Three-dimensional localization microscopy in live flowing cells

Abstract

Capturing the dynamics of live cell populations with nanoscale resolution poses a significant challenge, primarily owing to the speed-resolution trade-off of existing microscopy techniques. Flow cytometry would offer sufficient throughput, but lacks subsample detail. Here we show that imaging flow cytometry, in which the point detectors of flow cytometry are replaced with a camera to record 2D images, is compatible with 3D localization microscopy through point-spread-function engineering, which encodes the depth of the emitter into the emission pattern captured by the camera. The extraction of 3D positions from sub-cellular objects of interest is achieved by calibrating the depth-dependent response of the imaging system using fluorescent beads mixed with the sample buffer. This approach enables 4D imaging of up to tens of thousands of objects per minute and can be applied to characterize chromatin dynamics and the uptake and spatial distribution of nanoparticles in live cancer cells.

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Fig. 1: Device schematic and 3D calibration.
Fig. 2: Colocalization of fluorescent beads in six colours.
Fig. 3: Characterization of DNA nanorulers.
Fig. 4: Chromatin compaction states of the Gal locus in live yeast cells.
Fig. 5: Realtime (de)compaction of the Gal locus in live yeast cells.
Fig. 6: Localization of liposome nanoparticles in human T lymphocyte cells.
Fig. 7: Extended-depth imaging in flow using the Tetrapod PSF.

Data availability

The data that supports the plots within this paper and other findings of this study are available from the corresponding authors on reasonable request.

Code availability

The analysis scripts for image categorization, calibration, localization and 3D distance measurements were written in MATLAB 2018b (Mathworks) and are available from the corresponding authors on reasonable request.

References

  1. 1.

    Brown, M. & Wittwer, C. Flow cytometry: principles and clinical applications in hematology. Clin. Chem. 46, 1221–1229 (2000).

    CAS  Article  Google Scholar 

  2. 2.

    Luider, J., Cyfra, M., Johnson, P. & Auer, I. Impact of the New Beckman Coulter Cytomics FC 500 5-Color Flow Cytometer on a regional flow cytometry clinical laboratory service. Lab. Hematol. 10, 102–108 (2004).

    CAS  Article  Google Scholar 

  3. 3.

    Betters, D. M. Use of flow cytometry in clinical practice. J. Adv. Pract. Oncol. 6, 435–440 (2015).

    Google Scholar 

  4. 4.

    Chandler, W. L., Yeung, W. & Tait, J. F. A new microparticle size calibration standard for use in measuring smaller microparticles using a new flow cytometer. J. Thromb. Haemost. 9, 1216–1224 (2011).

    CAS  Article  Google Scholar 

  5. 5.

    Kay, D. B. & Wheeless, L. L. Laser stroboscopic photography. Technique for cell orientation studies in flow. J. Histochem. Cytochem. 24, 265–268 (1976).

    CAS  Article  Google Scholar 

  6. 6.

    Kay, D. B., Cambier, J. L. & Wheeless, L. L. Imaging in flow. J. Histochem. Cytochem. 27, 329–334 (1979).

    CAS  Article  Google Scholar 

  7. 7.

    Cambier, J. L., Kay, D. B. & Wheeless, L. L. A multidimensional slit-scan flow system. J. Histochem. Cytochem. 27, 321–324 (1979).

    CAS  Article  Google Scholar 

  8. 8.

    George, T. C. et al. Distinguishing modes of cell death using the ImageStream® multispectral imaging flow cytometer. Cytom. Part A. 59A, 237–245 (2004).

    Article  Google Scholar 

  9. 9.

    Basiji, D. A., Ortyn, W. E., Liang, L., Venkatachalam, V. & Morrissey, P. Cellular image analysis and imaging by flow cytometry. Clin. Lab. Med. 27, 653–670 (2007).

    Article  Google Scholar 

  10. 10.

    Gorthi, S. S. & Schonbrun, E. Phase imaging flow cytometry using a focus-stack collecting microscope. Opt. Lett. 37, 707 (2012).

    Article  Google Scholar 

  11. 11.

    Regmi, R., Mohan, K. & Mondal, P. P. High resolution light-sheet based high-throughput imaging cytometry system enables visualization of intra-cellular organelles. AIP Adv. 4, 097125 (2014).

    Article  Google Scholar 

  12. 12.

    Gualda, E. J., Pereira, H., Martins, G. G., Gardner, R. & Moreno, N. Three-dimensional imaging flow cytometry through light-sheet fluorescence microscopy. Cytom. Part A. 91, 144–151 (2017).

    Article  Google Scholar 

  13. 13.

    Toprak, E. & Selvin, P. R. New fluorescent tools for watching nanometer-scale conformational changes of single molecules. Annu. Rev. Biophys. Biomol. Struct. 36, 349–369 (2007).

    CAS  Article  Google Scholar 

  14. 14.

    Kao, H. P. & Verkman, A. S. Tracking of single fluorescent particles in three dimensions: use of cylindrical optics to encode particle position. Biophys. J. 67, 1291–1300 (1994).

