Automated calibration and control for polarization-resolved second harmonic generation on commercial microscopes

PLoS One. 2018 Apr 10;13(4):e0195027. doi: 10.1371/journal.pone.0195027. eCollection 2018.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Automation
  • Calibration
  • Equipment Design
  • Least-Squares Analysis
  • Linear Models
  • Microscopy, Confocal / methods
  • Microscopy, Polarization / instrumentation*
  • Microscopy, Polarization / methods*
  • Optics and Photonics
  • Reproducibility of Results
  • Second Harmonic Generation Microscopy / instrumentation*
  • Second Harmonic Generation Microscopy / methods*
  • Software

Grants and funding

Our research was funded by the Norwegian University of Science and Technology (NTNU). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.