Abstract
Polarization-resolved second-harmonic-generation (PR-SHG) microscopy is a powerful tool for investigating collagen fiber orientation quantitatively with low invasiveness. However, the waiting time for the mechanical polarization rotation makes it too sensitive to motion artifacts and hence has hampered its use in various applications in vivo. In the work described in this article, we constructed a motion-artifact-robust, PR-SHG microscope based on rapid polarization switching at every pixel with an electro-optic Pockells cell (PC) in synchronization with step-wise raster scanning of the focus spot and alternate data acquisition of a vertical-polarization-resolved SHG signal and a horizontal-polarization-resolved one. The constructed PC-based PR-SHG microscope enabled us to visualize orientation mapping of dermal collagen fiber in human facial skin in vivo without the influence of motion artifacts. Furthermore, it implied the location and/or age dependence of the collagen fiber orientation in human facial skin. The robustness to motion artifacts in the collagen orientation measurement will expand the application scope of SHG microscopy in dermatology and collagen-related fields.
Highlights
Second-harmonic-generation (SHG) is a nonlinear optical phenomenon resulting from the nonlinear interaction of a high-peak-power optical electric field with a material [1] and has been widely used for wavelength conversion of laser light with a nonlinear optical crystal
Polarization-resolved second-harmonic-generation (PR-SHG) microscopy is a powerful tool for investigating collagen fiber orientation quantitatively with low invasiveness
In the work described in this article, we constructed a motion-artifact-robust, PR-SHG microscope based on rapid polarization switching at every pixel with an electro-optic Pockells cell (PC) in synchronization with step-wise raster scanning of the focus spot and alternate data acquisition of a verticalpolarization-resolved SHG signal and a horizontal-polarization-resolved one
Summary
Second-harmonic-generation (SHG) is a nonlinear optical phenomenon resulting from the nonlinear interaction of a high-peak-power optical electric field with a material [1] and has been widely used for wavelength conversion of laser light with a nonlinear optical crystal. In vivo imaging of these collagen aggregates in tissues is an important biological application of SHG microscopy [5,6,7,8,9,10,11,12] because it is difficult to visualize collagen in vivo using conventional methods, including histological methods. The orientation of collagen fibers is closely related to the structural and functional properties of the tissues. For this reason, there is a considerable need for a reasonable method that can reveal the collagen orientation quantitatively in vivo in biological studies and clinical medicine. There have been no attempts to perform in vivo PR-SHG imaging without the help of anesthesia
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