    CAS  Article  Google Scholar 

  15. 15.

    Shtengel, G. et al. Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure. Proc. Natl Acad. Sci. USA 106, 3125–3130 (2009).

    CAS  Article  Google Scholar 

  16. 16.

    Juette, M. F. et al. Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples. Nat. Methods 5, 527–529 (2008).

    CAS  Article  Google Scholar 

  17. 17.

    Abrahamsson, S. et al. Fast multicolor 3D imaging using aberration-corrected multifocus microscopy. Nat. Methods 10, 60–63 (2012).

    Article  CAS  Google Scholar 

  18. 18.

    Backer, A. S. & Moerner, W. E. Extending single-molecule microscopy using optical Fourier processing. J. Phys. Chem. B. 118, 8313–8329 (2014).

    CAS  Article  Google Scholar 

  19. 19.

    Huang, B., Wang, W., Bates, M. & Zhuang, X. Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319, 810–813 (2008).

    CAS  Article  Google Scholar 

  20. 20.

    Mlodzianoski, M. J., Juette, M. F., Beane, G. L. & Bewersdorf, J. Experimental characterization of 3D localization techniques for particle-tracking and super-resolution microscopy. Opt. Express 17, 8264 (2009).

    CAS  Article  Google Scholar 

  21. 21.

    Dowski, E. R. & Cathey, W. T. Extended depth of field through wave-front coding. Appl. Opt. 34, 1859 (1995).

    Article  Google Scholar 

  22. 22.

    Ortyn, W. E. et al. Extended depth of field imaging for high speed cell analysis. Cytometry. A. 71, 215–231 (2007).

    Article  Google Scholar 

  23. 23.

    Shechtman, Y., Weiss, L. E., Backer, A. S., Sahl, S. J. & Moerner, W. E. Precise three-dimensional scan-free multiple-particle tracking overlarge axial ranges with tetrapod point spread functions. Nano Lett. 15, 4194–4199 (2015).

    CAS  Article  Google Scholar 

  24. 24.

    Cierpka, C., Rossi, M., Segura, R. & Kähler, C. J. On the calibration of astigmatism particle tracking velocimetry for microflows. Meas. Sci. Technol. 22, 015401 (2011).

    Article  CAS  Google Scholar 

  25. 25.

    Schmied, J. J. et al. DNA origami–based standards for quantitative fluorescence microscopy. Nat. Protoc. 9, 1367 (2014).

    CAS  Article  Google Scholar 

  26. 26.

    Finn, E. H. et al. Extensive heterogeneity and intrinsic variation in spatial genome organization. Cell 176, 1502–1515.e10 (2019).

    CAS  Article  Google Scholar 

  27. 27.

    Aylon, Y. & Kupiec, M. New insights into the mechanism of homologous recombination in yeast. Mutat. Res. 566, 231–248 (2004).

    CAS  Article  Google Scholar 

  28. 28.

    Dultz, E. et al. Quantitative imaging of chromatin decompaction in living cells. Mol. Biol. Cell 29, 1763–1777 (2018).

    CAS  Article  Google Scholar 

  29. 29.

    Khanna, N., Zhang, Y., Lucas, J. S., Dudko, O. K. & Murre, C. Chromosome dynamics near the sol-gel phase transition dictate the timing of remote genomic interactions. Nat. Commun. 10, 2771 (2019).

    Article  CAS  Google Scholar 

  30. 30.

    Even-Faitelson, L., Hassan-Zadeh, V., Baghestani, Z. & Bazett-Jones, D. P. Coming to terms with chromatin structure. Chromosoma 125, 95–110 (2016).

    CAS  Article  Google Scholar 

  31. 31.

    Hopper, J. E., Broach, J. R. & Rowe, L. B. Regulation of the galactose pathway in Saccharomyces cerevisiae: induction of uridyl transferase mRNA and dependency on GAL4 gene function. Proc. Natl Acad. Sci. USA 75, 2878–2882 (1978).

    CAS  Article  Google Scholar 

  32. 32.

    Dultz, E. et al. Global reorganization of budding yeast chromosome conformation in different physiological conditions. J. Cell Biol. 212, 321–334 (2016).

    CAS  Article  Google Scholar 

  33. 33.

    Shechtman, Y. et al. Observation of live chromatin dynamics in cells via 3D localization microscopy using Tetrapod point spread functions. Biomed. Opt. Express 8, 5735 (2017).

    CAS  Article  Google Scholar 

  34. 34.

    Sahay, G. et al. Efficiency of siRNA delivery by lipid nanoparticles is limited by endocytic recycling. Nat. Biotechnol. 31, 653–658 (2013).

    CAS  Article  Google Scholar 

  35. 35.

    Veiga, N. et al. Cell specific delivery of modified mRNA expressing therapeutic proteins to leukocytes. Nat. Commun. 9, 4493 (2018).

    Article  CAS  Google Scholar 

  36. 36.

    van der Meel, R. et al. Smart cancer nanomedicine. Nat. Nanotechnol. 14, 1007–1017 (2019).

