Abstract
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.
Highlights
Polarization-resolved second harmonic generation (P-SHG) is based on the dependency of the second harmonic generation (SHG) on the polarization of the excitation beam
The purpose of this study is to present a fully automated system for P-SHG analysis designed for commercial microscopes, with easy methods for calibration and assessment of polarization accuracy
This microscope was equipped with a Ti:Sapphire laser (Chameleon Vision-S, Coherent), which was tuned to a wavelength of 890 nm for second harmonic generation
Summary
Polarization-resolved second harmonic generation (P-SHG) is based on the dependency of the second harmonic generation (SHG) on the polarization of the excitation beam. The relation between the incoming electric field and the generated second harmonic field is governed by the second order susceptibility tensor This tensor has molecular specificity, and depends on the orientation of the molecule compared to the incoming electric field. P-SHG can be employed to distinguish between collagen types [2] and provide structural information, such as in-plane molecular orientation [3] and helical angles [4, 5], all at pixel level resolution [6].
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