Capturing the intracellular universe at near-native states and in 4D: the many uses of cryo-soft X-ray tomography for in-depth investigations of biological systems

Session

Poster Session 2

Authors

Dr Ilias Kounatidis (1), Dr Mohamed Koronfel (1), Dr Archana Jadhav (1), Dr Chidinma Okolo (1), Mr Jeffrey Irwin (2), Dr Maria Harkiolaki (1)

Affiliations

1. Diamond Light Source
2. Zeiss

Keywords

Cryo-soft X-ray tomography, host-pathogen interaction, cytotoxicity, biomedical imaging, multimodal imaging 

Abstract text

Summary

 

Cryo-Soft X-ray Tomography (cryo-SXT) is a 3D imaging technique that addresses the need for mesoscale imaging of cellular ultrastructure of relatively thick samples without the need for sectioning, staining, or chemical modification. The power of the technique is demonstrated here through a series of current biomedical projects with an emphasis on cellular interactions and their structural responses to environmental challenges within a near-physiological context. The potential of cryo-SXT as a part of emerging advanced multimodal imaging techniques is also presented. 

 

Introduction

 

Soft X-ray imaging has earned its place in the 3D high-resolution imaging hall of fame in recent years through the accumulated body of work that has given us a greater insight into the structures and processes that individual cells and cell populations employ in their maintenance and response mechanisms. Cryo-SXT allows imaging of whole cryo-preserved cells in the water-window X-ray energy range (284-543 eV) in which carbon-rich biological structures absorb X-rays more than the oxygen-rich media that surrounds them resulting in natural absorption contrast imaging through direct detection. At the UK synchrotron correlative cryo-imaging beamline B24 cryoSXT has matured into a well-documented, semi-automated, and amenable method that can generate 4D imaging data that faithfully captures spatial and temporal information within in vitro biological systems. The main technological challenges that present themselves within a facility once the microscopy physical infrastructure is optimised invariably involve applicability and in particular ease-of-access and ease-of-use. These parameters have been satisfied at B24 through the production of robust sample preparation protocols, intelligent data collection strategies and the development of user-friendly interfaces and in silico processes ¹. These advances have been exploited in a number of projects of biomedical relevance. In this work, we will present, as a case study for the benefits affronted by the B24 developments in SXT, advances in our understanding of the immune response with special emphasis. The ability of cryoSXT to interface with other imaging methods and provide correlated high-content composites that cannot be attained otherwise will also be presented.

 

Methods/Materials

 

Cryo-SXT imaging at beamline B24 is performed with an UltraXRM-S220C microscope (Carl Zeiss X-ray Microscopy) following an experimental workflow based on the following sequential steps: i) culture or deposition of cells on perforated carbon film-coated EM Grids, ii) exposure of cells to biological or/and chemical triggers, iii) addition of gold nanoparticles fiducials necessary for projection alignment, v) plunge freezing with the use of a Leica EM GP model. The sample preparation is followed by: vi) mapping and samples quality evaluation with a conventional microscope (Axioimager M2, Zeiss, coupled to a Linkam cryostage), vii) loading of samples into the X-ray microscope, viii) acquisition of 2D X-ray mosaics, ix) acquisition of tilt series projections and ix) X-ray data reconstruction into 3D tomographic volumes using the IMOD software. Visualisation, analyses and correlation is achieved through a number of freely available software packages such as Fiji, ICY and Chimera.

Results 

Understanding the structural manifestations of cellular responses and how these propagate within a population of cells is an absolute requirement when trying to understand aspects of the immune response. Here we present a select panel of projects that take us through (a) a pathogenic challenge in a cell population and two of the ways professional killer immune cells can react to clear perceived threats, (b) the production of cytotoxic substances and (c) the engulfment of pathogens. 

The infection process we present here involves a member of the Herpesviridae family which induces Marek’s disease in avian species. The virus propagates through cell contacts and is highly tumourogenic. We have used cryoSXT to document massive virus-induced membrane remodeling in host cells and track the progress of the virus from the nucleus of infected cells through a number of envelopment stages in the cytoplasm and finally its cross-over to healthy cells where a new infection is established. We will present new structural data that links this virus with clear cytopathic events in the host cell population. As an exemplar of the use of cryoSXT in the understanding of cytotoxic substance generation, we will present work that resulted in the discovery of a new membrane-less organelle that functions as a molecular bomb on the surface of infected cells ². And finally, the act of close-contact clearance through phagocytosis will be documented in high-resolution 3D snapshots of primary professional macrophages responding to pathogens and exhausting themselves while ingesting perceived assailants for the benefit of their community.

The correlative potential of the method will be discussed and explore through its use in existing projects that harness the power of more than one high-resolution multidimensional imaging methods such as electron tomography and hard X-ray elemental analysis. In specific a recent SARS-Cov2 project that brought together cryoSXT, serial cryoFIB/SEM volume imaging and cell lamellae-based cryo-electron tomography (cryoET) will be highlighted ³.

Conclusion 

Here, we present a wide range of biomedical projects that have benefited from the use of cryo-SXT at the correlative cryo-imaging beamline B24. This technology is now fully commissioned, easily accessible and serving the wider biomedical community.

References

1. Kounatidis I, Stanifer ML, Phillips MA, Paul-Gilloteaux P, Heiligenstein X, Wang H, Okolo CA, Fish TM, Spink MC, Stuart DI, Davis I, Boulant S, Grimes JM, Dobbie IM, Harkiolaki M. 3D Correlative Cryo-Structured Illumination Fluorescence and Soft X-ray Microscopy Elucidates Reovirus Intracellular Release Pathway. Cell. 2020 Jul 23;182(2):515-530.e17. doi: 10.1016/j.cell.2020.05.051. 

2. Bálint Š, Müller S, Fischer R, Kessler BM, Harkiolaki M, Valitutti S, Dustin ML. Supramolecular attack particles are autonomous killing entities released from cytotoxic T cells. Science. 2020 May 22;368(6493):897-901. doi: 10.1126/science.aay9207. 

3. Mendonca L, Howe A, Gilchrist J, Sun D, Knight M, Zanetti-Domingues L, Bateman B, Krebs AS, Chen L, Radecke J, Sheng Y, Li V, Ni T, Kounatidis I, Koronfel M, Szynkiewicz M, Harkiolaki M, Martin-Fernandez M, James W. Zhang P. Correlative Multi-scale Cryo-imaging Unveils SARS-CoV-2 Assembly and Egress. Res Sq [Preprint]. 2021 Jan 19:rs.3.rs-134794. doi: 10.21203/rs.3.rs-134794/v1.