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

Area of Science:

• Biophysics
• Microscopy
• Biomedical Imaging

Background:

• Polarization-resolved second harmonic generation (P-SHG) microscopy offers subresolution insights into biological macromolecular structure and orientation.
• Wider adoption requires seamless integration with commercial laser scanning microscopes, alongside automated calibration and accuracy assessment.

Purpose of the Study:

• To present an automated P-SHG setup easily integrated onto commercial multiphoton microscopes.
• To enable rapid calibration and automated imaging for various polarization states.
• To quantify measurement accuracy and system-dependent calibration settings.

Main Methods:

• Developed a P-SHG setup for commercial multiphoton microscopes.
• Implemented a fully automated system for calibration and imaging.
• Included automated linear and circular polarization measurements of the excitation beam.
• Utilized freely available software for calibration.

Main Results:

• Demonstrated easy integration of the P-SHG setup onto commercial microscopes.
• Showcased automated calibration and imaging capabilities.
• Highlighted that calibration settings are highly system-dependent.
• Quantified polarization control accuracy, showing it varies between systems.
• Visualized result accuracy in real sample images.

Conclusions:

• The automated P-SHG system facilitates rapid calibration and accurate imaging.
• System-dependent calibration and polarization accuracy can be quantified and improved.
• This development is expected to increase the adoption of P-SHG in biomedical applications.