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Precision super-resolution cryo-correlative light and electron microscopy for rapid in situ structural analyses of optogenetically-positioned organell…
Precision super-resolution cryo-correlative light and electron microscopy for rapid in situ structural analyses of optogenetically-positioned organell…
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Length:
20 minutes
Released:
Nov 23, 2022
Format:
Podcast episode
Description
Link to bioRxiv paper:
http://biorxiv.org/cgi/content/short/2022.11.22.516823v1?rss=1
Authors: Redpath, G. M. I., Rae, J. A., Yao, Y., Ruan, J., Cagigas, M. L., Whan, R., Hardeman, E. C., Gunning, P. W., Ananthanarayanan, V., Parton, R. G., Ariotti, N.
Abstract:
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.
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http://biorxiv.org/cgi/content/short/2022.11.22.516823v1?rss=1
Authors: Redpath, G. M. I., Rae, J. A., Yao, Y., Ruan, J., Cagigas, M. L., Whan, R., Hardeman, E. C., Gunning, P. W., Ananthanarayanan, V., Parton, R. G., Ariotti, N.
Abstract:
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.
Copy rights belong to original authors. Visit the link for more info
Podcast created by Paper Player, LLC
Released:
Nov 23, 2022
Format:
Podcast episode
Titles in the series (100)
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