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Second harmonic generation (SHG) is a widely used nonlinear optical imaging technique that reveals collagen orientation in biological tissues. However, the orientation of the collagen fibers is not spatially resolved. In addition, collagen types and orientations vary from one tissue to another. Sum frequency generation (SFG) spectroscopy has proven to be a powerful nonlinear optical technique that enables simultaneous probing of proteins and lipids in tissue. However, the ability to image specific proteins or lipids while using SFG spectroscopy has been limited. In this study, we investigate the use of a quasi-simultaneous SHG/SFG imaging technique based on the spectral domain of the tunable laser scanning microscope for simultaneous mapping of collagen fibers and proteins in biological tissue. This technique improves upon existing methods of simultaneous SHG/SFG microscopy by combining the strengths of both of these techniques. In the analysis of these two microscopy techniques in biological samples, the quasi-simultaneous method was found to improve significantly the visualization of collagen fibers compared to standard SHG microscopy and SFG microscopy. Furthermore, the quasi-simultaneous method was shown to be able to simultaneously imaging the spatial distribution of proteins. We show the ability to use this technique to visualize the spatial distribution of collagen in the dermis and of specific proteins in the skin and muscle of rats. To our knowledge, this is the first demonstration of simultaneous SHG and SFG imaging in biological tissue.The Man Who Never Was
In the early days of the army, Henry Ford was a first sergeant, one of the rank-and-file in the 15th