5 June 2017 Effects of laser polarization on responses of the fluorescent Ca2+ indicator X-Rhod-1 in neurons and myelin
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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.
© 2017 Society of Photo-Optical Instrumentation Engineers (SPIE)
Ileana Micu, Craig Brideau, Li Lu, Peter K. Stys, "Effects of laser polarization on responses of the fluorescent Ca2+ indicator X-Rhod-1 in neurons and myelin," Neurophotonics 4(2), 025002 (5 June 2017). https://doi.org/10.1117/1.NPh.4.2.025002 . Submission: Received: 8 March 2017; Accepted: 15 May 2017
Received: 8 March 2017; Accepted: 15 May 2017; Published: 5 June 2017
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