Research Papers

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

[+] Author Affiliations
Ileana Micu, Craig Brideau, Li Lu, Peter K. Stys

University of Calgary, Cumming School of Medicine, Department of Clinical Neurosciences, Hotchkiss Brain Institute, Calgary, Alberta, Canada

Neurophoton. 4(2), 025002 (Jun 05, 2017). doi:10.1117/1.NPh.4.2.025002
History: Received March 8, 2017; Accepted May 15, 2017
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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 Ca2+ 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 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 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.

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© 2017 Society of Photo-Optical Instrumentation Engineers

Citation

Ileana Micu ; Craig Brideau ; Li Lu and Peter K. Stys
"Effects of laser polarization on responses of the fluorescent Ca2+ indicator X-Rhod-1 in neurons and myelin", Neurophoton. 4(2), 025002 (Jun 05, 2017). ; http://dx.doi.org/10.1117/1.NPh.4.2.025002


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