Second harmonic and sum frequency generation imaging of fibrous astroglial filaments in ex vivo spinal tissues

Sum frequency generation (SFG) and second harmonic generation (SHG) were observed from helical fibrils in spinal cord white matter isolated from guinea pigs. By combining SFG with coherent anti-Stokes Raman scattering microscopy, which allows visualization of myelinated axons, these fibers were found to be distributed near the surface of the spinal cord, between adjacent axons, and along the blood vessels. Using 20-microm-thick tissue slices, the ratio of forward to backward SHG signal from large bundles was found to be much larger than that from small single fibrils, indicating a phase-matching effect in coherent microscopy. Based on the intensity profiles across fibrils and the size dependence of forward and backward signal from the same fibril, we concluded that the main SHG signal directly originates from the fibrils, but not from surface SHG effects. Further polarization analysis of the SHG signal showed that the symmetry property of the fibril could be well described with a cylindrical model. Colocalization of the SHG signal with two-photon excitation fluorescence (TPEF) from the immunostaining of glial fibrillary acidic protein demonstrated that SHG arises from astroglial filaments. This assignment was further supported by colocalization of the SHG contrast with TPEF signals from astrocyte processes labeled by a Ca(2+) indicator and sulforhodamine 101. This work shows that a combination of three nonlinear optical imaging techniques--coherent anti-Stokes Raman scattering, TPEF, and SHG (SFG) microscopy--allows simultaneous visualization of different structures in a complex biological system.