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

Autor: Titze VM; Centre of Biophotonics, School of Physics and Astronomy, University of St Andrews, St Andrews, UK. vmt1@st-andrews.ac.uk.; Humboldt Centre for Nano- and Biophotonics, University of Cologne, Cologne, Germany. vmt1@st-andrews.ac.uk., Caixeiro S; Humboldt Centre for Nano- and Biophotonics, University of Cologne, Cologne, Germany., Dinh VS; Centre of Biophotonics, School of Physics and Astronomy, University of St Andrews, St Andrews, UK.; Graduate Program in Applied Physics, Northwestern University, Evanston, Illinois, USA., König M; Humboldt Centre for Nano- and Biophotonics, University of Cologne, Cologne, Germany., Rübsam M; Department of Cell Biology of the Skin, University Hospital Cologne, University of Cologne, Cologne, Germany.; Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Disease (CECAD), University of Cologne, Cologne, Germany., Pathak N; Humboldt Centre for Nano- and Biophotonics, University of Cologne, Cologne, Germany., Schumacher AL; FACS & Imaging Core Facility, Max Planck Institute for Biology of Ageing, Cologne, Germany., Germer M; FACS & Imaging Core Facility, Max Planck Institute for Biology of Ageing, Cologne, Germany., Kukat C; FACS & Imaging Core Facility, Max Planck Institute for Biology of Ageing, Cologne, Germany., Niessen CM; Department of Cell Biology of the Skin, University Hospital Cologne, University of Cologne, Cologne, Germany.; Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Disease (CECAD), University of Cologne, Cologne, Germany., Schubert M; Centre of Biophotonics, School of Physics and Astronomy, University of St Andrews, St Andrews, UK. marcel.schubert@uni-koeln.de.; Humboldt Centre for Nano- and Biophotonics, University of Cologne, Cologne, Germany. marcel.schubert@uni-koeln.de., Gather MC; Centre of Biophotonics, School of Physics and Astronomy, University of St Andrews, St Andrews, UK. malte.gather@uni-koeln.de.; Humboldt Centre for Nano- and Biophotonics, University of Cologne, Cologne, Germany. malte.gather@uni-koeln.de.; Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Disease (CECAD), University of Cologne, Cologne, Germany. malte.gather@uni-koeln.de.
Jazyk: angličtina
Zdroj: Nature protocols [Nat Protoc] 2024 Mar; Vol. 19 (3), pp. 928-959. Date of Electronic Publication: 2024 Jan 18.
DOI: 10.1038/s41596-023-00924-6
Abstrakt: 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.
(© 2024. Springer Nature Limited.)
Databáze: MEDLINE
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