thicker than the front. To thin only the back portion of the
lamella, the stage should be tilted approximately +0.5
which
will expose the back for milling without disturbing the front
edge of the lamella [13].
16. Given the large chamber volume, poor vacuum compared to
the TEM, and cryogenic temperatures, amorphous ice buildup
is inevitable inside the cryo-FIB/SEM. This can be minimized
with proper instrument maintenance, but in our experience,
long operating times of more than 6–8 h lead to ice buildup
from the chamber and from milling redeposition. Before sample retrieval, it is recommended to perform a short 5-min
lamella cleaning step with a low FIB current for all lamellae
immediately prior to sample retrieval (Fig. 6). This step is
essentially identical to the fine milling step, but with the pattern set to skim over the surface of the lamella without removing too much material.
Acknowledgments
We would like to acknowledge the following people: Reika Watanabe and Miles Paszek for providing images of NIH3T3 and yeast
grids, respectively. Reika Watanabe, Kanika Khanna, and Sergey
Suslov for fruitful discussion of FIB/SEM. Mario Aguilera for
photography of microscope equipment.
This work was supported by an NIH Director’s New Innovator
Award 1DP2GM123494-01 (to E.V.) and NIH 5T32GM7240-40
(to V.L.). Images of cyanobacteria are from projects supported by
NIH R35GM118290 awarded to Susan S. Golden. This work was
performed in part at the San Diego Nanotechnology Infrastructure
(SDNI) of UCSD, a member of the National Nanotechnology
Coordinated Infrastructure, which is supported by the National
Science Foundation (Grant ECCS-1542148).
References
1. Koning RI, Koster AJ, Sharp TH (2018)
Advances in cryo-electron tomography for
biology and medicine. Annal Anatomy Anatomischer Anzeiger 217:82–96
2. Russo CJ, Passmore LA (2016) Progress
towards an optimal specimen support for electron cryomicroscopy. Curr Opin Struct Biol
37:81–89
3. Al-Amoudi A, Chang JJ, Leforestier A, McDowall AW, Michel Salamin L, Norle ´n L,
Richter K, Sartori Blanc N, Studer D, Dubochet J (2004) Cryo-electron microscopy of vitreous sections. EMBO J 23:3583–3588
4. Al-Amoudi A, Studer D, Dubochet J (2005)
Cutting artefacts and cutting process in vitreous sections for cryo-electron microscopy. J
Struct Biol 150(1):109–121
5. Mahamid J, Tegunov D, Maiser A, Arnold J,
Leonhardt H, Plitzko JM, Baumeister W
(2019) Liquid-crystalline phase transitions in
lipid droplets are related to cellular states and
specific organelle association. Proc Natl Acad
Sci 116(34):16866–16871
6. Chaikeeratisak V, Khanna K, Nguyen KT,
Sugie J, Egan ME, Erb ML, Vavilina A,
Nonejuie P, Nieweglowska E, Pogliano K,
80
Vinson Lam and Elizabeth Villa
lamella, the stage should be tilted approximately +0.5
which
will expose the back for milling without disturbing the front
edge of the lamella [13].
16. Given the large chamber volume, poor vacuum compared to
the TEM, and cryogenic temperatures, amorphous ice buildup
is inevitable inside the cryo-FIB/SEM. This can be minimized
with proper instrument maintenance, but in our experience,
long operating times of more than 6–8 h lead to ice buildup
from the chamber and from milling redeposition. Before sample retrieval, it is recommended to perform a short 5-min
lamella cleaning step with a low FIB current for all lamellae
immediately prior to sample retrieval (Fig. 6). This step is
essentially identical to the fine milling step, but with the pattern set to skim over the surface of the lamella without removing too much material.
Acknowledgments
We would like to acknowledge the following people: Reika Watanabe and Miles Paszek for providing images of NIH3T3 and yeast
grids, respectively. Reika Watanabe, Kanika Khanna, and Sergey
Suslov for fruitful discussion of FIB/SEM. Mario Aguilera for
photography of microscope equipment.
This work was supported by an NIH Director’s New Innovator
Award 1DP2GM123494-01 (to E.V.) and NIH 5T32GM7240-40
(to V.L.). Images of cyanobacteria are from projects supported by
NIH R35GM118290 awarded to Susan S. Golden. This work was
performed in part at the San Diego Nanotechnology Infrastructure
(SDNI) of UCSD, a member of the National Nanotechnology
Coordinated Infrastructure, which is supported by the National
Science Foundation (Grant ECCS-1542148).
References
1. Koning RI, Koster AJ, Sharp TH (2018)
Advances in cryo-electron tomography for
biology and medicine. Annal Anatomy Anatomischer Anzeiger 217:82–96
2. Russo CJ, Passmore LA (2016) Progress
towards an optimal specimen support for electron cryomicroscopy. Curr Opin Struct Biol
37:81–89
3. Al-Amoudi A, Chang JJ, Leforestier A, McDowall AW, Michel Salamin L, Norle ´n L,
Richter K, Sartori Blanc N, Studer D, Dubochet J (2004) Cryo-electron microscopy of vitreous sections. EMBO J 23:3583–3588
4. Al-Amoudi A, Studer D, Dubochet J (2005)
Cutting artefacts and cutting process in vitreous sections for cryo-electron microscopy. J
Struct Biol 150(1):109–121
5. Mahamid J, Tegunov D, Maiser A, Arnold J,
Leonhardt H, Plitzko JM, Baumeister W
(2019) Liquid-crystalline phase transitions in
lipid droplets are related to cellular states and
specific organelle association. Proc Natl Acad
Sci 116(34):16866–16871
6. Chaikeeratisak V, Khanna K, Nguyen KT,
Sugie J, Egan ME, Erb ML, Vavilina A,
Nonejuie P, Nieweglowska E, Pogliano K,
80
Vinson Lam and Elizabeth Villa