Correlative all-optical quantification of mass density and mechanics of sub-cellular compartments with fluorescence specificity

  1. Raimund Schlüßler  Is a corresponding author
  2. Kyoohyun Kim  Is a corresponding author
  3. Martin Nötzel
  4. Anna Taubenberger
  5. Shada Abuhattum
  6. Timon Beck
  7. Paul Müller
  8. Shovamaye Maharana
  9. Gheorghe Cojoc
  10. Salvatore Girardo
  11. Andreas Hermann
  12. Simon Alberti
  13. Jochen Guck  Is a corresponding author
  1. Technische Universität Dresden, Germany
  2. Max Planck Institute for the Science of Light, Germany
  3. University of Rostock, Germany

Abstract

Quantitative measurements of physical parameters become increasingly important for understanding biological processes. Brillouin microscopy (BM) has recently emerged as one technique providing the 3D distribution of viscoelastic properties inside biological samples - so far relying on the implicit assumption that refractive index (RI) and density can be neglected. Here, we present a novel method (FOB microscopy) combining BM with optical diffraction tomography and epi-fluorescence imaging for explicitly measuring the Brillouin shift, RI and absolute density with specificity to fluorescently labeled structures. We show that neglecting the RI and density might lead to erroneous conclusions. Investigating the nucleoplasm of wild-type HeLa cells, we find that it has lower density but higher longitudinal modulus than the cytoplasm. Thus, the longitudinal modulus is not merely sensitive to the water content of the sample - a postulate vividly discussed in the field. We demonstrate the further utility of FOB on various biological systems including adipocytes and intracellular membraneless compartments. FOB microscopy can provide unexpected scientific discoveries and shed quantitative light on processes such as phase separation and transition inside living cells.

Data availability

The data sets generated during and/or analyzed during the current study are available from figshare under the following link: https://doi.org/10.6084/m9.figshare.c.5347778

The following data sets were generated

Article and author information

Author details

  1. Raimund Schlüßler

    Center for Molecular and Cellular Bioengineering, Technische Universität Dresden, Dresden, Germany
    For correspondence
    raimund.schluessler@tu-dresden.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3752-2382
  2. Kyoohyun Kim

    Biological Optomechanics, Max Planck Institute for the Science of Light, Erlangen, Germany
    For correspondence
    kyoohyun.kim@mpl.mpg.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1808-775X
  3. Martin Nötzel

    Center for Molecular and Cellular Bioengineering, Technische Universität Dresden, Dresden, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6442-9899
  4. Anna Taubenberger

    Center for Molecular and Cellular Bioengineering, Technische Universität Dresden, Dresden, Germany
    Competing interests
    The authors declare that no competing interests exist.
  5. Shada Abuhattum

    Biological Optomechanics, Max Planck Institute for the Science of Light, Erlangen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  6. Timon Beck

    Biological Optomechanics, Max Planck Institute for the Science of Light, Erlangen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  7. Paul Müller

    Biological Optomechanics, Max Planck Institute for the Science of Light, Erlangen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  8. Shovamaye Maharana

    Center for Molecular and Cellular Bioengineering, Technische Universität Dresden, Dresden, Germany
    Competing interests
    The authors declare that no competing interests exist.
  9. Gheorghe Cojoc

    Center for Molecular and Cellular Bioengineering, Technische Universität Dresden, Dresden, Germany
    Competing interests
    The authors declare that no competing interests exist.
  10. Salvatore Girardo

    Biological Optomechanics, Max Planck Institute for the Science of Light, Erlangen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  11. Andreas Hermann

    Translational Neurodegeneration Section 'Albrecht Kossel', University of Rostock, Rostock, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7364-7791
  12. Simon Alberti

    Center for Molecular and Cellular Bioengineering, Technische Universität Dresden, Dresden, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4017-6505
  13. Jochen Guck

    Biological Optomechanics, Max Planck Institute for the Science of Light, Erlangen, Germany
    For correspondence
    jochen.guck@mpl.mpg.de
    Competing interests
    The authors declare that no competing interests exist.

