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.