3. Because continuous rotation of the sample stage during
MicroED is analogous to rotation of a mounted crystal on an
X-ray diffractometer, software originally developed for X-ray
crystallography can also process MicroED data. However,
numerous parameters and settings (e.g., the wavelength of
the incident radiation and the values of the relevant atomic
scattering factors) must be adjusted accordingly to account
for the discrepancies between X-rays and electrons.
4. To avoid redundancy, we minimize our description of procedures that may be more extensively covered by other chapters.
Please refer to other chapters in this volume for a more thorough explanation of these topics and/or procedures.
5. Due to liquid nitrogen’s relatively low heat capacity, samples
plunge-frozen in liquid nitrogen often suffer from
co-crystallization of adventitious ice. This is because interaction with the room-temperature sample causes an insulating
layer of nitrogen to evaporate immediately upon contact as
described by the Leidenfrost effect, prolonging the
flash-freezing process and allowing water sufficient time to
crystallize. Conversely, liquid ethane exhibits much higher
heat capacity and therefore functions as a ruthlessly efficient
cryogen that captures samples in vitreous ice. However, this
property also renders it far more hazardous. Liquid ethane
causes severe cryogenic burns immediately upon contact with
exposed skin. When condensing ethane, exercise extreme caution and wear appropriate personal protective equipment, such
as cryogenic gloves and eye protection.
6. Certain types of EM grids exhibit incompatibility with particular solvents. For example, formvar, a common plastic support
film, is soluble in chloroform, dichloroethane, and
1,4-dioxane. Also, copper and some other metal meshes are
highly susceptible to degradation by acids and bases.
7. Some cryo-protectants can increase the difficulty of an experiment by obstructing blotting efficiency. In general, an evaluation of solvent choice and sample compatibility with grids and
freezing conditions prior to sample deposition is critical. For
example, certain materials routinely used in X-ray diffraction
can be incompatible with conventional sample preparation for
MicroED. Common, non-volatile cryoprotectants (such as
paratone oil, sucrose, trimethylamine N-oxide (TMAO), lipidic
cubic phases, and perfluoropolyether oils) can cause particular
challenges for both freezing and sample interrogation by an
electron beam.
8. The size of the beam will determine the flux on the sample in
electrons per A ˚ ngstrom squared. An image of the electron
beam propagating through vacuum can help estimate the
dose at low electron counts.
342
Chih-Te Zee et al.
MicroED is analogous to rotation of a mounted crystal on an
X-ray diffractometer, software originally developed for X-ray
crystallography can also process MicroED data. However,
numerous parameters and settings (e.g., the wavelength of
the incident radiation and the values of the relevant atomic
scattering factors) must be adjusted accordingly to account
for the discrepancies between X-rays and electrons.
4. To avoid redundancy, we minimize our description of procedures that may be more extensively covered by other chapters.
Please refer to other chapters in this volume for a more thorough explanation of these topics and/or procedures.
5. Due to liquid nitrogen’s relatively low heat capacity, samples
plunge-frozen in liquid nitrogen often suffer from
co-crystallization of adventitious ice. This is because interaction with the room-temperature sample causes an insulating
layer of nitrogen to evaporate immediately upon contact as
described by the Leidenfrost effect, prolonging the
flash-freezing process and allowing water sufficient time to
crystallize. Conversely, liquid ethane exhibits much higher
heat capacity and therefore functions as a ruthlessly efficient
cryogen that captures samples in vitreous ice. However, this
property also renders it far more hazardous. Liquid ethane
causes severe cryogenic burns immediately upon contact with
exposed skin. When condensing ethane, exercise extreme caution and wear appropriate personal protective equipment, such
as cryogenic gloves and eye protection.
6. Certain types of EM grids exhibit incompatibility with particular solvents. For example, formvar, a common plastic support
film, is soluble in chloroform, dichloroethane, and
1,4-dioxane. Also, copper and some other metal meshes are
highly susceptible to degradation by acids and bases.
7. Some cryo-protectants can increase the difficulty of an experiment by obstructing blotting efficiency. In general, an evaluation of solvent choice and sample compatibility with grids and
freezing conditions prior to sample deposition is critical. For
example, certain materials routinely used in X-ray diffraction
can be incompatible with conventional sample preparation for
MicroED. Common, non-volatile cryoprotectants (such as
paratone oil, sucrose, trimethylamine N-oxide (TMAO), lipidic
cubic phases, and perfluoropolyether oils) can cause particular
challenges for both freezing and sample interrogation by an
electron beam.
8. The size of the beam will determine the flux on the sample in
electrons per A ˚ ngstrom squared. An image of the electron
beam propagating through vacuum can help estimate the
dose at low electron counts.
342
Chih-Te Zee et al.