Oblique illumination in microscopy: A quantitative evaluation

Abstract

Many biological objects are barely distinguished with the brightfield microscope because they appear transparent, translucent and colourless. One simple way to make such specimens visible without compromising contrast and resolution is by controlling the amount and the directionality of the illumination light. Oblique illumination is an old technique described by many scientists and microscopists that however has been largely neglected in favour of other alternative methods. Oblique lighting (OL) is created by illuminating the sample by only a portion of the light coming from the condenser. If properly used it can improve the resolution and contrast of transparent specimens such as diatoms. In this paper a quantitative evaluation of OL in brigthfield microscopy is presented. Several feature descriptors were selected for characterising contrast and sharpness showing that in general OL provides better performance for distinguishing minute details compared to other lighting modalities. Oblique lighting is capable to produce directionally shadowed differential contrast images allowing to observe phase details in a similar way to differential contrast images (DIC) but at lower cost. The main advantage of OL is that the resolution of the light microscope can be increased by effectively doubling the angular aperture. OL appears as a cost-effective technique both for the amateur and professional scientist that can be used as a replacement of DIC or phase contrast when resources are scarce.

Keywords: Contrast; Feature descriptors; Oblique illumination; Resolution.