Diffracted Light Contrast: Improving the Resolution of a Basic Light Microscope by an Order of Magnitude

Introduction The discovery that the diffracted light from a convex edge can be used to form a very high-quality, shadowcast image on any light microscope has led to a device and method, diffracted-light contrast (DLC), which will allow shadowcast imaging to be routinely performed on student/laboratory microscopes (Piekos, 1999, 2003). The surface lattice of Surirella gema was easily resolved, and micrographs comparing the subcellular details of buccal epithelial cells viewed with DLC vs. Nomarski DIC showed that, on the microscopes used, DLC was superior in both the detail it rendered and depth of field. Although the images presented revealed DLC to be an excellent technique, the full capabilities of the technique were not known at the time. DLC differs from the centuries-old technique of oblique illumination in that it uses an edge plate, inserted at the level of the field stop, as a source of partially coherent, diffracted light while oblique illumination uses some sort of beam stop to create an off-axis beam of illumination (Wright, 1913; Ott, 1924; Diggins, 1940). The shape of the edge has a great influence on the quality of the DLC image with a convex edge generating the highest contrast, most threedimensional image (Piekos, 1999). This observation indicates that the edge itself is actively affecting the beam, via diffraction, and it is this light which is generating the image. The use of diffracted light as a source of illumination is the basis of schlieren imaging—a common technique used to image refractive, phase-altering samples and one which has, in the past, been applied to light microscopes (Saylor, 1935; Axelrod, 1980). A schlieren apparatus generally consists of two components: a slit aperture, used to generate the Fraunhofer diffraction pattern, and a second beam stop, or knife, inserted into a conjugate plane and used to eliminate zero-order light, thereby providing a darkened background. DLC differs from this common arrangement in that it uses a single convex edge in order to produce the diffraction pattern and it does not require a second knife. With DLC, the coherent rays produced by the single edge will not interfere with rays from an opposing edge, as is the case when using a slit aperture. When using a convex edge to generate the diffracted light, each point along the edge serves as a point source of diffracted light. Consequently, the sample is actually being illuminated by an infinite number of separate, partially coherent rays, each approaching the specimen from a slightly different direction and interacting with the sample (phase shifting) in a slightly different way (Piekos, 2003). Interference of these rays only occurs at the image plane and will result in a subtle pattern of interference, which, in turn, gives rise to the delicate, graded pattern of shadowing and a quasi-three dimensional appearance. Consequently, DLC appears to be a form of multiple-beam interference–a technique widely used in interferometry and known to give much more precise interference patterns than is achievable with standard two-beam interference optics. Materials and Methods All images contained in this report were made with a standard Leitz Diaplan microscope (ca. 1985) using either a 40/0.70 or 100/1.32 PL Fluotar lens. The microscope was set to its most basic brightfield configuration, i.e., polarizers, DIC prisms and analyzers were removed and both the field stop and condenser iris diaphragms were left wide open. The microscope was fitted with a small diffracting edge plate, located at the level of the field stop, and this was used as previously described (Piekos, 1999). The edge plate is a circular, opaque plate (~40 mm diameter) that is inserted into the optical path until a convex edge is visible within the field of view. The condenser is then defocused from its usual Koehler position so that a broad band of diffracted light (the defocused edge of the edge plate) is cast onto the specimen plane. Excellent