35. Information on how to set up and use grid montaging and low
dose imaging can be found at https://bio3d.colorado.edu/
SerialEM/hlp/html/about_low_dose.htm,
https://www.
youtube.com/watch?v¼N6A_BVR13Gc
36. If feature of interest is not detectable by this approach, you may
consider using a correlative light and electron microscopy
approach. Here, the feature of interest can be identified by
fluorescence light microscopy first (see [46]).
37. To check the image stack file has the correct pixel size, type
“header filename.st” at the command line. If the pixel size is
wrong (as can happen if MotionCor2 is used), type “alterheader filename.st” and use the “del” option to change the pixel
value.
38. Instructions on how to use the drawing tool plugin for 3dmod
can be found at https://www.youtube.com/watch?
v¼BsNSVLIQ-cE.
Acknowledgments
This work was supported in part by the Office of Science of the US
Department of Energy DE-AC02-O5CH11231 (K.M.D.), the
Human Frontier Science Program fellowship LT000234/2018-L
(D.S.) and UCB Start-up funds (K.M.D.).
References
1. Porter KR, Claude A, Fullam EF (1945) A
study of tissue culture cells by Electron microscopy. J Exp Med 81:233–246. https://doi.
org/10.1084/jem.81.3.233
2. Palade GE (1952) The fine structure of mitochondria. Anat Rec 114:427–451. https://doi.
org/10.1002/ar.1091140304
3. Dalton AJ, Felix MD (1954) Cytologic and
cytochemical characteristics of the Golgi substance of epithelial cells of the epididymis–in
situ, in homogenates and after isolation. Am J
Anat 94:171–207. https://doi.org/10.1002/
aja.1000940202
4. Geuze HJ (1999) A future for electron microscopy in cell biology? Trends Cell Biol 9:92–93.
https://doi.org/10.1016/S0962-8924(98)
01493-7
5. Heuser J (2002) Whatever happened to the
‘microtrabecular
concept’?
Biol
Cell
94:561–596.
https://doi.org/10.1016/
S0248-4900(02)00013-8
6. Small JV (1981) Organization of actin in the
leading edge of cultured cells: influence of
osmium tetroxide and dehydration on the
ultrastructure of actin meshworks. J Cell Biol
91:695–705
7. Maupin-Szamier P, Pollard TD (1978) Actin
filament destruction by osmium tetroxide. J
Cell Biol 77:837–852
8. Dubochet J, McDowall AW, Menge B et al
(1983) Electron microscopy of frozenhydrated bacteria. J Bacteriol 155:381–390
9. Dubochet J, McDowall AW (1981) Vitrification of pure water for Electron microscopy. J
Microsc 124:3–4. https://doi.org/10.1111/j.
1365-2818.1981.tb02483.x
10. Dubochet J, Adrian M, Chang J-J et al (1988)
Cryo-electron microscopy of vitrified specimens. Q Rev Biophys 21:129–228. https://
doi.org/10.1017/S0033583500004297
11. Medalia O, Weber I, Frangakis AS et al (2002)
Macromolecular architecture in eukaryotic cells
visualized by Cryoelectron tomography. Science 298:1209–1213. https://doi.org/10.
1126/science.1076184
Cryo-ET of Cellular Structures
21
dose imaging can be found at https://bio3d.colorado.edu/
SerialEM/hlp/html/about_low_dose.htm,
https://www.
youtube.com/watch?v¼N6A_BVR13Gc
36. If feature of interest is not detectable by this approach, you may
consider using a correlative light and electron microscopy
approach. Here, the feature of interest can be identified by
fluorescence light microscopy first (see [46]).
37. To check the image stack file has the correct pixel size, type
“header filename.st” at the command line. If the pixel size is
wrong (as can happen if MotionCor2 is used), type “alterheader filename.st” and use the “del” option to change the pixel
value.
38. Instructions on how to use the drawing tool plugin for 3dmod
can be found at https://www.youtube.com/watch?
v¼BsNSVLIQ-cE.
Acknowledgments
This work was supported in part by the Office of Science of the US
Department of Energy DE-AC02-O5CH11231 (K.M.D.), the
Human Frontier Science Program fellowship LT000234/2018-L
(D.S.) and UCB Start-up funds (K.M.D.).
References
1. Porter KR, Claude A, Fullam EF (1945) A
study of tissue culture cells by Electron microscopy. J Exp Med 81:233–246. https://doi.
org/10.1084/jem.81.3.233
2. Palade GE (1952) The fine structure of mitochondria. Anat Rec 114:427–451. https://doi.
org/10.1002/ar.1091140304
3. Dalton AJ, Felix MD (1954) Cytologic and
cytochemical characteristics of the Golgi substance of epithelial cells of the epididymis–in
situ, in homogenates and after isolation. Am J
Anat 94:171–207. https://doi.org/10.1002/
aja.1000940202
4. Geuze HJ (1999) A future for electron microscopy in cell biology? Trends Cell Biol 9:92–93.
https://doi.org/10.1016/S0962-8924(98)
01493-7
5. Heuser J (2002) Whatever happened to the
‘microtrabecular
concept’?
Biol
Cell
94:561–596.
https://doi.org/10.1016/
S0248-4900(02)00013-8
6. Small JV (1981) Organization of actin in the
leading edge of cultured cells: influence of
osmium tetroxide and dehydration on the
ultrastructure of actin meshworks. J Cell Biol
91:695–705
7. Maupin-Szamier P, Pollard TD (1978) Actin
filament destruction by osmium tetroxide. J
Cell Biol 77:837–852
8. Dubochet J, McDowall AW, Menge B et al
(1983) Electron microscopy of frozenhydrated bacteria. J Bacteriol 155:381–390
9. Dubochet J, McDowall AW (1981) Vitrification of pure water for Electron microscopy. J
Microsc 124:3–4. https://doi.org/10.1111/j.
1365-2818.1981.tb02483.x
10. Dubochet J, Adrian M, Chang J-J et al (1988)
Cryo-electron microscopy of vitrified specimens. Q Rev Biophys 21:129–228. https://
doi.org/10.1017/S0033583500004297
11. Medalia O, Weber I, Frangakis AS et al (2002)
Macromolecular architecture in eukaryotic cells
visualized by Cryoelectron tomography. Science 298:1209–1213. https://doi.org/10.
1126/science.1076184
Cryo-ET of Cellular Structures
21