Department of Physics, Norwegian University of Science and Technology, Trondheim, Norway.
PLoS One. 2018 Apr 10;13(4):e0195027. doi: 10.1371/journal.pone.0195027. eCollection 2018.
Polarization-resolved second harmonic generation (P-SHG) microscopy has evolved as a promising technique to reveal subresolution information about the structure and orientation of ordered biological macromolecules. To extend the adoption of the technique, it should be easily integrated onto commercial laser scanning microscopes. Furthermore, procedures for easy calibration and assessment of measurement accuracy are essential, and measurements should be fully automated to allow for analysis of large quantities of samples. In this paper we present a setup for P-SHG which is readily incorporated on commercial multiphoton microscopes. The entire system is completely automated which allows for rapid calibration through the freely available software and for automated imaging for different polarization measurements, including linear and circular polarization of the excitation beam. The results show that calibration settings are highly system dependent. We also show that the accuracy of the polarization control is easily quantified and that it varies between systems. The accuracy can be tuned by iterative alignment of optics or a more fine-grained calibration procedure. Images of real samples show that the red accuracy of the results is easily visualized with the automated setup. Through this system we believe that P-SHG could develop a wider adoption in biomedical applications.
偏振分辨二次谐波产生（P-SHG）显微镜已经发展成为一种很有前途的技术，可以揭示关于有序生物大分子的结构和取向的亚分辨率信息。为了推广该技术，它应该很容易集成到商业激光扫描显微镜上。此外，易于校准和评估测量精度的程序是必不可少的，并且测量应该完全自动化，以允许对大量样品进行分析。在本文中，我们提出了一种易于集成到商业多光子显微镜上的 P-SHG 装置。整个系统完全自动化，可通过免费提供的软件进行快速校准，并可进行不同偏振测量的自动化成像，包括激发光束的线性和圆偏振。结果表明，校准设置高度依赖于系统。我们还表明，偏振控制的精度很容易量化，并且在系统之间有所不同。可以通过迭代对准光学器件或更细粒度的校准程序来调整精度。真实样品的图像表明，自动化设置可以轻松可视化结果的红色精度。通过该系统，我们相信 P-SHG 在生物医学应用中可能会得到更广泛的应用。