Funding

Deutsche Forschungsgemeinschaft (419138906)

  • Simon Alberti
  • Jochen Guck

Volkswagen Foundation (92847)

  • Simon Alberti
  • Jochen Guck

Alexander von Humboldt-Stiftung

  • Jochen Guck

NOMIS Stiftung

  • Andreas Hermann

Hermann und Lilly Schilling-Stiftung

  • Andreas Hermann

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Rohit V Pappu, Washington University in St Louis, United States

Version history

  1. Preprint posted: October 30, 2020 (view preprint)
  2. Received: March 17, 2021
  3. Accepted: January 8, 2022
  4. Accepted Manuscript published: January 10, 2022 (version 1)
  5. Version of Record published: February 4, 2022 (version 2)

Copyright

© 2022, Schlüßler 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.

Metrics

  • 2,357
    Page views
  • 435
    Downloads
  • 24
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Raimund Schlüßler
  2. Kyoohyun Kim
  3. Martin Nötzel
  4. Anna Taubenberger
  5. Shada Abuhattum
  6. Timon Beck
  7. Paul Müller
  8. Shovamaye Maharana
  9. Gheorghe Cojoc
  10. Salvatore Girardo
  11. Andreas Hermann
  12. Simon Alberti
  13. Jochen Guck
(2022)
Correlative all-optical quantification of mass density and mechanics of sub-cellular compartments with fluorescence specificity
eLife 11:e68490.
https://doi.org/10.7554/eLife.68490

Share this article

https://doi.org/10.7554/eLife.68490

Further reading

    1. Cell Biology
    2. Immunology and Inflammation
    Chinky Shiu Chen Liu, Tithi Mandal ... Dipyaman Ganguly
    Research Article

    T cells are crucial for efficient antigen-specific immune responses and thus their migration within the body, to inflamed tissues from circulating blood or to secondary lymphoid organs, plays a very critical role. T cell extravasation in inflamed tissues depends on chemotactic cues and interaction between endothelial adhesion molecules and cellular integrins. A migrating T cell is expected to sense diverse external and membrane-intrinsic mechano-physical cues, but molecular mechanisms of such mechanosensing in cell migration are not established. We explored if the professional mechanosensor Piezo1 plays any role during integrin-dependent chemotaxis of human T cells. We found that deficiency of Piezo1 in human T cells interfered with integrin-dependent cellular motility on ICAM-1-coated surface. Piezo1 recruitment at the leading edge of moving T cells is dependent on and follows focal adhesion formation at the leading edge and local increase in membrane tension upon chemokine receptor activation. Piezo1 recruitment and activation, followed by calcium influx and calpain activation, in turn, are crucial for the integrin LFA1 (CD11a/CD18) recruitment at the leading edge of the chemotactic human T cells. Thus, we find that Piezo1 activation in response to local mechanical cues constitutes a membrane-intrinsic component of the ‘outside-in’ signaling in human T cells, migrating in response to chemokines, that mediates integrin recruitment to the leading edge.

    1. Cell Biology
    2. Chromosomes and Gene Expression
    Carolline Ascenção, Jennie R Sims ... Marcus B Smolka
    Research Article

    Meiotic sex chromosome inactivation (MSCI) is a critical feature of meiotic prophase I progression in males. While the ATR kinase and its activator TOPBP1 are key drivers of MSCI within the specialized sex body (SB) domain of the nucleus, how they promote silencing remains unclear given their multifaceted meiotic functions that also include DNA repair, chromosome synapsis, and SB formation. Here we report a novel mutant mouse harboring mutations in the TOPBP1-BRCT5 domain. Topbp1B5/B5 males are infertile, with impaired MSCI despite displaying grossly normal events of early prophase I, including synapsis and SB formation. Specific ATR-dependent events are disrupted, including phosphorylation and localization of the RNA:DNA helicase Senataxin. Topbp1B5/B5 spermatocytes initiate, but cannot maintain ongoing, MSCI. These findings reveal a non-canonical role for the ATR-TOPBP1 signaling axis in MSCI dynamics at advanced stages in pachynema and establish the first mouse mutant that separates ATR signaling and MSCI from SB formation.