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10 July 2003 Second harmonic generation imaging of endogenous structural proteins
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We find that several key endogenous structural proteins including collagen, acto-myosin, and tubulin give rise to intense second harmonic generation (SHG) and that these structures can be imaged in intact tissues on a laser-scanning microscope. Because SHG is a non-resonant process, this modality suffers little inherent photobleaching or toxicity. In this study we demonstrate the clarity of SHG optical sectioning within unfixed, unstained thick specimens, including fish scales, C. elegans, and mouse muscle, where penetration into tissue upwards of 600 microns was achieved. The simultaneous use of SHG and two-photon excited GFP fluorescence allows for the inference of the molecular isoform that gives rise to SHG from the myofilament lattice in C. elegans. The physical origin of SHG within these tissues is addressed and is attributed to the laser interaction with dipolar protein structures. SHG polarization anisotropy is also used to determine the extent of dipolar order and radial symmetry in the helical structures. Comparisons are drawn between SHG and other forms of microscopy including polarization and fluorescence microscopy, highlighting the advantages and disadvantages.
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Paul J. Campagnola, William H. Mohler, Sergey Plotnikov, and Andrew C. Millard "Second harmonic generation imaging of endogenous structural proteins", Proc. SPIE 4963, Multiphoton Microscopy in the Biomedical Sciences III, (10 July 2003);

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