Effects of laser polarization on responses of the fluorescent Ca2+ indicator X-Rhod-1 in neurons and myelin

Ileana Micu, Craig Brideau, Lu Li, Peter K. Stys

    Research output: Contribution to journalArticle

    1 Citation (Scopus)

    Abstract

    Laser-scanning optical microscopes generally do not control the polarization of the exciting laser field. We show that laser polarization and imaging mode (confocal versus two photon) exert a profound influence on the ability to detect Ca 2 + changes in both cultured neurons and living myelin. With two-photon excitation, increasing ellipticity resulted in a ≈ 50 % reduction in resting X-Rhod-1 fluorescence in homogeneous bulk solution, cell cytoplasm, and myelin. In contrast, varying the angle of a linearly polarized laser field only had appreciable effects on dyes that partitioned into myelin in an ordered manner. During injury-induced Ca 2 + increases, larger ellipticities resulted in a significantly greater injury-induced signal increase in neurons, and particularly in myelin. Indeed, the traditional method of measuring Ca 2 + changes using one-photon confocal mode with linearly polarized continuous wave laser illumination produced no appreciable X-Rhod-1 signal increase in ischemic myelin, compared to a robust ≈ 50 % fluorescence increase with two-photon excitation and optimized ellipticity with the identical injury paradigm. This underscores the differences in one- versus two-photon excitation and, in particular, the under-appreciated effects of laser polarization on the behavior of certain Ca 2 + reporters, which may lead to substantial underestimates of the real Ca 2 + fluctuations in various cellular compartments.
    LanguageEnglish
    Article number 28612034
    JournalNeurophotonics
    Volume4
    Issue number2
    DOIs
    Publication statusPublished - 05 Jun 2017

    Fingerprint

    Myelin Sheath
    Photons
    Lasers
    Neurons
    Wounds and Injuries
    Fluorescence
    Lighting
    Cytoplasm
    Coloring Agents

    Keywords

    • Myelin
    • polarization
    • multi-photon microscopy

    Cite this

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    abstract = "Laser-scanning optical microscopes generally do not control the polarization of the exciting laser field. We show that laser polarization and imaging mode (confocal versus two photon) exert a profound influence on the ability to detect Ca 2 + changes in both cultured neurons and living myelin. With two-photon excitation, increasing ellipticity resulted in a ≈ 50 {\%} reduction in resting X-Rhod-1 fluorescence in homogeneous bulk solution, cell cytoplasm, and myelin. In contrast, varying the angle of a linearly polarized laser field only had appreciable effects on dyes that partitioned into myelin in an ordered manner. During injury-induced Ca 2 + increases, larger ellipticities resulted in a significantly greater injury-induced signal increase in neurons, and particularly in myelin. Indeed, the traditional method of measuring Ca 2 + changes using one-photon confocal mode with linearly polarized continuous wave laser illumination produced no appreciable X-Rhod-1 signal increase in ischemic myelin, compared to a robust ≈ 50 {\%} fluorescence increase with two-photon excitation and optimized ellipticity with the identical injury paradigm. This underscores the differences in one- versus two-photon excitation and, in particular, the under-appreciated effects of laser polarization on the behavior of certain Ca 2 + reporters, which may lead to substantial underestimates of the real Ca 2 + fluctuations in various cellular compartments.",
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    Effects of laser polarization on responses of the fluorescent Ca2+ indicator X-Rhod-1 in neurons and myelin. / Micu, Ileana; Brideau, Craig ; Li, Lu; Stys, Peter K.

    In: Neurophotonics, Vol. 4, No. 2, 28612034 , 05.06.2017.

    Research output: Contribution to journalArticle

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