13. The lesser the angle of the knife, the less compression artifacts
(crevassing) will occur on the cryosections, and the more fragile the blade will be. While a 25
cryo-dry knife will give the
best results, it should be reserved for experts as it is fragile. The
35
knife is a good compromise between sturdiness and compression artifacts.
14. The devitrification temperature of water is À135
C. Above
this temperature, the sample will devitrify and cubic ice will
nucleate and spread. The state of the ice in cryosections can be
checked by cryo-electron diffraction.
15. For the cutting to be smooth, everything in the chamber needs
to be tightened in order to eliminate sources of vibration
during the process.
16. In order to optimize trimming time, it is good to trim as much
metal as possible during the first trimming session. This way,
when the sample block-face needs to be cleared after several
section collection sessions, one will only need to trim 50 μm
around the existing block-face, instead of going through the
whole process again.
17. For high-resolution imaging and to prevent premature radiation damage, we recommend that each image of the montage
be collected at a low-dose (0.5 e
À /Å
2 ) and far from focus
(À40 μm) to maintain good contrast of the outline and thin
edges of the cells.
18. Before collecting the polygon montage, align the whole-grid
200 Â montage with the microscope X and Y stage coordinates
using the “shift to marker” function in SerialEM.
Acknowledgments
Cryo-ET work in the Jensen lab is supported by NIH grants R35
GM122588, RO1 A127401, and P50 AI150464 and by the
Howard Hughes Medical Institute and performed in the Beckman
Institute Resource Center for Transmission Electron Microscopy.
M.K. acknowledges a Rubicon postdoctoral fellowship from De
Nederlandse Organisatie voor Wetenschappelijk Onderzoek
(NWO). We thank Catherine M Oikonomou for reading and editing the manuscript.
References
1. Sigal YM, Zhou R, Zhuang X (2018) Visualizing and discovering cellular structures with
super-resolution
microscopy.
Science
361:880–887
2. H€ ansel R, Luh LM, Corbeski I, Trantirek L,
Do ¨tsch V (2014) In-cell NMR and EPR
spectroscopy of biomacromolecules. Angew
Chem Int Ed 53:10300–10314
3. Kaplan M et al (2015) Probing a cellembedded megadalton protein complex by
DNP-supported solid-state NMR. Nat Methods 12:649–652
Methods in Cryo-Electron Tomography
109
(crevassing) will occur on the cryosections, and the more fragile the blade will be. While a 25
cryo-dry knife will give the
best results, it should be reserved for experts as it is fragile. The
35
knife is a good compromise between sturdiness and compression artifacts.
14. The devitrification temperature of water is À135
C. Above
this temperature, the sample will devitrify and cubic ice will
nucleate and spread. The state of the ice in cryosections can be
checked by cryo-electron diffraction.
15. For the cutting to be smooth, everything in the chamber needs
to be tightened in order to eliminate sources of vibration
during the process.
16. In order to optimize trimming time, it is good to trim as much
metal as possible during the first trimming session. This way,
when the sample block-face needs to be cleared after several
section collection sessions, one will only need to trim 50 μm
around the existing block-face, instead of going through the
whole process again.
17. For high-resolution imaging and to prevent premature radiation damage, we recommend that each image of the montage
be collected at a low-dose (0.5 e
À /Å
2 ) and far from focus
(À40 μm) to maintain good contrast of the outline and thin
edges of the cells.
18. Before collecting the polygon montage, align the whole-grid
200 Â montage with the microscope X and Y stage coordinates
using the “shift to marker” function in SerialEM.
Acknowledgments
Cryo-ET work in the Jensen lab is supported by NIH grants R35
GM122588, RO1 A127401, and P50 AI150464 and by the
Howard Hughes Medical Institute and performed in the Beckman
Institute Resource Center for Transmission Electron Microscopy.
M.K. acknowledges a Rubicon postdoctoral fellowship from De
Nederlandse Organisatie voor Wetenschappelijk Onderzoek
(NWO). We thank Catherine M Oikonomou for reading and editing the manuscript.
References
1. Sigal YM, Zhou R, Zhuang X (2018) Visualizing and discovering cellular structures with
super-resolution
microscopy.
Science
361:880–887
2. H€ ansel R, Luh LM, Corbeski I, Trantirek L,
Do ¨tsch V (2014) In-cell NMR and EPR
spectroscopy of biomacromolecules. Angew
Chem Int Ed 53:10300–10314
3. Kaplan M et al (2015) Probing a cellembedded megadalton protein complex by
DNP-supported solid-state NMR. Nat Methods 12:649–652
Methods in Cryo-Electron Tomography
109