solutions (e.g., high-molecular weight PEGs), dilution in the
presence of less viscous (e.g., low-molecular weight PEGs) is
required. This is generally a trial-and-error process where the
user optimizes the ability to blot the solution with the stability
of the microcrystals in these solutions. For sensitive crystals,
pipetting should be kept as gentle as possible. If crystals are
stable yet too large, there are protocols for fragmenting by
pipetting, and other means, available [6].
5. Blotting time is another variable that must be screened to
obtain optimal samples. Ideally, the crystals will have a thin
layer of vitrified solution surrounding them, and the rest of the
grid will be easily penetrated by the electron beam. If blotting
time is too long, too much solution may be removed, and
crystal quality can be affected. If blotting time is too short,
the grid may be too thick and the electron beam will not
penetrate. It is recommended to try a wide range of blotting
time (e.g., 2–20 s) and multiple blotting procedures, where the
sample is blotted more than once before plunge freezing.
6. It is critical that the microscope be well aligned for the collection of high-quality MicroED data. Also, the mediummagnification search mode must be aligned with the diffraction
data collection mode so that when a crystal is located in
medium-magnification search, it can be exposed by the diffraction mode and data can be collected.
7. The rotation rate of the microscope is a parameter that can be
adjusted depending on the crystal samples being studied. By
adjusting the rotation rate along with the integration time on
the high-speed detector, the user can control how many
degrees of rotation occurs for each diffraction frame in the
data set. For example, if the rotation rate on the stage is
0.1
/s and the frame rate of the detector is set to 4 s, each
frame in the data set will consist of a 0.4
sampling of reciprocal
space.
Acknowledgments
The Nannenga lab is supported by the National Institutes of Health
grant R01GM124152 and R21GM135784, the Air Force Office of
Scientific Research grant FA9550-18-1-0012, and the National
Science Foundation award 1942084. We acknowledge the use of
facilities within the LeRoy Eyring Center for Solid State Science at
Arizona State University, Tempe, AZ, specifically the use of the
Titan Krios and the funding of this instrument by NSF MRI
1531991.
294
Guanhong Bu and Brent L. Nannenga
presence of less viscous (e.g., low-molecular weight PEGs) is
required. This is generally a trial-and-error process where the
user optimizes the ability to blot the solution with the stability
of the microcrystals in these solutions. For sensitive crystals,
pipetting should be kept as gentle as possible. If crystals are
stable yet too large, there are protocols for fragmenting by
pipetting, and other means, available [6].
5. Blotting time is another variable that must be screened to
obtain optimal samples. Ideally, the crystals will have a thin
layer of vitrified solution surrounding them, and the rest of the
grid will be easily penetrated by the electron beam. If blotting
time is too long, too much solution may be removed, and
crystal quality can be affected. If blotting time is too short,
the grid may be too thick and the electron beam will not
penetrate. It is recommended to try a wide range of blotting
time (e.g., 2–20 s) and multiple blotting procedures, where the
sample is blotted more than once before plunge freezing.
6. It is critical that the microscope be well aligned for the collection of high-quality MicroED data. Also, the mediummagnification search mode must be aligned with the diffraction
data collection mode so that when a crystal is located in
medium-magnification search, it can be exposed by the diffraction mode and data can be collected.
7. The rotation rate of the microscope is a parameter that can be
adjusted depending on the crystal samples being studied. By
adjusting the rotation rate along with the integration time on
the high-speed detector, the user can control how many
degrees of rotation occurs for each diffraction frame in the
data set. For example, if the rotation rate on the stage is
0.1
/s and the frame rate of the detector is set to 4 s, each
frame in the data set will consist of a 0.4
sampling of reciprocal
space.
Acknowledgments
The Nannenga lab is supported by the National Institutes of Health
grant R01GM124152 and R21GM135784, the Air Force Office of
Scientific Research grant FA9550-18-1-0012, and the National
Science Foundation award 1942084. We acknowledge the use of
facilities within the LeRoy Eyring Center for Solid State Science at
Arizona State University, Tempe, AZ, specifically the use of the
Titan Krios and the funding of this instrument by NSF MRI
1531991.
294
Guanhong Bu and Brent L. Nannenga