4f Koehler Transmitted Illumination Condenser for Teaching and Low-Cost Microscopic Imaging

Abstract

Transmitted light imaging is an important tool in biophysics for applications that include sample analysis, recording samples whose viability is compromised by high levels of illumination (e.g., live cell tracking), and finding regions of interest in a sample. Koehler transillumination is a powerful illumination method used in commercial microscopes; yet commercial Koehler condensers are expensive, are difficult to integrate into tabletop systems, and make learning the concepts of Koehler illumination difficult because of their closed-box nature. Here, we show a protocol to build a simple 4f Koehler illumination system that offers advantages with respect to commercial condensers in terms of simplicity, cost, and compatibility with tabletop systems, such as open-source light sheet fluorescence microscopes. We include step-by-step instructions that can be followed by advanced undergraduate or graduate students without experience in optics on how to align and assemble the illuminator, along with a list of the necessary parts for assembly. We also include supplemental material that describes 4 supporting educational activities students can conduct with the apparatus and helps in the understanding of key concepts relevant to Koehler illumination and optics. The performance of the system is comparable to that of commercial condensers and significantly better, in terms of illumination homogeneity and depth of field (optical sections are possible), than that of LED flashlights, such as those found in low-cost diagnostic devices and tabletop systems.