RT Journal Article
SR Electronic
T1 Precision super-resolution cryo-correlative light and electron microscopy for rapid <em>in situ</em> structural analyses of optogenetically-positioned organelles
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2022.11.22.516823
DO 10.1101/2022.11.22.516823
A1 G.M.I. Redpath
A1 J. Rae
A1 Y. Yao
A1 J. Ruan
A1 M.L. Cagigas
A1 R. Whan
A1 E.C. Hardeman
A1 P.W. Gunning
A1 V. Ananthanarayanan
A1 R.G. Parton
A1 N.A. Ariotti
YR 2022
UL http://biorxiv.org/content/early/2022/11/23/2022.11.22.516823.abstract
AB Unambiguous targeting of cellular structures for in situ cryo-electron microscopy in the heterogeneous, dense, and compacted environment of the cytoplasm remains challenging. Here we have developed a novel cryogenic correlative light and electron microscopy (cryo-CLEM) workflow which combines thin cells grown on a mechanically defined substratum to rapidly analyse organelles and macromolecular complexes in the cell by cryo-electron tomography (cryo-ET). We coupled these advancements with optogenetics to redistribute perinuclear-localised organelles to the cell periphery for cryo-ET. This reliable and robust workflow allows for fast in situ analyses without the requirement for cryo-focused ion beam milling. We have developed a protocol where cells can be frozen, imaged by cryo-fluorescence microscopy and ready for batch cryo-ET within a day.Competing Interest StatementThe authors have declared no competing interest.