SPECTROSCOPY, IMAGING, OTHER TECHNIQUES |
Second Harmonic and Sum Frequency Generation Imaging of
Fibrous Astroglial Filaments in ex vivo Spinal Tissues
Yan Fu 1, Haifeng Wang 1, Riyi Shi 1 and Ji-Xin Cheng 1*
1 Purdue University
* To whom correspondence should be addressed. E-mail: jcheng{at}purdue.edu.
Submitted on May 14, 2006
Revised on July 5, 2006
Accepted on 16 January 2007
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Abstract |
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Sum frequency generation (SFG) and second harmonic generation (SHG) are observed from helical fibrils in spinal cord white matter isolated from guinea pigs. By combining SFG with coherent anti-Stokes Raman scattering (CARS) microscopy which allows visualization of myelinated axons, these fibers are found to be distributed near the surface of spinal cord, between adjacent axons, and along the blood vessels. Using 20-µm thick tissue slices, the ratio of forward to backward SHG signal from large bundles is found much larger than that from small single fibrils, indicating the 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 conclude that the main SHG signal directly originates from the fibrils but not from surface SHG effects. Further polarization analysis of the SHG signal shows that the symmetry property of the fibril can be well described with a cylindrical model. Colocalization of the SHG signal with the two-photon excitation fluorescence (TPEF) from the immunostaining of glial fibrillary acidic protein demonstrates that SHG arises from astroglial filaments. This assignment is further supported by colocalization of the SHG contrast with TPEF signals from astrocyte processes labeled by Ca2+ indicator and SR101. The current work shows that combination of three nonlinear optical imaging techniques --- CARS, TPEF, and SHG (SFG) microscopy, allows simultaneous visualization of different structures in a complex biological system.
Key Words:
GFAP, SFG, SHG, astrocyte, intermediate filament, multiphoton microscopy
