Microscopy Conference 2023

Prof. Dr.

Plenary chair

Prof.

From cryoEM, cryoET to ptychography – an overview

Abstract text (incl. figure legends and references) Cryo-electron microscopy (cryoEM) is a powerful method for the high-resolution three-dimensional structural characterization of a wide range of biological samples in a close-to-native, frozen-hydrated state [1]. Such biological samples, preserved in vitrified ice, are extremely radiation sensitive, therefore images of these have low signal-to-noise ratios [2] and low contrast [3,4]. Recent development in microscope instrumentation, direct electron detector, microscope automation and high throughput imaging, and advanced software for data processing and image reconstruction, has revolutionized the field of structural biology, allowing protein structures to be determined at the atomic resolution, especially using cryoEM SPA method (Fig. 1). For studying macromolecular complexes that are intrinsically flexible and dynamic, and often function in higher-order assemblies that are difficult to purify, cryoET and subtomogram averaging (cryoET STA) has emerged as a potent tool to obtain structures of these at near-atomic resolution (Fig. 1). The study of such complexes and assemblies in situ using cryoET STA, coupled with cryoFIB and correlative and integrative imaging, opens a new frontier in structural cell biology [5]. While cryoEM SPA and cryoET STA rely on the use of conventional phase-contrast images that require the correction for the contrast transfer function, cryo-electron ptychography (cryoEPt) [6] (Fig. 2) is a new alternative technique that is based on dose-efficient diffractive imaging [7]. Ptychography uses a defocused probe to scan over a specimen with highly overlapping probe positions and computationally reconstructs the wavefunction of the sample. Such an approach has been widely employed in physical and material sciences, and now shows great promise for radiation-sensitive biological samples. I will present an overview of these cryo-electron microscopy modalities, using examples of our recent studies on human viruses, including HIV-1, SARS-CoV-2, and Rotavirus [8-10], to demonstrate the power of each method to image vitrified native biological samples. Figure legends: Figure 1. Schematic workflow of cryoEM using three imaging modalities, cryoEM single particle analysis (cryoEM SPA), cryo-electron tomography and subtomogram averaging (cryoET STA) and cryo-electron diffraction (cryoED). The workflow involves 1) sample preparation, 2) sample vitrification, 3) data acquisition, and 4) 3D reconstruction. A representative structure from each modality is shown. Figure 2. Schematic optical configuration diagram for cryo-ptychography (a), array of diffraction patterns as a function of probe positions (b), and reconstructed phase of rotavirus double-layered particles (c). Scale bars: 100 nm References: [1] R. Henderson et al., Journal of Molecular Biology 213, 899 (1990). [2] R. Henderson, Quarterly Reviews of Biophysics 28, 171 (2009). [3] R. Danev et al., Ultramicroscopy 88, 243 (2001). [4] R. Danev et al., Proceedings of the National Academy of Sciences 111, 15635 (2014). [5] P. Zhang, Curr Opin Struct Biol. doi: 10.1016/j.sbi.2019.05.021. (2019) [6] L. Zhou, et al., Nature Communications 11, 2773 (2020). [7] J.M. Rodenburg, Advances in Imaging and Electron Physics 150, 87-184 (2008). [8] T. Ni et al., Sci Adv 7(47):eabj5715 (2022) [9] L. Mendonça et al., Nat Commun. 12(1):4629 (2021) [10] L. Zhou et al., Nat Commun 11 (1):2773 (2020)

Plenary 2
Cryo-EM, HRTEM, ptychography

Appointment

Plenary chair

Programme

Plenary 2Cryo-EM, HRTEM, ptychography

Appointment

Plenary chair

Programme

Plenary 2
Cryo-EM, HRTEM, ptychography