samples, the user will have collected several high-quality
MicroED data sets that can be processed by standard programs
and procedures [29, 30, 35].
4 Notes
1. There is not one specific plunge freezing device required for
MicroED. Many types of automated or manual plunge freezing
devices can be used for vitrification of samples for MicroED.
Users are encouraged to use whichever device they prefer.
2. In principle, any cryo-TEM capable of low-dose data collection
can be used for MicroED. Much of the previous work on
MicroED has used Thermo Fisher cryo-TEMs (e.g., TF-20,
Titan Krios); however, other manufactures such as JEOL can
be used as well. One of the required features of the cryo-TEM
is the ability to load vitrified samples into the grid either with an
autoloader system or a compatible cryo-holder. The second
required feature is the ability to collect data under very
low-dose conditions and to be able to switch between the
three aligned low-dose modes: low-magnification search,
medium-magnification search, and diffraction data collection
mode. Low-magnification search should be approximately
100–200Â and will be used to visualize large areas of the grid
at once. Medium-magnification mode will be around
500–1000Â (enough to see one grid square) and is used to
identify crystals on the grid. Alternatively, mediummagnification search can be performed in over-focused diffraction mode. Both search modes should be set up to have the
absolute minimum amount of dose required to visualize crystals on the grid. The third mode, diffraction data collection
mode, is performed while the microscope is in diffraction mode
and should be set such that the exposure is on the order of 0.01
e
À
/A ˚ 2 /s. It is critical that the diffraction data collection mode
is very well aligned with the medium-magnification search
mode, so that when a crystal is centered in the search mode,
it is also centered in the beam for diffraction data
collection mode.
3. A requirement for continuous rotation MicroED data collection is that the detector used must have a high dynamic range,
be sensitive, and be able to collect high-speed data. Several
camera systems have been used for MicroED including highspeed scintillator-based CMOS detectors [7, 8, 16, 17], direct
electron detectors [35], and hybrid-pixel detectors [10, 36,
37].
4. In some cases, crystals must be diluted using the precipitant
solution. In the cases where the crystals grow in highly viscous
MicroED Sample Preparation for Proteins
293
MicroED data sets that can be processed by standard programs
and procedures [29, 30, 35].
4 Notes
1. There is not one specific plunge freezing device required for
MicroED. Many types of automated or manual plunge freezing
devices can be used for vitrification of samples for MicroED.
Users are encouraged to use whichever device they prefer.
2. In principle, any cryo-TEM capable of low-dose data collection
can be used for MicroED. Much of the previous work on
MicroED has used Thermo Fisher cryo-TEMs (e.g., TF-20,
Titan Krios); however, other manufactures such as JEOL can
be used as well. One of the required features of the cryo-TEM
is the ability to load vitrified samples into the grid either with an
autoloader system or a compatible cryo-holder. The second
required feature is the ability to collect data under very
low-dose conditions and to be able to switch between the
three aligned low-dose modes: low-magnification search,
medium-magnification search, and diffraction data collection
mode. Low-magnification search should be approximately
100–200Â and will be used to visualize large areas of the grid
at once. Medium-magnification mode will be around
500–1000Â (enough to see one grid square) and is used to
identify crystals on the grid. Alternatively, mediummagnification search can be performed in over-focused diffraction mode. Both search modes should be set up to have the
absolute minimum amount of dose required to visualize crystals on the grid. The third mode, diffraction data collection
mode, is performed while the microscope is in diffraction mode
and should be set such that the exposure is on the order of 0.01
e
À
/A ˚ 2 /s. It is critical that the diffraction data collection mode
is very well aligned with the medium-magnification search
mode, so that when a crystal is centered in the search mode,
it is also centered in the beam for diffraction data
collection mode.
3. A requirement for continuous rotation MicroED data collection is that the detector used must have a high dynamic range,
be sensitive, and be able to collect high-speed data. Several
camera systems have been used for MicroED including highspeed scintillator-based CMOS detectors [7, 8, 16, 17], direct
electron detectors [35], and hybrid-pixel detectors [10, 36,
37].
4. In some cases, crystals must be diluted using the precipitant
solution. In the cases where the crystals grow in highly viscous
MicroED Sample Preparation for Proteins
293