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.
Myelination, i.e. the wrapping of axons in multiple layers of lipid-rich membrane, is a unique phenomenon in the
nervous systems of both vertebrates and invertebrates, that greatly increases the speed and efficiency of signal
transmission. In turn, disruption of axo-myelinic integrity underlies disability in numerous clinical disorders. The
dependence of myelin physiology on nanometric organization of its lamellae makes it difficult to accurately study this
structure in the living state. We expected that fluorescent probes might become highly oriented when partitioned into the
myelin sheath, and in turn, this anisotropy could be interrogated by controlling the polarization state of the exciting laser
field used for 2-photon excited fluorescence (TPEF). Live ex vivo myelinated rodent axons were labeled with a series of
lipohilic and hydrophilic fluorescenct probes, and TPEF images acquired while laser polarization was varied at the
sample over a broad range of ellipticities and orientations of the major angle [see Brideau, Micu & Stys, abstract this
meeting]. We found that most probes exhibited strong dependence on both the major angle of polarization, and perhaps
more surprisingly, on ellipticity as well. Lipophilic vs. hydrophilic probes exhibited distinctly different behavior. We
propose that polarization-dependent TPEF microscopy represents a powerful tool for probing the nanostructural
architecture of both myelin and axonal cytoskeleton in a domain far below the resolution limit of visible light
microscopy. By selecting probes with different sizes and physicochemical properties, distinct aspects of cellular
nanoarchitecture can be accurately interrogated in real-time in living tissue.