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Imaging Considerations for Cryo-Tomography of Organelles and Whole Cells at High Accelerating Voltage
Published online by Cambridge University Press: 02 July 2020
Extract
Cryo-electron tomography can be used to obtain highly-detailed 3-D reconstructions of micrometer-sized biological structures which are unfixed, unstained and embedded in vitreous ice. Important practical considerations for acquiring the requisite tilt-series images are the choice of electron dose and defocus value, and the effects of contamination and ice thickness.
It is essential to reduce the total dose enough to prevent excessive ice radiolysis and damage to the specimen itself. To reduce the dose, a sensitive cooled CCD camera and microscope automation system are all but required. Because the specimens are thick, it is advantageous to use a high accelerating voltage, and/or energy filtering to reduce inelastic scattering. At high accelerating voltage, it is important to keep the dose high enough so that the signal is above the “scintillator noise” generated by the interaction of the high-energy electrons with the phosphor scintillator of the CCD camera. Initial test reconstructions indicate that scintillator noise, unlike electron shot noise, cannot be mitigated using dose fractionation. Fortunately, we find that large objects embedded in a thick (ca. 1 μm) vitrified ice layer are more resistant to beam damage (as well as charging) compared to smaller objects embedded in ice 100-200nm thick.
- Type
- Cryotechniques, Immunocytochemistry, and Electron Microscopy I. Molecular Approach
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- Copyright © Microscopy Society of America
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