Enhancing optical microscopy illumination to enable quantitative imaging

There has been an increasing push to derive quantitative measurements using optical microscopes. While several aspects of microscopy have been identified to enhance quantitative imaging, non-uniform angular illumination asymmetry (ANILAS) across the field-of-view is an important factor that has been largely overlooked. Non-uniform ANILAS results in loss of imaging precision and can lead to, for example, less reliability in medical diagnoses. We use ANILAS maps to demonstrate that objective lens design, illumination wavelength and location of the aperture diaphragm are significant factors that contribute to illumination aberrations. To extract the best performance from an optical microscope, the combination of all these factors must be optimized for each objective lens. This requires the capability to optimally align the aperture diaphragm in the axial direction. Such optimization enhances the quantitative imaging accuracy of optical microscopes and can benefit applications in important areas such as biotechnology, optical metrology, and nanotechnology.