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Hyperspectral light sheet microscopy

Author

Listed:
  • Wiebke Jahr

    (Max Planck Institute of Molecular Cell Biology and Genetics
    DIGS-BB, Technische Universität Dresden)

  • Benjamin Schmid

    (Max Planck Institute of Molecular Cell Biology and Genetics)

  • Christopher Schmied

    (Max Planck Institute of Molecular Cell Biology and Genetics)

  • Florian O. Fahrbach

    (Max Planck Institute of Molecular Cell Biology and Genetics)

  • Jan Huisken

    (Max Planck Institute of Molecular Cell Biology and Genetics)

Abstract

To study the development and interactions of cells and tissues, multiple fluorescent markers need to be imaged efficiently in a single living organism. Instead of acquiring individual colours sequentially with filters, we created a platform based on line-scanning light sheet microscopy to record the entire spectrum for each pixel in a three-dimensional volume. We evaluated data sets with varying spectral sampling and determined the optimal channel width to be around 5 nm. With the help of these data sets, we show that our setup outperforms filter-based approaches with regard to image quality and discrimination of fluorophores. By spectral unmixing we resolved overlapping fluorophores with up to nanometre resolution and removed autofluorescence in zebrafish and fruit fly embryos.

Suggested Citation

  • Wiebke Jahr & Benjamin Schmid & Christopher Schmied & Florian O. Fahrbach & Jan Huisken, 2015. "Hyperspectral light sheet microscopy," Nature Communications, Nature, vol. 6(1), pages 1-7, November.
    • Handle: RePEc:nat:natcom:v:6:y:2015:i:1:d:10.1038_ncomms8990
    DOI: 10.1038/ncomms8990
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    Cited by:

    1. Gareth O. S. Williams & Elvira Williams & Neil Finlayson & Ahmet T. Erdogan & Qiang Wang & Susan Fernandes & Ahsan R. Akram & Kev Dhaliwal & Robert K. Henderson & John M. Girkin & Mark Bradley, 2021. "Full spectrum fluorescence lifetime imaging with 0.5 nm spectral and 50 ps temporal resolution," Nature Communications, Nature, vol. 12(1), pages 1-9, December.
    2. Xiaomeng Han & Xiaotang Lu & Peter H. Li & Shuohong Wang & Richard Schalek & Yaron Meirovitch & Zudi Lin & Jason Adhinarta & Karl D. Murray & Leah M. MacNiven & Daniel R. Berger & Yuelong Wu & Tao Fan, 2024. "Multiplexed volumetric CLEM enabled by scFvs provides insights into the cytology of cerebellar cortex," Nature Communications, Nature, vol. 15(1), pages 1-18, December.
    3. Jonghee Yoon & Alexandru Grigoroiu & Sarah E Bohndiek, 2020. "A background correction method to compensate illumination variation in hyperspectral imaging," PLOS ONE, Public Library of Science, vol. 15(3), pages 1-21, March.

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