Resolving the molecular architecture of the photoreceptor active zone with 3D-MINFLUX

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Abstract

Cells assemble macromolecular complexes into scaffoldings that serve as substrates for catalytic processes. Years of molecular neurobiology research indicate that neurotransmission depends on such optimization strategies. However, the molecular topography of the presynaptic active zone (AZ), where transmitter is released upon synaptic vesicle (SV) fusion, remains to be visualized. Therefore, we implemented MINFLUX optical nanoscopy to resolve the AZ of rod photoreceptors. This was facilitated by a novel sample immobilization technique that we name heat-assisted rapid dehydration (HARD), wherein a thin layer of rod synaptic terminals (spherules) was transferred onto glass coverslips from fresh retinal slices. Rod ribbon AZs were readily immunolabeled and imaged in 3D with a precision of a few nanometers. Our 3D-MINFLUX results indicate that the SV release site in rods is a molecular complex of bassoon–RIM2–ubMunc13-2–Ca _v1.4, which repeats longitudinally on both sides of the ribbon.

Abstract

3D-MINFLUX nanoscopy of heat-immobilized retinal sections reveals the molecular topography of rod photoreceptor active zones.

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Most cited references 61

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Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes

Francisco Balzarotti, Yvan Eilers, Klaus C. Gwosch … (2017)

We introduce MINFLUX, a concept for localizing photon emitters in space. By probing the emitter with a local intensity minimum of excitation light, MINFLUX minimizes the fluorescence photons needed for high localization precision. In our experiments, 22 times fewer fluorescence photons are required as compared to popular centroid localization. In superresolution microscopy, MINFLUX attained ~1-nm precision, resolving molecules only 6 nanometers apart. MINFLUX tracking of single fluorescent proteins increased the temporal resolution and the number of localizations per trace by a factor of 100, as demonstrated with diffusing 30S ribosomal subunits in living Escherichia coli As conceptual limits have not been reached, we expect this localization modality to break new ground for observing the dynamics, distribution, and structure of macromolecules in living cells and beyond.

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Release probability of hippocampal glutamatergic terminals scales with the size of the active zone

Noemi Holderith, Andrea Lorincz, Gergely Katona … (2012)

Cortical synapses display remarkable structural, molecular and functional heterogeneity. Our knowledge regarding the relationship between the ultrastructural and functional parameters is still fragmented. Here we asked how the release probability and presynaptic [Ca2+] transients relate to the ultrastructure of rat hippocampal glutamatergic axon terminals. Two-photon Ca2+ imaging-derived optical quantal analysis and correlated electron microscopic reconstructions revealed a tight correlation between the release probability and the active zone area. The peak amplitude of [Ca2+] transients in single boutons also positively correlated with the active zone area. Freeze-fracture immunogold labeling revealed that the voltage-gated Ca2+ channel subunit Cav2.1 and the presynaptic protein Rim1/2 are confined to the active zone and their numbers scale linearly with the active zone area. Gold particles for Cav2.1 showed a nonrandom distribution within the active zones. Our results demonstrate that the number of several active zone proteins, including presynaptic Ca2+ channels, docked vesicles and the release probability scales linearly with the active zone area.

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MINFLUX nanoscopy delivers 3D multicolor nanometer resolution in cells

Klaus C. Gwosch, Jasmin Pape, Francisco Balzarotti … (2020)

The ultimate goal of biological super-resolution fluorescence microscopy is to provide three-dimensional resolution at the size scale of a fluorescent marker. Here we show that by localizing individual switchable fluorophores with a probing donut-shaped excitation beam, MINFLUX nanoscopy can provide resolutions in the range of 1 to 3 nm for structures in fixed and living cells. This progress has been facilitated by approaching each fluorophore iteratively with the probing-donut minimum, making the resolution essentially uniform and isotropic over scalable fields of view. MINFLUX imaging of nuclear pore complexes of a mammalian cell shows that this true nanometer-scale resolution is obtained in three dimensions and in two color channels. Relying on fewer detected photons than standard camera-based localization, MINFLUX nanoscopy is poised to open a new chapter in the imaging of protein complexes and distributions in fixed and living cells.

