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

Autor: Okolo CA; Beamline B24, Diamond Light Source, Harwell Science and Innovation Campus, Didcot, UK., Kounatidis I; Beamline B24, Diamond Light Source, Harwell Science and Innovation Campus, Didcot, UK., Groen J; Beamline 09-MISTRAL, ALBA Synchrotron, Barcelona, Spain., Nahas KL; Beamline B24, Diamond Light Source, Harwell Science and Innovation Campus, Didcot, UK.; Division of Virology, Department of Pathology, University of Cambridge, Cambridge, UK., Balint S; Kennedy Institute of Rheumatology, University of Oxford, Oxford, UK., Fish TM; Beamline B24, Diamond Light Source, Harwell Science and Innovation Campus, Didcot, UK., Koronfel MA; Beamline B24, Diamond Light Source, Harwell Science and Innovation Campus, Didcot, UK., Cortajarena AL; Center for Cooperative Research in Biomaterials (CIC biomaGUNE), Basque Research and Technology Alliance (BRTA), Donostia San Sebastián, Spain.; Ikerbasque, Basque Foundation for Science, Bilbao, Spain., Dobbie IM; Micron Advanced Imaging Consortium, Department of Biochemistry, University of Oxford, Oxford, UK., Pereiro E; Beamline 09-MISTRAL, ALBA Synchrotron, Barcelona, Spain., Harkiolaki M; Beamline B24, Diamond Light Source, Harwell Science and Innovation Campus, Didcot, UK. maria.harkiolaki@diamond.ac.uk.
Jazyk: angličtina
Zdroj: Nature protocols [Nat Protoc] 2021 Jun; Vol. 16 (6), pp. 2851-2885. Date of Electronic Publication: 2021 May 14.
DOI: 10.1038/s41596-021-00522-4
Abstrakt: 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).
Databáze: MEDLINE
Externí odkaz: