In situ, back-focal-plane-based determination of the numerical apertures in optical microscopes

Area of Science:

• Optical microscopy
• Metrology
• Diffraction optics

Background:

• Accurate numerical aperture (NA) determination is crucial for optical microscopy performance.
• Conventional methods for NA measurement often rely on external setups, which may not reflect the actual performance within the microscope system.
• System-intrinsic apertures can alter objective and condenser NAs, necessitating in situ measurement techniques.

Purpose of the Study:

• To develop and validate an in situ technique for determining the numerical apertures (NAs) of optical microscopes.
• To extend the method for simultaneous measurement of both imaging and illumination NAs.
• To improve the precision of NA determination compared to manufacturer specifications.

Main Methods:

• Utilized calibrated diffraction gratings for in situ NA determination within the microscope.
• Employed the technique to measure imaging NA for objectives with nominal NAs of 0.55 and 0.9.
• Assessed illumination NA for circular aperture diaphragms ranging from 10 µm to 500 µm in diameter.

Main Results:

• All determined NA values closely matched their nominal values within experimental uncertainties.
• The in situ method achieved a reduction in uncertainty by approximately one order of magnitude compared to manufacturer specifications.
• Successfully validated the technique across multiple microscope objectives and illumination aperture sizes.

Conclusions:

• The developed in situ method provides a precise and reliable approach for measuring optical microscope NAs.
• This technique offers significant advantages in accuracy and reduced uncertainty for both imaging and illumination NAs.
• The findings contribute to enhanced performance verification and calibration of optical microscopy systems.