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Background Electron cryomicroscopy of biological molecules has recently undergone a revolution (detailed in Reference 1), resulting in hundreds of new structures that were previously undeterminable by any method. Electron cryomicroscopy methods have been used for many years,2 but remain under development as we reach a deeper understanding of the radiation physics and electron imaging process involved. A recent synopsis contains a comprehensive review of many aspects of electron cryomicroscopy, from detectors to specimen preparation, to data processing and analysis.3 Reviews of radiation damage in the electron microscope have also recently been published.4–6 Here, we point out some key ideas about radiation damage that underpin the success of electron cryomicroscopy for biological specimens, describe how these might be applied to radiation-sensitive specimens in materials science, and look to important remaining questions about radiation damage in the transmission electron microscope or scanning transmission electron microscope.

Radiation damage in the electron microscope: Types of damage and when to worry Beam-sensitive specimens in an electron microscope are damaged as a result of inelastic scattering of primary electrons, which excite atomic electrons to higher energy levels with a probability determined by a total-inelastic cross section.

The atom or molecule quickly loses its excess energy, but might not return to its original ground state, resulting in the breakage of chemical bonds and a permanent change in structure known as ionization damage or radiolysis. In the case of a crystal, structural change can be monitored from the electron diffraction pattern (Figure 1).7 The fluence (electrons per unit area) at which a particular diffraction spot fades to a fraction of its original intensity (by a factor of e = 2.718) is called a characteristic fluence (or dose) and is the reciprocal of a damage cross section. Longer-range atomic motion leads to the escape of volatilized material from the specimen’s surface into vacuum, known as mass loss. Cooling a specimen below room temperature reduces the radiolysis efficiency (or damage cross section) and thereby increases the characteristic dose for these processes (Table I).8,9 In electrically conducting materials (metals and semiconductors), the abundance of free electrons quenches radiolysis, leaving high momentum transfer elastic scattering as the only damage mechanism. Most elastic collisions involve small scattering angles and negligible energy transfer, which does not create damage, but provides diffraction patterns and amplitude or phase contrast in transmission electron microscopy (TEM) images. Large-angle collisions are rare (low elastic cross section), but they can transfer several eV of energy to atomic nuclei, resulting in atom displacement and so-called knock-on damage. This process requires a primary-beam

C.J. Russo, Structural Studies Division, Medical Research Council Laboratory of Molecular Biology, UK; [email protected] R.F. Egerton, University