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Author and article information

Contributors

Chad P. Grabner:

ORCID: https://orcid.org/0000-0001-7885-7627

Role: ConceptualizationRole: Formal analysisRole: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: SupervisionRole: ValidationRole: VisualizationRole: Writing - original draftRole: Writing - review & editing

Isabelle Jansen:

ORCID: https://orcid.org/0000-0002-4333-1868

Role: InvestigationRole: MethodologyRole: ValidationRole: VisualizationRole: Writing - review & editing

Jakob Neef:

ORCID: https://orcid.org/0000-0002-4757-9385

Role: Data curationRole: Formal analysisRole: MethodologyRole: SoftwareRole: ValidationRole: VisualizationRole: Writing - review & editing

Tobias Weihs:

ORCID: https://orcid.org/0000-0003-4509-7873

Role: Formal analysis

Roman Schmidt:

ORCID: https://orcid.org/0000-0002-6588-3778

Role: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: ResourcesRole: SoftwareRole: Writing - review & editing

Dietmar Riedel:

ORCID: https://orcid.org/0000-0003-2970-6894

Role: InvestigationRole: MethodologyRole: Visualization

Christian A. Wurm:

ORCID: https://orcid.org/0000-0002-6157-2032

Role: InvestigationRole: Project administrationRole: ResourcesRole: SupervisionRole: ValidationRole: VisualizationRole: Writing - review & editing

Tobias Moser:

ORCID: https://orcid.org/0000-0001-7145-0533

Role: ConceptualizationRole: Funding acquisitionRole: Project administrationRole: SupervisionRole: ValidationRole: Writing - original draftRole: Writing - review & editing

Journal

Journal ID (nlm-ta): Sci Adv

Journal ID (iso-abbrev): Sci Adv

Journal ID (publisher-id): sciadv

Journal ID (hwp): advances

Title: Science Advances

Publisher: American Association for the Advancement of Science

ISSN (Electronic): 2375-2548

Publication date Collection: July 2022

Publication date (Electronic, pub): 15 July 2022

Volume: 8

Issue: 28

Electronic Location Identifier: eabl7560

Affiliations

[ ¹ ]Institute for Auditory Neuroscience, University Medical Center Göttingen, 37075 Göttingen, Germany.

[ ² ]Auditory Neuroscience and Synaptic Nanophysiology Group, Max Planck Institute for Multidisciplinary Sciences, 37077 Göttingen, Germany.

[ ³ ]Collaborative Research Center 1286, University of Göttingen, Göttingen, Germany.

[ ⁴ ]Cluster of Excellence “Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells”, University of Göttingen, 37075 Göttingen, Germany.

[ ⁵ ]Abberior Instruments, Hans-Adolf-Krebs-Weg 1, 37077 Göttingen, Germany.

[ ⁶ ]Laboratory of Electron Microscopy, Max Planck Institute for Multidisciplinary Sciences, 37077 Göttingen, Germany.

Author notes

[* ]Corresponding author. Email: chadgrabner@ 123456gmail.com (C.P.G.); c.wurm@ 123456abberior-instruments.com (C.A.W.); tmoser@ 123456gwdg.de (T.M.)

[†]

These authors contributed equally to this work.

Author information

Chad P. Grabner https://orcid.org/0000-0001-7885-7627

Isabelle Jansen https://orcid.org/0000-0002-4333-1868

Jakob Neef https://orcid.org/0000-0002-4757-9385

Tobias Weihs https://orcid.org/0000-0003-4509-7873

Roman Schmidt https://orcid.org/0000-0002-6588-3778

Dietmar Riedel https://orcid.org/0000-0003-2970-6894

Christian A. Wurm https://orcid.org/0000-0002-6157-2032

Tobias Moser https://orcid.org/0000-0001-7145-0533

Article

Publisher ID: abl7560

DOI: 10.1126/sciadv.abl7560

PMC ID: 9286502

PubMed ID: 35857490

SO-VID: 22a29970-88ae-44ab-ada7-3e5a6ffc55e2

Copyright © Copyright © 2022 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).

License:

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.

History

Date received : 05 August 2021

Date accepted : 02 June 2022

Funding

Funded by: FundRef http://dx.doi.org/10.13039/501100001659, Deutsche Forschungsgemeinschaft;

Award ID: EXC 2067/1

Funded by: FundRef http://dx.doi.org/10.13039/501100001659, Deutsche Forschungsgemeinschaft;

Award ID: CRC1286

Funded by: FundRef http://dx.doi.org/10.13039/501100002347, Bundesministerium für Bildung und Forschung;

Award ID: 13N14122

Funded by: FundRef http://dx.doi.org/10.13039/501100004189, Max-Planck-Gesellschaft;

Award ID: Max-Planck-Fellowship

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Resolving the molecular architecture of the photoreceptor active zone with 3D-MINFLUX

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Most cited references 61

Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes

Release probability of hippocampal glutamatergic terminals scales with the size of the active zone

MINFLUX nanoscopy delivers 3D multicolor nanometer resolution in cells

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Cited by 8

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