Chapter 15
Low-Dose Data Collection and Radiation Damage in MicroED
Johan Hattne
Abstract
Microcrystal electron diffraction (MicroED) is a technique for structure determination that relies on the
strong interaction of electrons with a minuscule, crystalline sample. While some of the electrons used to
probe the crystal interact without altering the crystal, others deposit energy which changes the sample
through a series of damage events. It follows that the sample cannot be observed without damaging it, and
the frames obtained at the beginning of data collection reflect a crystal that differs from the one that yields
the last frames of the dataset. Data acquisition at cryogenic temperatures has been found to reduce the rate
of damage progression and is routinely used to increase the dose tolerance of the crystal, allowing more
useful data to be obtained before the sample is destroyed. Low-dose data collection can further prolong the
lifetime of the crystal, such that less damage is inflicted over the course of data acquisition. Ideally, lower
doses increase the measurable volume of a single-crystal lattice by reducing the damage caused by probing
electrons. However, the information that can be recovered from a diffraction image is directly related to the
number of electrons used to probe the sample. The signal from a weakly exposed crystal runs the risk of
being lost in the noise contributed by solvent, crystal disorder, and the electron detection process. This
work focuses on obtaining the best possible data from a MicroED measurement, which requires considering
several aspects such as sample, dose, and camera type.
Key words MicroED, Data collection, Dose, Radiation damage, Cryo-EM
1 Introduction
Microcrystal electron diffraction (MicroED) is a diffraction method
in cryo-electron microscopy (cryo-EM) [1]. It exploits the strong
interaction of electrons with matter to recover the signal diffracted
from tiny, three-dimensional crystals. One of the key insights that
made MicroED possible is low-dose data collection [2] and its
implication on radiation damage. In earlier work on electron diffraction from two-dimensional crystals, it was often found that
crystals were destroyed by radiation damage before an interpretable
dataset could be obtained. Crystals would frequently only last for a
single diffraction image, which even with today’s data-processing
algorithms results in a formidable data-analysis challenge when the
crystal’s unit cell is unknown with hundreds or even thousands of
Tamir Gonen and Brent L. Nannenga (eds.), CryoEM: Methods and Protocols, Methods in Molecular Biology, vol. 2215,
https://doi.org/10.1007/978-1-0716-0966-8_15, © Springer Science+Business Media, LLC, part of Springer Nature 2021
309
Low-Dose Data Collection and Radiation Damage in MicroED
Johan Hattne
Abstract
Microcrystal electron diffraction (MicroED) is a technique for structure determination that relies on the
strong interaction of electrons with a minuscule, crystalline sample. While some of the electrons used to
probe the crystal interact without altering the crystal, others deposit energy which changes the sample
through a series of damage events. It follows that the sample cannot be observed without damaging it, and
the frames obtained at the beginning of data collection reflect a crystal that differs from the one that yields
the last frames of the dataset. Data acquisition at cryogenic temperatures has been found to reduce the rate
of damage progression and is routinely used to increase the dose tolerance of the crystal, allowing more
useful data to be obtained before the sample is destroyed. Low-dose data collection can further prolong the
lifetime of the crystal, such that less damage is inflicted over the course of data acquisition. Ideally, lower
doses increase the measurable volume of a single-crystal lattice by reducing the damage caused by probing
electrons. However, the information that can be recovered from a diffraction image is directly related to the
number of electrons used to probe the sample. The signal from a weakly exposed crystal runs the risk of
being lost in the noise contributed by solvent, crystal disorder, and the electron detection process. This
work focuses on obtaining the best possible data from a MicroED measurement, which requires considering
several aspects such as sample, dose, and camera type.
Key words MicroED, Data collection, Dose, Radiation damage, Cryo-EM
1 Introduction
Microcrystal electron diffraction (MicroED) is a diffraction method
in cryo-electron microscopy (cryo-EM) [1]. It exploits the strong
interaction of electrons with matter to recover the signal diffracted
from tiny, three-dimensional crystals. One of the key insights that
made MicroED possible is low-dose data collection [2] and its
implication on radiation damage. In earlier work on electron diffraction from two-dimensional crystals, it was often found that
crystals were destroyed by radiation damage before an interpretable
dataset could be obtained. Crystals would frequently only last for a
single diffraction image, which even with today’s data-processing
algorithms results in a formidable data-analysis challenge when the
crystal’s unit cell is unknown with hundreds or even thousands of
Tamir Gonen and Brent L. Nannenga (eds.), CryoEM: Methods and Protocols, Methods in Molecular Biology, vol. 2215,
https://doi.org/10.1007/978-1-0716-0966-8_15, © Springer Science+Business Media, LLC, part of Springer Nature 2021
309