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A 3D Cartographic Description of the Cell by Cryo Soft X-ray Tomography

Imaging techniques are fundamental in order to understand cell organization and machinery in biological research and the related fields. Among these ...

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Cryo-Structured Illumination Microscopic Data Collection from Cryogenically Preserved Cells

Three-dimensional (3D) structured illumination microscopy (SIM) allows imaging of fluorescently labelled cellular structures at higher resolution than ...

Diamond Light Source, Harwell Science and Innovation Campus, Didcot, UK

Beamline B24, Diamond Light Source

In Drosophila, it is thought that peptidoglycan recognition proteins (PGRPs) SA and LC structurally discriminate between bacterial peptidoglycans with lysine (Lys) or diaminopimelic (DAP) acid, respectively, thus inducing differential antimicrobial transcription response. Here, we find that accessibility to PG at the cell wall plays a central role in immunity to infection. When wall teichoic acids (WTAs) are genetically removed from S. aureus (Lys type) and Bacillus subtilis (DAP type), thus increasing accessibility, the binding of both PGRPs to either bacterium is increased. PGRP-SA and -LC double mutant flies are more susceptible to infection with both WTA-less bacteria. In addition, WTA-less bacteria grow better in PGRP-SA/-LC double mutant flies. Finally, infection with WTA-less bacteria abolishes any differential activation of downstream antimicrobial transcription. Our results indicate that accessibility to cell wall PG is a major factor in PGRP-mediated immunity and may be the cause for discrimination between classes of pathogens.

Accessibility to Peptidoglycan Is Important for the Recognition of Gram-Positive Bacteria in Drosophila.

The organoiridium complex Ir[(C,N)(O,O)] (1) where C, N = 1-phenylisoquinoline and O,O = 2,2,6,6-tetramethyl-3,5-heptanedionate is a promising photosensitiser for Photo-Dynamic Therapy (PDT). 1 is not toxic to cells in the dark. However, irradiation of the compound with one-photon blue or two-photon red light generates high levels of singlet oxygen (O) (in Zhang et al. Angew Chem Int Ed Engl 56 (47):14898-14902 https://doi.org/10.1002/anie.201709082,2017), both within cell monolayers and in tumour models. Moreover, photo-excited 1 oxidises key proteins, causing metabolic alterations in cancer cells with potent antiproliferative activity. Here, the tomograms obtained by cryo-Soft X-ray Tomography (cryo-SXT) of human PC3 prostate cancer cells treated with 1, irradiated with blue light, and cryopreserved to maintain them in their native state, reveal that irradiation causes extensive and specific alterations to mitochondria, but not other cellular components. Such new insights into the effect of O generation during PDT using iridium photosensitisers on cells contribute to a detailed understanding of their cellular mode of action.

X-ray tomography of cryopreserved human prostate cancer cells: mitochondrial targeting by an organoiridium photosensitiser.

Imaging of biological matter across resolution scales entails the challenge of preserving the direct and unambiguous correlation of subject features from the macroscopic to the microscopic level. Here, we present a correlative imaging platform developed specifically for imaging cells in 3D under cryogenic conditions by using X-rays and visible light. Rapid cryo-preservation of biological specimens is the current gold standard in sample preparation for ultrastructural analysis in X-ray imaging. However, cryogenic fluorescence localization methods are, in their majority, diffraction-limited and fail to deliver matching resolution. We addressed this technological gap by developing an integrated, user-friendly platform for 3D correlative imaging of cells in vitreous ice by using super-resolution structured illumination microscopy in conjunction with soft X-ray tomography. The power of this approach is demonstrated by studying the process of reovirus release from intracellular vesicles during the early stages of infection and identifying intracellular virus-induced structures.

3D Correlative Cryo-Structured Illumination Fluorescence and Soft X-ray Microscopy Elucidates Reovirus Intracellular Release Pathway.

This protocol illustrates the steps necessary to deposit correlated 3D cryo-imaging data from cryo-structured illumination microscopy and cryo-soft X-ray tomography with the BioStudies and EMPIAR deposition databases of the European Bioinformatics Institute. There is currently a real need for a robust method of data deposition to ensure unhindered access to and independent validation of correlative light and X-ray microscopy data to allow use in further comparative studies, educational activities, and data mining. For complete details on the use and execution of this protocol, please refer to Kounatidis et al. (2020).

Data-deposition protocols for correlative soft X-ray tomography and super-resolution structured illumination microscopy applications.

3D correlative microscopy methods have revolutionized biomedical research, allowing the acquisition of multidimensional information to gain an in-depth understanding of biological systems. With the advent of relevant cryo-preservation methods, correlative imaging of cryogenically preserved samples has led to nanometer resolution imaging (2-50 nm) under harsh imaging regimes such as electron and soft X-ray tomography. These methods have now been combined with conventional and super-resolution fluorescence imaging at cryogenic temperatures to augment information content from a given sample, resulting in the immediate requirement for protocols that facilitate hassle-free, unambiguous cross-correlation between microscopes. We present here sample preparation strategies and a direct comparison of different working fiducialization regimes that facilitate 3D correlation of cryo-structured illumination microscopy and cryo-soft X-ray tomography. Our protocol has been tested at two synchrotron beamlines (B24 at Diamond Light Source in the UK and BL09 Mistral at ALBA in Spain) and has led to the development of a decision aid that facilitates experimental design with the strategic use of markers based on project requirements. This protocol takes between 1.5 h and 3.5 d to complete, depending on the cell populations used (adherent cells may require several days to grow on sample carriers).

Sample preparation strategies for efficient correlation of 3D SIM and soft X-ray tomography data at cryogenic temperatures.

Correlation of 3D images acquired on different microscopes can be a daunting prospect even for experienced users. This protocol describes steps for registration of images from soft X-ray absorption contrast imaging and super-resolution fluorescence imaging of hydrated biological materials at cryogenic temperatures. Although it is developed for data generated at synchrotron beamlines that offer the above combination of microscopies, it is applicable to all analogous imaging systems where the same area of a sample is examined using successive non-destructive imaging techniques. For complete details on the use and execution of this protocol, please refer to Kounatidis et al. (2020).

ArticleTotal : 2	Year
Publication title Cited by 17	2021
Publication title Cited by 18	2021

Article	Year
Accessibility to Peptidoglycan Is Important for the Recognition of Gram-Positive Bacteria in Drosophila. Cell reports\| PubMed ID: 31116990	2019
X-ray tomography of cryopreserved human prostate cancer cells: mitochondrial targeting by an organoiridium photosensitiser. Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry\| PubMed ID: 32124100	2020
3D Correlative Cryo-Structured Illumination Fluorescence and Soft X-ray Microscopy Elucidates Reovirus Intracellular Release Pathway. Cell\| PubMed ID: 32610083	2020
Data-deposition protocols for correlative soft X-ray tomography and super-resolution structured illumination microscopy applications. STAR protocols\| PubMed ID: 33490973	2021
Sample preparation strategies for efficient correlation of 3D SIM and soft X-ray tomography data at cryogenic temperatures. Nature protocols\| PubMed ID: 33990802	2021
Protocol for image registration of correlative soft X-ray tomography and super-resolution structured illumination microscopy images. STAR protocols\| PubMed ID: 34027487	2021

Maria Harkiolaki

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