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Second harmonic and sum frequency generation imaging of fibrous astroglial filaments in ex vivo spinal tissues
Yan Fu1, Haifeng Wang, Riyi Shi
1Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana 47907, USA.
Abstract:
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.
Insights
Nonlinear optical microscopy revealed astroglial filaments in guinea pig spinal cord white matter. Sum frequency generation (SFG) and second harmonic generation (SHG) microscopy visualized these structures alongside myelinated axons.
Area of Science:
- Neuroscience
- Biophysics
- Microscopy
Background:
- Spinal cord white matter contains complex structures including myelinated axons and astroglial filaments.
- Nonlinear optical microscopy techniques offer high-resolution imaging of biological tissues.
Purpose of the Study:
- To investigate the origin and distribution of helical fibrils in spinal cord white matter using nonlinear optical microscopy.
- To demonstrate the capability of combining multiple nonlinear optical techniques for simultaneous visualization of diverse cellular components.
Main Methods:
- Sum frequency generation (SFG) and second harmonic generation (SHG) microscopy.
- Coherent anti-Stokes Raman scattering (CARS) microscopy for visualizing myelinated axons.
- Two-photon excitation fluorescence (TPEF) microscopy for immunostaining and labeling.
Main Results:
- SFG and SHG signals were observed from helical fibrils in spinal cord white matter.
- SHG signal originated from astroglial filaments, confirmed by colocalization with glial fibrillary acidic protein and astrocyte markers.
- Phase-matching effects were observed in SHG signal intensity with fibril size.
- A cylindrical model described the symmetry properties of the fibrils.
Conclusions:
- Nonlinear optical microscopy, particularly the combination of SFG, SHG, and CARS, enables simultaneous visualization of different structures in complex biological systems.
- SHG microscopy is a valuable tool for identifying astroglial filaments within the spinal cord white matter.
