Universal Variable Brightfield–Darkfield Contrast: A Variant Technique for Improved Imaging of Problematic Specimens in Light Microscopy

Abstract

Several problematic specimens, especially when composed of a complex three-dimensional architecture or very high or ultralow ranges in regional thickness and density, can be observed in improved clarity and precision when universal variable brightfield-darkfield contrast (UVBDC) is used. In this method, two different partial images are optically superimposed and interfere with each other, contributing to complementary visual information: a brightfield and a darkfield image. These images can be generated with concentric-peripheral, paraxial, or axial illuminating light. In all variants, variable transitions between bright- and darkfield are achievable. By use of a pancratic condenser (zoom system), the illuminating light can be universally adjusted and optimally adapted to each type of specimen and each type of objective (glass and mirror lenses). The concentric-peripheral variant is preferably carried out with normal glass lenses, the axial variant with mirror lenses. Glass lenses can also be used for UVBDC based on axial or paraxial light when combined with a special contrast tube, which is described in detail. Which technical variant of UVBDC might lead to the best result may be determined by the particular properties of the specimen, but all techniques described promise significant improvements in image quality and visual information.