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

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.

Maturation of the Meniscal Collagen Structure Revealed by Polarization-Resolved and Directional Second Harmonic Generation Microscopy

“…A perfect linear polarization is needed for P-SHG, so the polarization was calibrated with the routine developed in Romijn et al . 56 , using a modified version of their MatLab code 57 . This enables to use a varying linear polarization (from 0 to 180°) that stays linear even if the waveplates are placed before the input of the commercial microscope.…”

Label-Free Non-linear Multimodal Optical Microscopy—Basics, Development, and Applications

“…In the past few years, polarization-resolved susceptibility based SHG microscopy has been used for several biomedical applications such as characterization of breast cancer, skin, muscle, and engineered cartilage tissues [135,136,138]. Automated control for the linearly-polarized excitation light to perform susceptibility based SHG microscopy on commercial microscopes is demonstrated recently [139]. Collagen is one of the commonly found structural proteins in ECM in a human body that is modified in structure or content in several disorders, e.g., connective tissue diseases, cardiovascular diseases, autoimmune diseases, ovarian diseases, etc.…”

“…In general, susceptibility based SHG microscopy can quantitatively discriminate several harmonophores (e.g., collagen type I, II, III, muscles etc.) and thus could emerge as a powerful tool for detecting early-stage ECM modification at molecular level with high sensitivity, and can contribute in understanding various associated pathophysiological process [130,133,139,140].…”

Real-Time Polarization-Resolved Imaging of Living Tissues Based on Two-Photon Excitation Spinning-Disk Confocal Microscopy

“…The incident excitation light beam was introduced at the pupil of the water immersion objective lens (Plan Apo IR 60X, numerical aperture: 1.27, working distance: 0.17 mm; Nikon) and focused on multiple points of a specimen. In order to adjust the polarization state of the beam at the position of the objective lens, a half-wave plate and a quarter wave plate were placed in the optical path of the excitation laser light beam [17]. To measure the polarization state of the incident light beam at the specimen, a linear polarizer film was placed just after the objective lens, and angle dependent throughput was measured and plotted in polar coordinates ( Figures 1B-F).…”