Abstract

The throughput of cell mechanical characterization has recently approached that of conventional flow cytometers. However, this very sensitive, label-free approach still lacks the specificity of molecular markers. Here we developed an approach that combines real-time 1D-imaging fluorescence and deformability cytometry in one instrument (RT-FDC), thus opening many new research avenues. We demonstrated its utility by using subcellular fluorescence localization to identify mitotic cells and test for mechanical changes in those cells in an RNA interference screen.

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Acknowledgements

We thank D. Klaue, M. Urbanska, F. Rosendahl, and I. Richter for helpful discussions and technical support. We thank M. Schürmann for refractive index measurements of agarose. We thank M. Piel (Institute Curie, Paris, France) for donation of the HeLa H2B–mCherry cell line. We thank M. Bornhäuser (Department of Hematology and Oncology, University Clinic Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany) for providing the apheresis blood samples. We received support from the Light Microscopy Facility for confocal microscopy, and from the Microstructure Facility for microfluidic chip production; both are core facilities of the Center for Molecular and Cellular Bioengineering (CMCB) at Technische Universität Dresden. We gratefully acknowledge financial support from the Alexander-von-Humboldt Stiftung (Humboldt Professorship to J.G.), Sächsisches Ministerium für Wissenschaft und Kunst (TG70 grant to O.O. and J.G.; European Fund for Regional Development–EFRE to S.G. and J.G.), the ERC (starting grant “LightTouch” 282060 to J.G.), the DFG Center for Regenerative Medicine of the Technische Universität Dresden (seed grant to J.G.), the DFG KFO249 (GU 612/2-2 grant to J.G.), DKMS ('Mechthild Harf Research Grant' DKMS-SLS-MHG-2016-02 to A.J.), and Cancer Research UK (C1529/A17343 grant to B.B.).

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Author notes

    • Philipp Rosendahl
    •  & Katarzyna Plak

    These authors contributed equally to this work.

Affiliations

  1. Biotechnology Center, Center for Molecular and Cellular Bioengineering, Technische Universität Dresden, Dresden, Germany.

    • Philipp Rosendahl
    • , Katarzyna Plak
    • , Angela Jacobi
    • , Martin Kraeter
    • , Nicole Toepfner
    • , Oliver Otto
    • , Christoph Herold
    • , Maria Winzi
    • , Maik Herbig
    • , Yan Ge
    • , Katrin Wagner
    •  & Jochen Guck
  2. MRC Laboratory for Molecular and Cellular Biology, University College London, London, UK.

    • Katarzyna Plak
    •  & Buzz Baum
  3. Medical Clinic I, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

    • Martin Kraeter
  4. Department of Pediatrics, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

    • Nicole Toepfner
  5. Microstructure Facility, Center for Molecular and Cellular Bioengineering, Technische Universität Dresden, Dresden, Germany.

    • Salvatore Girardo

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Contributions

J.G. conceived the project. P.R. built the experimental setup. O.O. and C.H. supported the optics design. P.R. programmed the real-time data-evaluation software. K.P. designed and performed experiments on Kc167 and HeLa cells and analyzed the data with B.B.'s support. P.R. and N.T. performed experiments with RBCs. A.J., M.K., and P.R. performed experiments on HSPCs and analyzed the data. P.R. performed and analyzed experiments for device characterization. M.H., O.O., and Y.G. developed the linear mixed model algorithm for statistical evaluation. K.W. and S.G. produced fluorescent beads for device characterization. M.W. performed reference measurements with calibration beads on FCM. S.G. produced masters for microfluidic chips. J.G. and B.B. supervised the project. P.R., K.P., and J.G. wrote the manuscript. All authors reviewed the manuscript.

Competing interests

P.R., O.O., and C.H. are shareholders and employees of the company Zellmechanik Dresden GmbH, which sells devices based on RT-DC and RT-FDC technology. All other authors declare no competing interests.

Corresponding author

Correspondence to Jochen Guck.

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DOI

https://doi.org/10.1038/nmeth.4639