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Cryo-structured illumination microscopic data collection from cryogenically preserved cells

DOI: 10.3791/62274 DOI Help

Authors: Nina Vyas (Diamond Light Source) , Nina Perry (Diamond Light Source) , Chidinma A. Okolo (Diamond Light Source) , Ilias Kounatidis (Diamond Light Source) , Thomas M. Fish (Diamond Light Source) , Kamal L. Nahas (Dimond Light Source; University of Cambridge) , Archana Jadhav (Diamond Light Source) , Mohamed A. Koronfel (Diamond Light Source) , Johannes Groen (ALBA Synchrotron) , Eva Pereiro (ALBA Synchrotron) , Ian M. Dobbie (University of Oxford) , Maria Harkiolaki (Diamond Light Source)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of Visualized Experiments

State: Published (Approved)
Published: May 2021
Diamond Proposal Number(s): 25512

Open Access Open Access

Abstract: Three-dimensional (3D) structured illumination microscopy (SIM) allows imaging of fluorescently labelled cellular structures at higher resolution than conventional fluorescence microscopy. This super-resolution (SR) technique enables visualization of molecular processes in whole cells and has the potential to be used in conjunction with electron microscopy and X-ray tomography to correlate structural and functional information. A SIM microscope for cryogenically preserved samples (cryoSIM) has recently been commissioned at the correlative cryo-imaging beamline B24 at the UK synchrotron. It was designed specifically for 3D imaging of biological samples at cryogenic temperatures in a manner compatible with subsequent imaging of the same samples by X-ray microscopy methods such as cryo-soft X-ray tomography. This video article provides detailed methods and protocols for successful imaging using the cryoSIM. In addition to instructions on the operation of the cryoSIM microscope, recommendations have been included regarding the choice of samples, fluorophores, and parameter settings. The protocol is demonstrated in U2OS cell samples whose mitochondria and tubulin have been fluorescently labelled.

Subject Areas: Biology and Bio-materials, Technique Development


Instruments: B24-Cryo Soft X-ray Tomography

Discipline Tags:

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

Technical Tags:

Microscopy X-ray Microscopy Cryo X-ray Microscopy