Strategies to Find Where's Waldo (Wally) in Biology using 4D STEM (2024)

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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.

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