Abstract
Imaging of biomolecules by ionizing radiation, such as electrons, causes radiation damage which introduces structural and compositional changes of the specimen. The total number of high-energy electrons per surface area that can be used for imaging in cryogenic electron microscopy (cryo-EM) is severely restricted due to radiation damage, resulting in low signal-to-noise ratios (SNR). High resolution details are dampened by the transfer function of the microscope and detector, and are the first to be lost as radiation damage alters the individual molecules which are presumed to be identical during averaging. As a consequence, radiation damage puts a limit on the particle size and sample heterogeneity with which electron microscopy (EM) can deal. Since a transmission EM (TEM) image is formed from the scattering process of the electron by the specimen interaction potential, radiation damage is inevitable. However, we can aim to maximize the information transfer for a given dose and increase the SNR by finding alternatives to the conventional phase-contrast cryo-EM techniques. Here some alternative transmission electron microscopy techniques are reviewed, including phase plate, multi-pass transmission electron microscopy, off-axis holography, ptychography and a quantum sorter. Their prospects for providing more or complementary structural information within the limited lifetime of the sample are discussed.
Highlights
Cryogenic electron microscopy has become a powerful tool for structural biologists to study the structure– function relationships of their biomolecules of interest
We review TEM techniques, such as phase plate, multi-pass transmission electron microscopy (MPTEM) and off-axis holography, as well as STEM techniques, such as ptychography, and a quantum sorter
This is in contrast to of the grid (Ravelli et al, 2020), split-illumination holography conventional TEM, where the contrast is enhanced by might become possible for a wider range of biological appliapplying a high defocus at the cost of fast damping of contrast transfer function (CTF) and cations
Summary
Cryogenic electron microscopy (cryo-EM) has become a powerful tool for structural biologists to study the structure– function relationships of their biomolecules of interest. TEM has the power to image individual atoms, its performance is limited by the radiation sensitivity of aqueous biological specimens and we need to minimize exposure to the electron beam. In TEM, the radiation dose is traditionally defined as charge density (C mÀ2) or electrons per unit area (eÀ A À2) (Williams & Carter, 2009). One wishes to image each particle individually, as each particle itself will define one point within the multi-dimensional conformation space a macromolecule can adapt This is done in single-particle analysis (SPA), with a fluence of, typically, 40 eÀ A À2 at 300 keV, which would correspond to 149 MGy. Doseweighting schemes are nowadays routinely applied to account for the loss of high-resolution information at such high doses (Scheres, 2014; Grant & Grigorieff, 2015). There is no amplitude contribution and the information transfer function becomes the contrast transfer function (CTF) multiplied by an aperture function and an envelope function (Reimer & Hohl, 2008; Williams & Carter, 2009),
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