Use of a refractive index–coupled diffuser to both generate and measure high‐numerical aperture illumination for light microscopy

While numerical aperture of transillumination at or above 1.25 can be achieved with a substage oiled Abbe condenser, such immersion‐capable condensers can be expensive limiting their use in resource poor settings. Also the measurement of numerical aperture generated by illuminators has received relatively little attention in the literature compared to methods for measuring the numerical aperture of acceptance by objectives. In this article, I show how an inexpensive paper diffuser with refractive‐index coupling to the sample slide can generate illumination of a numerical aperture of over 1.4 at a small fraction of the cost of oiled dioptric condensers of comparable numerical aperture. In addition, I present two ways in which a diffuser may also be used to measure the numerical aperture generated by an illuminator using either a calibrated index‐coupled paper diffuser to implement an interpolative variation of the Horsfall method or a diffuser as a detector screen coupled to a self‐built microscope slide‐based illumination system apertometer. The highest magnifications of light microscopy require immersion objectives that, in turn, require extremely wide‐angle illumination of the specimen to make maximum use of the spatial resolving power of these objectives. However, providing such illumination involves the use of expensive condenser lens arrangements. This article shows how a similar wide angle of illumination can be achieved using a diffuser made of paper that is optically coupled to the specimen slide with an immersion medium. Furthermore, measuring the capabilities of a wide‐angle illuminating device usually involves expensive specialist optical laboratory equipment. This article also shows how such measurements can be made using simple inexpensive self‐built apparatus with the aid of a diffuser. These techniques make the study and use of high‐magnification, high‐resolution light microscopy more accessible to those with limited resources.