Variable Phase Dark-Field Contrast—A Variant Illumination Technique for Improved Visualizations of Transparent Specimens

Abstract

Variable phase dark-field contrast has been developed as an illumination technique in light microscopy, which promises significant improvements and a higher variability in imaging of several transparent specimens. In this method, a phase contrast image is optically superimposed on a dark-field image so that a partial image based on the principal zeroth-order maximum (phase contrast) interferes with an image that is based on the secondary maxima (dark field). The background brightness and character of the resulting image can be continuously modulated from a phase-contrast-dominated to a dark-field-dominated character. The condenser aperture diaphragm can be used for modulations of the image's appearance. Specimens can either be illuminated concentrically or obliquely (eccentrically) when parts of the illuminating light beams are covered and blocked. Moreover, a bright-field-like partial image can be added. In this way, the illumination can be optimally adjusted to the specific properties of the specimen. The techniques described can lead to improved visual information especially in biological specimens consisting of phase structures and additional light-absorbing or -reflecting components. Moreover, the specimen's three-dimensionality can be accentuated with improved clarity because the illuminating light beams associated with phase contrast and dark field run to the specimen at different angles of incidence.

Condenser‐free contrast methods for transmitted‐light microscopy

“…The integration of several allied contrast methods into a single instrument composed of one or more rings complemented with central LED illumination further promises to allow multimodal or combinatorial imaging, several configurations of which have been recently demonstrated but requisite illumination system using conventional imaging is highly specialized and complex (Piper & Piper, , , , , ). Using the simple system detailed here, the intensity and spectral composition of each illuminating component is easily and arbitrarily variable to bring out the best in particular samples.…”

Phase contrast without phase plates and phase rings—optical solutions for improved imaging of phase structures

“…Alternative solutions for improvements of phase contrast imagery can be achieved when phase contrast is combined with complementary illumination techniques. Thus, phase contrast can be combined with darkfield carried out in peripheral or axial light (Piper and Piper, and ) or simultaneously carried out with brightfield (Piper and Piper, ). Such superimpositions of phase contrast and bright‐ or darkfield illumination can only lead to adequate results, when both illuminating light components associated with phase contrast and the respective complementary method are separated from each other so that they run to the specimen at different angles.…”

Chip-Based Cytometry Illuminated by a Blade-Shape Continuous Light for Multispectral Detection

“…Dark-field illumination is generally used for the detection and tracking of small or transparent particles in a solution near the surface [31,32]. The use of dark-field illumination in accomplishing multi-color fluorescence detection on a single microfluidic chip for capillary electrophoresis applications was also successfully demonstrated [33,34].…”