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Fast, multicolor 3-D imaging of brain organoids with a new single-objective two-photon virtual light-sheet microscope

Irina Rakotoson, Brigitte Delhomme, Philippe Djian, Andreas Deeg, Maia Brunstein, Christian Seebacher, Rainer Uhl, Clément Ricard, View ORCID ProfileMartin Oheim
doi: https://doi.org/10.1101/461335
Irina Rakotoson
1CNRS UMR 8118, Brain Physiology Laboratory, Paris F-75006, France
2Federation de Recherche en Neurosciences CNRS FR 3636, Paris F-75006, France
3Faculte de Sciences Fondamentales et Biomedicales, Universite Paris Descartes, PRES Sorbonne Paris Cite, F-75006 Paris, France
4Master programme: BCPP (Biologie cellulaire physiologie et pathologie), specialite Neurosciences
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Brigitte Delhomme
1CNRS UMR 8118, Brain Physiology Laboratory, Paris F-75006, France
2Federation de Recherche en Neurosciences CNRS FR 3636, Paris F-75006, France
3Faculte de Sciences Fondamentales et Biomedicales, Universite Paris Descartes, PRES Sorbonne Paris Cite, F-75006 Paris, France
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Philippe Djian
1CNRS UMR 8118, Brain Physiology Laboratory, Paris F-75006, France
2Federation de Recherche en Neurosciences CNRS FR 3636, Paris F-75006, France
3Faculte de Sciences Fondamentales et Biomedicales, Universite Paris Descartes, PRES Sorbonne Paris Cite, F-75006 Paris, France
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Andreas Deeg
5TILL.id, Am Klopferspitz 19, D-82153 Planegg/Martinsried, Munich, Germany
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Maia Brunstein
1CNRS UMR 8118, Brain Physiology Laboratory, Paris F-75006, France
2Federation de Recherche en Neurosciences CNRS FR 3636, Paris F-75006, France
3Faculte de Sciences Fondamentales et Biomedicales, Universite Paris Descartes, PRES Sorbonne Paris Cite, F-75006 Paris, France
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Christian Seebacher
5TILL.id, Am Klopferspitz 19, D-82153 Planegg/Martinsried, Munich, Germany
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Rainer Uhl
5TILL.id, Am Klopferspitz 19, D-82153 Planegg/Martinsried, Munich, Germany
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Clément Ricard
1CNRS UMR 8118, Brain Physiology Laboratory, Paris F-75006, France
2Federation de Recherche en Neurosciences CNRS FR 3636, Paris F-75006, France
3Faculte de Sciences Fondamentales et Biomedicales, Universite Paris Descartes, PRES Sorbonne Paris Cite, F-75006 Paris, France
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Martin Oheim
1CNRS UMR 8118, Brain Physiology Laboratory, Paris F-75006, France
2Federation de Recherche en Neurosciences CNRS FR 3636, Paris F-75006, France
3Faculte de Sciences Fondamentales et Biomedicales, Universite Paris Descartes, PRES Sorbonne Paris Cite, F-75006 Paris, France
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  • ORCID record for Martin Oheim
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ABSTRACT

Human inducible pluripotent stem cells (hiPSCs) hold a large potential for disease modeling. hiPSC-derived human astrocyte and neuronal cultures permit investigations of neural signaling pathways with subcellular resolution. Combinatorial cultures, and three-dimensional (3-D) embryonic bodies enlarge the scope of investigations to multi-cellular phenomena. A the highest level of complexity, brain organoids that – in many aspects – recapitulate anatomical and functional features of the developing brain permit the study of developmental and morphological aspects of human disease. An ideal microscope for 3-D tissue imaging at these different scales would combine features from both confocal laser-scanning and light-sheet microscopes: a micrometric optical sectioning capacity and sub-micrometric spatial resolution, a large field of view and high frame rate, and a low degree of invasiveness, i.e., ideally, a better photon efficiency than that of a confocal microscope. In the present work, we describe such an instrument that belongs to the class of two-photon (2P) light-sheet microsocpes. Its particularity is that – unlike existing two- or three-lens designs – it is using a single, low-magnification, high-numerical aperture objective for the generation and scanning of a virtual light sheet. The microscope builds on a modified Nipkow-Petran spinning-disk scheme for achieving wide-field excitation. However, unlike the common Yokogawa design that uses a tandem disk, our concept combines micro lenses, dichroic mirrors and detection pinholes on a single disk. This design, advantageous for 2P excitation circumvents problems arising with the tandem disk from the large wavelength-difference between the infrared excitation light and visible fluorescence. 2P fluorescence excited in by the light sheet is collected by the same objective and imaged onto a fast sCMOS camera. We demonstrate three-dimensional imaging of TO-PRO3-stained embryonic bodies and of brain organoids, under control conditions and after rapid (partial) transparisation with triethanolamine and /ormamide (RTF) and compare the performance of our instrument to that of a confocal microscope having a similar numerical aperture. 2P-virtual light-sheet microscopy permits one order of magnitude faster imaging, affords less photobleaching and permits better depth penetration than a confocal microscope with similar spatial resolution.

Footnotes

  • ↵+ co-first authors

  • ↵§ co-last authors

  • ↵* Elements of this work has been published in the Master thesis of I.R. [1]

  • The authors declare not conflict of interest. Rainer Uhl is the founder and owner of TILL.id, Andreas Deeg and Christian Seebacher are employees of TILL.id.

  • The funders of this study had no hands on the outcome or interpretation of the obtained results.

  • List of abbreviations

    2P
    two-photon
    3-D
    three-dimensional
    AOI
    angle of incidence
    BSA
    bovine serum albumin
    CLSM
    confocal laser scanning microscope
    DMEM
    Dulbecco’s modified Eagle medium
    EB
    embryonic body
    FWHM
    full-width at half maximumhi
    PSC
    human inducible pluripotent stem cell
    LP
    long-pass (filter)
    MG
    Magnesium Green
    NA
    numerical aperture
    OASIS
    On-axis 2-photon light-sheet generation in-vivoimaging system
    OCT
    opimal cutting temperature
    PBS
    phosphate-buffered solution
    PEG
    polyethylene glycol
    RI
    refractive index
    ROI
    region of interest
    RTF
    Rapid clearing method based on Triethanolamine and Formamide
    sCMOS
    scientific Complementary Metal Oxide Semiconductor
    SD
    standard deviation
    SPIM
    selective-plane illumination
    TDE
    2,2’-thiodi ethanol
    TO-PRO-3
    a carbocyanine monomer nucleic acid stain with red excitation and far-red fluorescence (642 nm/661 nm) similar to Alexa Fluor 647 or Cy5. It is among the highest-sensitivity probes for nucleic acid detection.
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    Posted November 03, 2018.
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    Fast, multicolor 3-D imaging of brain organoids with a new single-objective two-photon virtual light-sheet microscope
    Irina Rakotoson, Brigitte Delhomme, Philippe Djian, Andreas Deeg, Maia Brunstein, Christian Seebacher, Rainer Uhl, Clément Ricard, Martin Oheim
    bioRxiv 461335; doi: https://doi.org/10.1101/461335
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    Fast, multicolor 3-D imaging of brain organoids with a new single-objective two-photon virtual light-sheet microscope
    Irina Rakotoson, Brigitte Delhomme, Philippe Djian, Andreas Deeg, Maia Brunstein, Christian Seebacher, Rainer Uhl, Clément Ricard, Martin Oheim
    bioRxiv 461335; doi: https://doi.org/10.1101/461335

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