electron microscopy (EM) grid (e.g., Quantifoil) and excess solution is blotted away using filter paper. Following blotting, the
crystal-loaded EM grid is immediately vitrified in liquid ethane
and either loaded into the cryo-TEM or stored under liquid nitrogen for future use. While the protocols presented here will focus on
more standard sample preparation methods, there are also other
procedures that can be used including crystal fragmentation [6]
and cryo-focus ion beam (cryo-FIB) milling [24, 25]. Readers are
encouraged to refer to other sources for details on how to use these
other methods for sample preparation.
Once the sample is inserted into the microscope, the quality of
the sample is screened, and data is collected from the best diffracting crystals on the grid. While there are recent methods for automated data collection [26–28] and new user interfaces for
MicroED data collection, this chapter will focus on the manual
method for MicroED data collection that does not require additional software besides the standard cryo-TEM control interface.
Data collection begins by screening the grid for the presence of
suitable microcrystals on the grid. Suitable crystals are those which
have a defined shape and are not too close to the EM grid bars or
other crystals, which would limit the range of data that could be
collected (see Fig. 1). Following the identification of a suitable
crystal, an initial diffraction pattern is collected, and its quality is
assessed. If the diffraction is of high quality (see Fig. 2), a continuous rotation data set [7] should be collected from this crystal. This
involves tilting the stage of the cryo-TEM to high-tilt and then
starting a steady and continuous rotation of the stage. Diffraction
data are recorded using a high-speed detector as the crystal continuously tilts in the beam. These diffraction data sets are then processed using standard data processing workflows [29, 30].
2 Materials
1. Holey carbon or continuous carbon-coated EM grids (copper;
200–400 mesh).
2. Protein microcrystals.
3. Glow discharge system.
4. Clean glass microscope slides.
5. Plunge freezing apparatus (see Note 1).
6. Locking fine-tip tweezers for plunge freezing apparatus.
7. Blotting paper for plunge freezing apparatus.
8. Liquid Nitrogen.
9. Liquid Ethane.
10. Pipettes capable of dispensing in the range of 1–20 μL.
288
Guanhong Bu and Brent L. Nannenga
crystal-loaded EM grid is immediately vitrified in liquid ethane
and either loaded into the cryo-TEM or stored under liquid nitrogen for future use. While the protocols presented here will focus on
more standard sample preparation methods, there are also other
procedures that can be used including crystal fragmentation [6]
and cryo-focus ion beam (cryo-FIB) milling [24, 25]. Readers are
encouraged to refer to other sources for details on how to use these
other methods for sample preparation.
Once the sample is inserted into the microscope, the quality of
the sample is screened, and data is collected from the best diffracting crystals on the grid. While there are recent methods for automated data collection [26–28] and new user interfaces for
MicroED data collection, this chapter will focus on the manual
method for MicroED data collection that does not require additional software besides the standard cryo-TEM control interface.
Data collection begins by screening the grid for the presence of
suitable microcrystals on the grid. Suitable crystals are those which
have a defined shape and are not too close to the EM grid bars or
other crystals, which would limit the range of data that could be
collected (see Fig. 1). Following the identification of a suitable
crystal, an initial diffraction pattern is collected, and its quality is
assessed. If the diffraction is of high quality (see Fig. 2), a continuous rotation data set [7] should be collected from this crystal. This
involves tilting the stage of the cryo-TEM to high-tilt and then
starting a steady and continuous rotation of the stage. Diffraction
data are recorded using a high-speed detector as the crystal continuously tilts in the beam. These diffraction data sets are then processed using standard data processing workflows [29, 30].
2 Materials
1. Holey carbon or continuous carbon-coated EM grids (copper;
200–400 mesh).
2. Protein microcrystals.
3. Glow discharge system.
4. Clean glass microscope slides.
5. Plunge freezing apparatus (see Note 1).
6. Locking fine-tip tweezers for plunge freezing apparatus.
7. Blotting paper for plunge freezing apparatus.
8. Liquid Nitrogen.
9. Liquid Ethane.
10. Pipettes capable of dispensing in the range of 1–20 μL.
288
Guanhong Bu and Brent L. Nannenga