TY - JOUR
T1 - Correlative cryo-imaging of the cellular universe with soft X-rays and laser light used to track F-actin structures in mammalian cells
AU - Koronfel, Mohamed
AU - Kounatidis, Ilias
AU - Mwangangi, Dennis M.
AU - Vyas, Nina
AU - Okolo, Chidinma
AU - Jadhav, Archana
AU - Fish, Tom
AU - Chotchuang, Phatcharin
AU - Schulte, Albert
AU - Robinson, Robert C.
AU - Harkiolaki, Maria
N1 - Funding Information:
This work was carried out with the support of the Diamond Light Source, instrument B24 (proposals BI23033 and BI23073). This work was also supported by A*STAR, Singapore and VISTEC and the Thailand Science Research and Innovation (TSRI) public funding agency through a grant within the Global Partnership Program, Thailand.
Publisher Copyright:
© 2021 International Union of Crystallography. All rights reserved.
PY - 2021/12/1
Y1 - 2021/12/1
N2 - Imaging of actin filaments is crucial due to the integral role that they play in many cellular functions such as intracellular transport, membrane remodelling and cell motility. Visualizing actin filaments has so far relied on fluorescence microscopy and electron microscopy/tomography. The former lacks the capacity to capture the overall local ultrastructure, while the latter requires rigorous sample preparation that can lead to potential artefacts, and only delivers relatively small volumes of imaging data at the thinnest areas of a cell. In this work, a correlative approach utilizing in situ super-resolution fluorescence imaging and cryo X-ray tomography was used to image bundles of actin filaments deep inside cells under near-native conditions. In this case, fluorescence 3D imaging localized the actin bundles within the intracellular space, while X-ray tomograms of the same areas provided detailed views of the local ultrastructure. Using this new approach, actin trails connecting vesicles in the perinuclear area and hotspots of actin presence within and around multivesicular bodies were observed. The characteristic prevalence of filamentous actin in cytoplasmic extensions was also documented.
AB - Imaging of actin filaments is crucial due to the integral role that they play in many cellular functions such as intracellular transport, membrane remodelling and cell motility. Visualizing actin filaments has so far relied on fluorescence microscopy and electron microscopy/tomography. The former lacks the capacity to capture the overall local ultrastructure, while the latter requires rigorous sample preparation that can lead to potential artefacts, and only delivers relatively small volumes of imaging data at the thinnest areas of a cell. In this work, a correlative approach utilizing in situ super-resolution fluorescence imaging and cryo X-ray tomography was used to image bundles of actin filaments deep inside cells under near-native conditions. In this case, fluorescence 3D imaging localized the actin bundles within the intracellular space, while X-ray tomograms of the same areas provided detailed views of the local ultrastructure. Using this new approach, actin trails connecting vesicles in the perinuclear area and hotspots of actin presence within and around multivesicular bodies were observed. The characteristic prevalence of filamentous actin in cytoplasmic extensions was also documented.
KW - Actin filaments
KW - Correlative imaging
KW - Soft X-ray tomography
KW - Structured illumination
KW - Super-resolution microscopy
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U2 - 10.1107/S2059798321010329
DO - 10.1107/S2059798321010329
M3 - Article
C2 - 34866605
AN - SCOPUS:85121430556
SN - 0907-4449
VL - 77
SP - 1479
EP - 1485
JO - Acta Crystallographica Section D: Structural Biology
JF - Acta Crystallographica Section D: Structural Biology
ER -