    Article  CAS  Google Scholar 

  37. 37.

    Tsang, M., Gantchev, J., Ghazawi, F. M. & Litvinov, I. V. Protocol for adhesion and immunostaining of lymphocytes and other non-adherent cells in culture. Biotechniques 63, 230–233 (2017).

    Article  Google Scholar 

  38. 38.

    Shechtman, Y., Sahl, S. J., Backer, A. S. & Moerner, W. E. Optimal point spread function design for 3D imaging. Phys. Rev. Lett. 113, 133902 (2014).

    Article  CAS  Google Scholar 

  39. 39.

    Nehme, E. et al. DeepSTORM3D: dense three dimensional localization microscopy and point spread function design by deep learning. Preprint at https://arxiv.org/abs/1906.09957 (2019).

  40. 40.

    Boyd, N., Jonas, E., Babcock, H. P. & Recht, B. DeepLoco: fast 3D localization microscopy using neural networks. Preprint at https://doi.org/10.1101/267096 (2018).

  41. 41.

    Nitta, N. et al. Intelligent image-activated cell sorting. Cell 175, 266–276.e13 (2018).

    CAS  Article  Google Scholar 

  42. 42.

    Egner, A. & Hell, S. W. in Handbook of Biological Confocal Microscopy 3rd edn (eds Egener, A. & Hell, S.W.) 404–413 (Springer, 2006).

  43. 43.

    Jia, S., Vaughan, J. C. & Zhuang, X. Isotropic three-dimensional super-resolution imaging with a self-bending point spread function. Nat. Photonics 8, 302–306 (2014).

    CAS  Article  Google Scholar 

  44. 44.

    Backer, A. S., Backlund, M. P., Von Diezmann, A. R., Sahl, S. J. & Moerner, W. E. A bisected pupil for studying single-molecule orientational dynamics and its application to three-dimensional super-resolution microscopy. Appl. Phys. Lett. 104, 193701 (2014).

    Article  CAS  Google Scholar 

  45. 45.

    Bergman, L. W., Saghbini, M., Hoekstra, D. & Gautsch, J. Growth and maintenance of yeast. Media formulations for various two-hybrid systems. Methods Mol. Biol. 177, 15–39 (2001).

    Google Scholar 

  46. 46.

    Hansen, A. S., Hao, N. & O’Shea, E. K. High-throughput microfluidics to control and measure signaling dynamics in single yeast cells. Nat. Protoc. 10, 1181–1197 (2015).

    CAS  Article  Google Scholar 

  47. 47.

    Sela, M. et al. Sequential phosphorylation of SLP-76 at tyrosine 173 is required for activation of T and mast cells. EMBO J. 30, 3160–3172 (2011).

    CAS  Article  Google Scholar 

  48. 48.

    Linkert, M. et al. Metadata matters: access to image data in the real world. J. Cell Biol. 189, 777–782 (2010).

    CAS  Article  Google Scholar 

  49. 49.

    Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

    CAS  Article  Google Scholar 

  50. 50.

    Ovesný, M., Křížek, P., Borkovec, J., Svindrych, Z. & Hagen, G. M. ThunderSTORM: a comprehensive ImageJ plug-in for PALM and STORM data analysis and super-resolution imaging. Bioinformatics 30, 2389–2390 (2014).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank E. Barak, Y. Lupu-Haber, M. Duvshani-Eshet and the Lorry I. Lokey Interdisciplinary Center for Life Sciences and Engineering for technical assistance with the imaging flow cytometer. The multicolour yeast cells were provided by K. Weis and E. Dultz and the Jurkat human T lymphocytes were provided by D. Yablonski, and verified with the assistance of the Genomic Center at the Technion Biomedical Core Facility. The Tetrapod phase mask was fabricated by M.Y. Lee. This work was partially supported by the European Research Council (ERC) under the European Union Horizon 2020 research and innovation programme to Y.S. (grant no. 802567), and an ERC Starting Grant to A.S. (ERC-STG-2015–680242), the Zuckerman foundation, the POLAK Fund for Applied Research at the Technion and the Israel Science Foundation (1421/17) to A.S. and (450/18) to Y.S.

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L.E.W. and Y.S. conceived of the approach. L.E.W., Y.S.E., O. Adir, A.S. and Y.S. designed the experiments. L.E.W., Y.S.E., S.G., O. Adir, B.F. and O. Alalouf performed the experiments. All authors contributed to data analysis and preparing the manuscript.

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Correspondence to Lucien E. Weiss or Yoav Shechtman.

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L.E.W. and Y.S. are inventors on a patent application (International Publication no. WO2019180705A1) concerning the described technology.

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Peer review information Nature Nanotechnology thanks Jörg Enderlein, Tom Misteli and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Weiss, L.E., Shalev Ezra, Y., Goldberg, S. et al. Three-dimensional localization microscopy in live flowing cells. Nat. Nanotechnol. 15, 500–506 (2020). https://doi.org/10.1038/s41565-020-0662-0

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