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Hyperspectral confocal imaging for high-throughput readout and analysis of bio-integrated microlasers

Nature Protocols volume 19, pages 928–959 (2024)Cite this article

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Abstract

Integrating micro- and nanolasers into live cells, tissue cultures and small animals is an emerging and rapidly evolving technique that offers noninvasive interrogation and labeling with unprecedented information density. The bright and distinct spectra of such lasers make this approach particularly attractive for high-throughput applications requiring single-cell specificity, such as multiplexed cell tracking and intracellular biosensing. The implementation of these applications requires high-resolution, high-speed spectral readout and advanced analysis routines, which leads to unique technical challenges. Here, we present a modular approach consisting of two separate procedures. The first procedure instructs users on how to efficiently integrate different types of lasers into living cells, and the second procedure presents a workflow for obtaining intracellular lasing spectra with high spectral resolution and up to 125-kHz readout rate and starts from the construction of a custom hyperspectral confocal microscope. We provide guidance on running hyperspectral imaging routines for various experimental designs and recommend specific workflows for processing the resulting large data sets along with an open-source Python library of functions covering the analysis pipeline. We illustrate three applications including the rapid, large-volume mapping of absolute refractive index by using polystyrene microbead lasers, the intracellular sensing of cardiac contractility with polystyrene microbead lasers and long-term cell tracking by using semiconductor nanodisk lasers. Our sample preparation and imaging procedures require 2 days, and setting up the hyperspectral confocal microscope for microlaser characterization requires <2 weeks to complete for users with limited experience in optical and software engineering.

Key points

  • The protocol describes the integration of microlasers into living cells and a workflow for obtaining lasing spectra with high spectral resolution using a customized hyperspectral confocal microscope.

  • Bio-integrated lasers enable high-throughput and highly multiplexed cell-tracking and biosensing, offering higher penetration depth and information density than alternative approaches.

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Fig. 1: Properties and applications of bio-integrated WGM laser particles.
Fig. 2: Schematic of the protocol workflow.
Fig. 3: Home-built confocal microscope.
Fig. 4: Controling internalization of LPs.
Fig. 5: Microscope overview.
Fig. 6: Coarse pinhole alignment.
Fig. 7: Typical results of reference measurements.
Fig. 8: Application examples of cell tracking and sensing with LPs.

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Data availability

The research data supporting this publication can be accessed at https://doi.org/10.17630/bec9180a-86e6-4157-822f-fa84249452dd56.

Code availability

A repository containing the supplementary code files can be found at https://github.com/GatherLab/sphyncs55. Custom hardware control software is available upon request.

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Acknowledgements

We thank Klara Voelckert and Manuel Neubauer for their contributions to lasing data acquisition, and Viktor Klippert and Thomas Michaelis for assistance with the design of custom adapters. This work received financial support from the Leverhulme Trust (RPG-2017-231), the European Union’s Horizon 2020 Framework Programme (FP/2014-2020)/ERC grant agreement no. 640012 (ABLASE), EPSRC (EP/P030017/1), the Humboldt Foundation (Alexander von Humboldt professorship) and the RS Macdonald Charitable Trust (St Andrews Seedcorn Fund for Neurological Research). M.S. acknowledges funding from the European Commission (Marie Skłodowska-Curie Individual Fellowship, 659213) and the Royal Society (Dorothy Hodgkin Fellowship, DH160102; Research Grant, RGF\R1\180070; Enhancement Award, RGF\EA\180051).

Author information

Author notes

  1. Vinh San Dinh

    Present address: Graduate Program in Applied Physics, Northwestern University, Evanston, Illinois, USA

Authors and Affiliations

  1. Centre of Biophotonics, School of Physics and Astronomy, University of St Andrews, St Andrews, UK

    Vera M. Titze, Vinh San Dinh, Marcel Schubert & Malte C. Gather

  2. Humboldt Centre for Nano- and Biophotonics, University of Cologne, Cologne, Germany

    Vera M. Titze, Soraya Caixeiro, Matthias König, Nachiket Pathak, Marcel Schubert & Malte C. Gather

  3. Department of Cell Biology of the Skin, University Hospital Cologne, University of Cologne, Cologne, Germany

    Matthias Rübsam & Carien M. Niessen

  4. Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Disease (CECAD), University of Cologne, Cologne, Germany

    Matthias Rübsam, Carien M. Niessen & Malte C. Gather

  5. FACS & Imaging Core Facility, Max Planck Institute for Biology of Ageing, Cologne, Germany

    Anna-Lena Schumacher, Maximilian Germer & Christian Kukat

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Contributions

V.M.T., M.S. and M.C.G. designed and planned the microscope. V.M.T. and V.S.D. constructed and automated the microscope. S.C., M.K., N.P. and M.S. prepared laser particles and developed the cell uptake assay. M.K., A.-L.S., M.G. and C.K. carried out cell and laser particle sorting experiments. C.M.N. and M.C.G. conceptualized the cell-tracking experiment, which was conducted by V.M.T., S.C. and M.R. V.M.T. and S.C. performed the high-throughput sensing experiment. V.M.T., S.C. and M.S. contributed to the analysis software. M.S. and M.C.G. supervised the project. V.M.T., M.S. and M.C.G. wrote the manuscript, with input from all authors.

Corresponding authors

Correspondence to Vera M. Titze, Marcel Schubert or Malte C. Gather.

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Nature Protocols thanks Paul Dannenberg and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Schubert, M. et al. Sci. Rep. 7, 40877 (2017): https://doi.org/10.1038/srep40877

Schubert, M. et al. Nat. Photonics 14, 452–458 (2020): https://doi.org/10.1038/s41566-020-0631-z

Titze, V. M. et al. ACS Photonics 9, 952–960 (2022): https://doi.org/10.1021/acsphotonics.1c01807

Supplementary information

Supplementary Information

Supplementary Notes 1–3, Figs. 1–15 and Tables 1–3

Reporting Summary

Supplementary Video

Dynamic refractive index sensing of glucose diffusion. x-y maximum intensity projection (MIP) (left) and x-z MIP (center) of the hyperspectral confocal z-stack, color-coded by fitted refractive index, with exemplary raw lasing spectra and calculated external refractive index changes (right). All panels are synchronized.

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Titze, V.M., Caixeiro, S., Dinh, V.S. et al. Hyperspectral confocal imaging for high-throughput readout and analysis of bio-integrated microlasers. Nat Protoc 19, 928–959 (2024). https://doi.org/10.1038/s41596-023-00924-6

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