Three-dimensional localization microscopy in live flowing cells


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.


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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.

Author information




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.

Corresponding authors

Correspondence to Lucien E. Weiss or Yoav Shechtman.

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Competing interests

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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Supplementary Figs. 1–8, figure captions, discussion and sample preparation details.

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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).

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