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, Judy S Kim The Rosalind Franklin Institute , Harwell Campus, Didcot, Oxfordshire , UK Department of Materials, University of Oxford , Oxfordshire , UK Corresponding author: judy.kim@rfi.ac.uk Search for other works by this author on: Oxford Academic Chen Huang The Rosalind Franklin Institute , Harwell Campus, Didcot, Oxfordshire , UK Search for other works by this author on: Oxford Academic Emanuela Liberti The Rosalind Franklin Institute , Harwell Campus, Didcot, Oxfordshire , UK Search for other works by this author on: Oxford Academic Marcus Gallagher-Jones The Rosalind Franklin Institute , Harwell Campus, Didcot, Oxfordshire , UK Search for other works by this author on: Oxford Academic Brian J Caffrey The Rosalind Franklin Institute , Harwell Campus, Didcot, Oxfordshire , UK Search for other works by this author on: Oxford Academic Adrián Pedrazo-Tardajos The Rosalind Franklin Institute , Harwell Campus, Didcot, Oxfordshire , UK Search for other works by this author on: Oxford Academic Mark Boyce Department of Structural Biology, University of Oxford , Oxfordshire , UK Search for other works by this author on: Oxford Academic Peng Wang Department of Physics, University of Warwick , Coventry , UK Search for other works by this author on: Oxford Academic Angus I Kirkland The Rosalind Franklin Institute , Harwell Campus, Didcot, Oxfordshire , UK Department of Structural Biology, University of Oxford , Oxfordshire , UK Search for other works by this author on: Oxford Academic
Microscopy and Microanalysis, Volume 30, Issue Supplement_1, July 2024, ozae044.900, https://doi.org/10.1093/mam/ozae044.900
Published:
24 July 2024
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Judy S Kim, Chen Huang, Emanuela Liberti, Marcus Gallagher-Jones, Brian J Caffrey, Adrián Pedrazo-Tardajos, Mark Boyce, Peng Wang, Angus I Kirkland, Strategies to Find Where's Waldo (Wally) in Biology using 4D STEM, Microscopy and Microanalysis, Volume 30, Issue Supplement_1, July 2024, ozae044.900, https://doi.org/10.1093/mam/ozae044.900
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Like the study of undomesticated animals, it is advantageous to study biological structures in their native environment. It gives scientists a more information-rich opportunity to study behavior, purpose and functionality based upon structure. Given that biological materials are notoriously radiation sensitive, it is difficult to get information at the nanometer scale at the same time as the wide field of view (FoV) necessary to understand the environment and cellular context needed by biologists and clinicians. Using TEM imaging, the resolution is inherently linked to the FoV by the Nyquist frequency, making high-resolution ultrastructural studies currently possible on smaller ∼800 nm x 800 tiles or volumes. Proximity to the surrounding environment is possible using correlated light and electron microscopy (CLEM) or FIB and SEM approaches.
Our work is now exploring different approaches to use 4D STEM technologies developed in physical sciences to find these elusive biological structures. By using this scanning technique, the FoV is not coupled to the resolution, and we are achieving 6.6 x 6.6 μm scans on cryo EM lamella specimens. Due to limitations of fluence (commonly called dose) needed to preserve the ultrastructure, we approach the problem with a defocused electron probe to scan the entire sample with a larger probe that can be reconstructed for a high resolution exit wave. This approach eliminates the need to further expose the sample while searching for regions of interest and reduces the need to perfectly focus prior to collecting data. Here we will show how this is beneficial to thick bacterial cells and targeting key structures such as viruses which have infected cells. If we collect the data that is of high quality and large FoV, we can take our time to find Waldo (Wally in UK) and see where he is, what he was doing and with whom.
© The Author(s) 2024. Published by Oxford University Press on behalf of the Microscopy Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.
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