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.

Filamented Light (FLight) Biofabrication of Highly Aligned Tissue‐Engineered Constructs

“…The assembly and alignment protocols were described in a previous study. [ 37 ] Light intensity in front of the cuvette was measured after each adjustment of field diameter and/or aperture diameter using a power meter.…”

“…[35,36] Here, microfilaments with varying diameter ranges were fabricated by adjusting the spatial coherence length using a standard Köhler illumination system consisting of a 405 nm light-emitting diode light source. [37] Specifically, this light-induced selforganization depends on spatial coherence of the light source and strength of the photoreactive material's RI nonlinearity. [26] The more spatially coherent the light beam (i.e., the longer the spatial coherence length l c of the light beam), the broader the diameter of the microfilaments.…”

Droplet dynamics affecting the shape of patterns formed spontaneously by transforming UV-curable emulsions

“…This observation system has the same principle of a fluorescence microscope, and it can project patterns and has a long working distance. The illumination system has a Köhler illumination 44 , 45 lens arrangement, and the imaging system has a reduction projection lens configuration 46 . A water-soluble fluorescent dye (eosin Y) was added to the emulsion to enable the differentiation between UV-pattern-exposed and -unexposed areas based on the fluorescence color (Table S2 ).…”

A Simple and Fast Compression‐Based Method to Fabricate Responsive Gold‐pNIPAM Hybrid Materials: From Thin Films to Anisotropic Microgels

“…More details about the implemented optical system can be found in ref. [80] As well as the original photopolymerization setup (Figure S1, Supporting Information), illumination occurs from the bottom to the top for employing the same fabrication procedure described above. Here, the photo-patterned Au-pNIPAM films were cured for 2 min and then thoroughly rinsed with Milli-Q water to get rid of the unreacted resin.…”