Design for a 10 keV multi-pass transmission electron microscope

Koppell, Stewart A.; Mankos, M.; Bowman, Adam J.; Israel, Yonatan; Juffmann, Thomas; Klopfer, Brannon B.; Kasevich, Mark A.

doi:10.1016/j.ultramic.2019.112834

Cited by 18 publications

(12 citation statements)

References 28 publications

(35 reference statements)

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“…The need for only a single un-tilted image greatly simplifies acquisition and makes cHRTM compatible with pulsed-beam (Spence 2017) and multi-pass imaging techniques (Juffmann et al 2017;Koppell et al 2019), both designed to increase the amount of highresolution information extracted before it is lost to radiation damage. Together with improved detectors, this might well boost the sensitivity of cHRTM enough to make the majority of proteins detectable.…”

mentioning

confidence: 99%

Label-free single-instance protein detection in vitrified cells

Choi

Lippincott‐Schwartz

et al. 2020

Preprint

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A general method to map molecular interactions and conformational states in structurally intact cells would find wide application in biochemistry and cell biology. We used a library of imagescalculated on the basis of known structural data-as search templates to detect targets as small as the "head" domain (350 kDa) of the ribosome's small subunit in single-tilt electron cryomicrographs by cellular high resolution template matching (cHRTM). Atomically precise position and orientation estimates reveal the conformation of individual ribosomes and enable the detection of specifically bound ligands down to 24 kDa. We show that highly head-swivelled states are likely to play a role in mRNA translocation in living cells. cHRTM outperforms cryoelectron tomography three-fold in sensitivity and completely avoids the vicissitudes of exogenous labelling. Main:Finding molecular components in intact cells has driven the development of biological imaging techniques, some of which use specific labels, such as fluorescent antibodies, while others rely on a target's intrinsic properties such as its shape (Oliver 1973) and structure (Rickgauer, Grigorieff, and Denk 2017). Labelling is frequently incomplete or nonspecific, can alter the target's interactions with other molecules, provides only moderate precision of localization and

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mentioning

confidence: 99%

Label-free single-instance protein detection in vitrified cells

Choi

Lippincott‐Schwartz

et al. 2020

Preprint

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“…After passing through the sample m times, the light leaves the cavity and is imaged using a microscope objective onto a camera. Analogous to light optical design, multi-pass microscopy was simulated (Juffmann et al, 2017) and designed for TEM (Koppell et al, 2019). Figure 3 shows a schematic view of a MPTEM setup (Koppell et al, 2019).…”

Section: Multi-pass Transmission Electron Microscopymentioning

confidence: 99%

“…Analogous to light optical design, multi-pass microscopy was simulated (Juffmann et al, 2017) and designed for TEM (Koppell et al, 2019). Figure 3 shows a schematic view of a MPTEM setup (Koppell et al, 2019).…”

Section: Multi-pass Transmission Electron Microscopymentioning

confidence: 99%

“…However, a high-voltage electron mirror, the key component for MPTEM, does not yet exist. A low-energy (10 keV) proof-of-concept MPTEM is currently under construction (Koppell et al, 2019), which could be the very first step to the application of MPTEM. Holography and ptychography have compelling advantages in dose-efficient phase retrieval of the exit wave.…”

Section: Figurementioning

confidence: 99%

“…In this case, the SNR will be three times higher at the same dose limit compared with conventional cryo-EM. This would enhance the resolution of a given sample (Koppell et al, 2019), or allow for SPA to be performed on smaller biomolecules, or enable the processing of highly heterogenous samples (Henderson, 1995;Hall et al, 2011;Zhang et al, 2020;Su et al, 2021).…”

Section: Multi-pass Transmission Electron Microscopymentioning

confidence: 99%

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Single-particle cryo-EM: alternative schemes to improve dose efficiency

Zhang¹,

Lu²,

Rotunno³

et al. 2021

J Synchrotron Radiat

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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.

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