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

DOI: 10.1038/s41596-021-00522-4 DOI Help

Authors: Chidinma Okolo (Diamond Light Source) , Ilias Kounatidis (Diamond Light Source) , Johannes Groen (ALBA Synchrotron) , Kamal Nahas (Dimond Light Source; University of Cambridge, Diamond Light Source) , Stefan Balint (University of Oxford) , Thomas M. Fish (Diamond Light Source) , Mohamed A. Koronfel (Diamond Light Source) , Aitziber L. Cortajarena (Center for Cooperative Research in Biomaterials (CIC biomaGUNE), Basque Research and Technology Alliance (BRTA); Ikerbasque, Basque Foundation for Science) , Ian M. Dobbie (University of Oxford) , Eva Pereiro (ALBA Synchrotron) , Maria Harkiolaki (Wellcome Trust Centre for Human Genetics, University of Oxford)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Nature Protocols , VOL 11

State: Published (Approved)
Published: May 2021
Diamond Proposal Number(s): 18737 , 20321 , 22274 , 23046 , 25162

Abstract: 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).

Journal Keywords: 3-D reconstruction; Fluorescence imaging; Structural biology; Super-resolution microscopy; X-ray tomography

Subject Areas: Technique Development, Biology and Bio-materials


Instruments: B24-Cryo Soft X-ray Tomography

Other Facilities: BL09 at ALBA

Discipline Tags:

Life Sciences & Biotech Structural biology Technique Development - Life Sciences & Biotech

Technical Tags:

Imaging Tomography