A cryogenic, coincident fluorescence, electron and ion beam microscope
- Delft University of Technology, Netherlands
- California Institute of Technology, United States
- Delmic B.V., Netherlands
- Leiden University Medical Center, Netherlands
- Max Planck Institute of Biochemistry, Germany
- Max Planck Institute of Molecular Physiology, Germany
- University of Queensland, Australia
Abstract
Cryogenic electron tomography (cryo-ET) combined with sub-tomogram averaging, allows in-situ visualization and structure determination of macromolecular complexes at sub-nanometre resolution. Cryogenic focused ion beam (cryo-FIB) micromachining is used to prepare a thin lamella-shaped sample out of a frozen-hydrated cell for cryo-ET imaging, but standard cryo-FIB fabrication is blind to the precise location of the structure or proteins of interest. Fluorescence-guided focused ion beam (FIB) milling at target locations requires multiple sample transfers prone to contamination, and relocation and registration accuracy is often insufficient for 3D targeting. Here, we present in-situ fluorescence microscopy-guided FIB fabrication of a frozen-hydrated lamella to address this problem: we built a coincident 3-beam cryogenic correlative microscope by retrofitting a compact cryogenic microcooler, custom positioning stage, and an inverted widefield fluorescence microscope (FM) on an existing focused ion-beam scanning electron microscope (FIB-SEM). We show FM controlled targeting at every milling step in the lamella fabrication process, validated with transmission electron microscope (TEM) tomogram reconstructions of the target regions. The ability to check the lamella during and after the milling process results in a higher success rate in the fabrication process and will increase the throughput of fabrication for lamellae suitable for high-resolution imaging.
Data availability
The data underlying the publication can be found at international data repository service 4TU.ResearchData, https://doi.org/10.4121/20787274
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Data underlying the publication: A cryogenic, coincident fluorescence, electron and ion beam microscope4TU.ResearchData, doi:10.4121/20787274.
Article and author information
Author details
Ernest B van der Wee
Funding
Nederlandse Organisatie voor Wetenschappelijk Onderzoek (TTW No 17152)
- Jacob P Hoogenboom
National Institutes of Health (RO1 AI127401)
- Grant J Jensen
European Commission (SME2 No 879673)
- Sander den Hoedt
Eurostars (No E13008)
- Stefan Raunser
- Sander den Hoedt
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Suzanne R Pfeffer, Stanford University, United States
Publication history
- Received: August 23, 2022
- Preprint posted: September 3, 2022 (view preprint)
- Accepted: October 25, 2022
- Accepted Manuscript published: October 28, 2022 (version 1)
- Version of Record published: December 1, 2022 (version 2)
- Version of Record updated: April 20, 2023 (version 3)
Copyright
© 2022, Boltje et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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A cryogenic, coincident fluorescence, electron and ion beam microscope
eLife 11:e82891.
https://doi.org/10.7554/eLife.82891
Further reading
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- Structural Biology and Molecular Biophysics
Formation of membraneless organelles or biological condensates via phase separation and related processes hugely expands the cellular organelle repertoire. Biological condensates are dense and viscoelastic soft matters instead of canonical dilute solutions. To date, numerous different biological condensates have been discovered; but mechanistic understanding of biological condensates remains scarce. In this study, we developed an adaptive single molecule imaging method that allows simultaneous tracking of individual molecules and their motion trajectories in both condensed and dilute phases of various biological condensates. The method enables quantitative measurements of concentrations, phase boundary, motion behavior and speed of molecules in both condensed and dilute phases as well as the scale and speed of molecular exchanges between the two phases. Notably, molecules in the condensed phase do not undergo uniform Brownian motion, but instead constantly switch between a (class of) confined state(s) and a random diffusion-like motion state. Transient confinement is consistent with strong interactions associated with large molecular networks (i.e., percolation) in the condensed phase. In this way, molecules in biological condensates behave distinctly different from those in dilute solutions. The methods and findings described herein should be generally applicable for deciphering the molecular mechanisms underlying the assembly, dynamics and consequently functional implications of biological condensates.
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- Structural Biology and Molecular Biophysics
Single-molecule tweezers, such as magnetic tweezers, are powerful tools for probing nm-scale structural changes in single membrane proteins under force. However, the weak molecular tethers used for the membrane protein studies have limited the observation of long-time, repetitive molecular transitions due to force-induced bond breakage. The prolonged observation of numerous transitions is critical in reliable characterizations of structural states, kinetics, and energy barrier properties. Here, we present a robust single-molecule tweezer method that uses dibenzocyclooctyne (DBCO) cycloaddition and traptavidin binding, enabling the estimation of the folding 'speed limit' of helical membrane proteins. This method is >100 times more stable than a conventional linkage system regarding the lifetime, allowing for the survival for ~12 h at 50 pN and ~1000 pulling cycle experiments. By using this method, we were able to observe numerous structural transitions of a designer single-chained transmembrane (TM) homodimer for 9 h at 12 pN, and reveal its folding pathway including the hidden dynamics of helix-coil transitions. We characterized the energy barrier heights and folding times for the transitions using a model-independent deconvolution method and the hidden Markov modeling (HMM) analysis, respectively. The Kramers rate framework yields a considerably low speed limit of 21 ms for a helical hairpin formation in lipid bilayers, compared to μs scale for soluble protein folding. This large discrepancy is likely due to the highly viscous nature of lipid membranes, retarding the helix-helix interactions. Our results offer a more valid guideline for relating the kinetics and free energies of membrane protein folding.
