Resolution in Light Microscopy

Abstract The resolution in conventional light microscopy was defined by Abbe as: Wavelength (λ) and Numerical Aperture (NA) have a direct influence while the factor (c) is dependent upon the geometry of the illumination and observation beam paths. Some typical examples are: High illumination apertures in darkfield or oblique illumination or specific ratios between illumination and observation aperture can substantially improve resolution. Various shapes of the illumination aperture, annular, point, centered or off axis at different azimuths directly influence numerical aperture and coherence and with this resolution, contrast and depth. In mirror objectives low apertures become vignetted, the resolution is enhanced at the expense of contrast. In studies of Hopkins and Barham the factor “c” can range from 0.5 to 0.8. For the practically attainable condition, where illumination and observation aperture are identical, “c” becomes 0.61 and we have partial coherence. Exceeding the observation aperture on the illumination side reduces “c” further but diffraction significantly reduces contrast.