Second-harmonic generation (SHG) is a coherent nonlinear optical process occurring in non-centrosymmetric molecules, including microtubule (MT). MT is a cytoskeleton playing many important roles in a variety of cellular processes depending on the cell type, and the conformation is crucial for the function. Here we present the use of SHG process to probe MT cytoskeleton in living neuronal tissue. Polarization-resolved SHG (p-SHG) imaging and the second-order tensor analysis were performed on the retinal nerve fibers in order to probe the structure of MTs in axon. The polar anisotropy of tubulins was determined at the molecular level. The effect of MT-stabilizing drug Taxol was also examined and the induced changes were not detectable by p-SHG. Our results demonstrate SHG as a novel optical method to measure conformational changes of MTs in the native cellular context. The technique could be employed in conjunction with existing atomic-resolution methods of structural biology to improve our understanding of cytoskeleton dynamics in vivo.
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