = "English (US)",
volume = "23",
pages = "3235--3242",
journal = "Biomacromolecules",
issn = "1525-7797",
publisher = "American Chemical Society",
number = "8",
}