Journal
NEUROPHOTONICS
Volume 4, Issue 2, Pages -Publisher
SPIE-SOC PHOTO-OPTICAL INSTRUMENTATION ENGINEERS
DOI: 10.1117/1.NPh.4.2.025002
Keywords
myelin; neuron; Ca2+ imaging; confocal; two-photon; polarization
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Funding
- MS Society of Canada, Canadian Institutes for Health Research
- Canada Foundation for Innovation and Canada Research Chairs
- AI-HS Scientist Award
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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 Ca2+ changes in both cultured neurons and living myelin. With two-photon excitation, increasing ellipticity resulted in a approximate to 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 Ca2+ increases, larger ellipticities resulted in a significantly greater injury-induced signal increase in neurons, and particularly in myelin. Indeed, the traditional method of measuring Ca2+ 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 approximate to 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 Ca2+ reporters, which may lead to substantial underestimates of the real Ca2+ fluctuations in various cellular compartments. (C) 2017 Society of Photo-Optical Instrumentation Engineers (SPIE)
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