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Variable Phase Bright-Field Contrast—An Alternative Illumination Technique for Improved Imaging in Transparent Specimens

Published online by Cambridge University Press:  14 December 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

In variable phase bright-field contrast, a bright-field image based on axial or concentric-peripheral light is optically superimposed with a phase-contrast image, so that typical details that are imminent in one or the other technique contribute to the resulting composite image. In particular, complex structured specimens consisting of high-density light absorbing details and additional low-density phase shifting components can be observed with improved clarity. As both partial images interfere with each other, fine details within thin specimens can be highlighted further by additional contrast effects based on interference. Haloing and shade-off are significantly reduced when compared with phase contrast carried out stand-alone. Our method is characterized by several technical means that are relevant for the high image quality that can be achieved: both illuminating light components associated with bright field and phase contrast are filtered at different colors and separated from each other so that they meet the specimen at different angles of incidence. The intensities of the phase-contrast- and bright-field-producing light can be selectively regulated so that the final image can be dominated by phase contrast or bright field, or be equalized. The condenser aperture diaphragm can be used for modulations of the image's appearance.

Keywords

phase contrastbright fieldvariable phase bright-field contrast (VPBC)haloingshade-offlight absorptionphase shiftinterference
Type
Software, Techniques and Equipment Development
Information
Microscopy and Microanalysis , Volume 19 , Issue 1 , February 2013 , pp. 11 - 21
Copyright
Copyright © Microscopy Society of America 2013

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