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

Published online by Cambridge University Press:  02 March 2012

Timm Piper  and
Jörg Piper
Timm Piper
Affiliation:
Laboratory for Applied Microscopy Research–Light Microscopy, Marienburgstr. 23, Bullay, RLP-56859, Germany
Jörg Piper*
Affiliation:
Clinic “Meduna”, Department for Internal Medicine, Clara Viebig Road No 4, D-56864 Bad Bertrich, Germany
*
Corresponding author. E-mail: [email protected]
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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.

Keywords

phase contrastdark fieldvariable phase dark-field contrast (VPDC)illuminationfocal depthhaloingbloomingscattering
Type
Techniques and Software Development
Information
Microscopy and Microanalysis , Volume 18 , Issue 2 , April 2012 , pp. 343 - 352
Copyright
Copyright © Microscopy Society of America 2012

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