Phase-contrast versus off-axis illumination: is a more complex microscope always more powerful?

Abstract

In this article, a practical demonstration suitable for any biology college classroom is presented. With the examples of a complex biological specimen (slug's radula) and a simple reference specimen (electron microscopical grid imprint in gelatin), both of which can be easily prepared, the capabilities of two imaging modes commonly used in optical microscopy are demonstrated. The results obtained under phase contrast (a rather sophisticated method, 1953 Nobel Prize to Zernike) and off-axis illumination (a very simple method) are compared. The off-axis illumination setup is capable of delivering noticeably better microscopic images of these two particular specimens, yet it can be easily assembled in a laboratory classroom. The outcome of such a demonstration is expected to be the realization on the part of the students that one needs to carefully choose the apparatus to address a given biological problem, with the “bottom line” being that a more complex one may not necessarily yield better results. An attempt to explain this “paradox” is presented, in the particular case presented here, partly from the physiology of vision perspective (the shape-from-shading problem). The overall aim of the present article is to induce in students critical thinking about the capabilities of a laboratory equipment in general and about data interpretation.