Experimental validation of numerical point spread function calculation including aberration estimation.

Area of Science:

• Optical microscopy
• Image reconstruction
• Computational optics

Background:

• Accurate image reconstruction in fluorescence microscopy relies on precise knowledge of the optical system's impulse response, known as the point spread function (PSF).
• Aberrations significantly impact PSF accuracy and, consequently, image quality.

Purpose of the Study:

• To experimentally validate numerical methods for calculating realistic vector Fourier-based point spread functions (PSFs).
• To assess the accuracy and performance of these validated methods in fluorescence microscopy image reconstruction.

Main Methods:

• Development of a MATLAB toolbox for calculating vector Fourier-based PSFs, incorporating optical aberrations.
• Experimental validation of the numerical PSF calculation methods using fluorescence microscopy.
• Quantitative assessment of simulation-experiment agreement using normalized cross-correlation.

Main Results:

• Simulated PSFs demonstrated high accuracy, fitting experimental data with a normalized cross-correlation above 0.97 under various conditions.
• The validated methods enable significant improvements in image contrast, reaching up to 95% relative contrast.
• Successful experimental validation confirms the reliability of the numerical PSF calculation approach.

Conclusions:

• The developed numerical methods for PSF calculation are experimentally validated and highly accurate for fluorescence microscopy.
• These validated methods significantly enhance image reconstruction fidelity and contrast.
• The approach provides a robust tool for optical system characterization and improved imaging performance.