Metrology of Multiphoton Microscopes Using Second Harmonic Generation Nanoprobes

Area of Science:

• Optics and Photonics
• Biomedical Imaging
• Materials Science

Background:

• Multiphoton microscopy is increasingly using the near-infrared spectrum, necessitating new performance standards.
• Current methods for evaluating microscope performance are often complex or limited in wavelength range.

Purpose of the Study:

• To introduce a novel, efficient method for characterizing multiphoton microscope imaging properties across a wide excitation wavelength range.
• To establish a standardized approach for quantifying and comparing multiphoton microscope performance.

Main Methods:

• Utilized second harmonic generation (SHG) nanoprobes, specifically KTiOPO4 nanocrystals, for characterization.
• Mapped spatial resolution, field curvature, and chromatic aberrations using SHG nanoprobes from 850-1100 nm.
• Applied the method to assess and compare performance of various multiphoton microscope objectives.

Main Results:

• Achieved precise measurements of imaging properties below the diffraction limit.
• Demonstrated unique advantages of SHG nanoprobes over traditional fluorescence-based methods.
• Successfully extended the SHG nanoprobe approach to post-acquisition chromatic aberration correction in multicolor imaging.

Conclusions:

• Second harmonic generation (SHG) nanoprobes provide a uniquely suitable and efficient method for standardizing multiphoton microscope metrology.
• This technique facilitates accurate performance assessment and comparison across diverse excitation wavelengths.
• The method offers a pathway for improving multicolor multiphoton imaging through aberration correction.