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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: webmaster@prof-piper.com
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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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References

Chen, H.., Wang, H, Slipchenko, M.N., Jung, Y.K., Shi, Y., Zhu, J., Buhman, K.K. & Cheng, J.X. (2009). A multimodal platform for nonlinear optical microscopy and microspectroscopy. Opt Exp 17(3), 1282–1290.CrossRefGoogle ScholarPubMed
Curl, C.L., Bellair, C.J., Harris, P.J., Allman, B.E., Roberts, A., Nugent, K.A. & Delbridge, L.M.D. (2004). Quantitative phase microscopy—A new tool for investigating the structure and function of unstained live cells. Proceedings of the Australian Physiological and Pharmacological Society 34, 121–127.Google Scholar
Determann, H. & Lepusch, F. (1981). Darkfield microscopy, phase contrast microscopy, interference contrast microscopy. In The Microscope and Its Applications, pp. 18–24. Factory print. Wetzlar, Germany: E. Leitz Wetzlar Company.Google Scholar
E. Leitz Wetzlar Company (1969). Vertical illuminators. In Imaging and Illuminating Optics in Microscopes. Factory print. Wetzlar, Germany: E. Leitz Wetzlar Company.Google Scholar
E. Leitz Wetzlar Company (1970). Devices for Phase Contrast, pp. 5–6. The Heine Condenser, Factory print. Wetzlar, Germany: E. Leitz Wetzlar Company. Google Scholar
Garrett, N.L., Lalatsa, A., Ijeoma Uchegbu, I., Schätzlein, A. & Moger, J. (2012). Exploring uptake mechanisms of oral nanomedicines using multimodal nonlinear optical microscopy. J Biophoton 5(5-6), 458–468.Google Scholar
Glückstad, J., Mogensen, P.C. & Eriksen, R.L. (2001). The generalised phase contrast method and its applications. DOPS-NYT 1, pp. 49–54. Available from http://www.dops.dk/pictures/pdf/archive/2001/01_1_art1.pdf Google Scholar
Huff, T.B., Shi, Y., Fu, Y., Wang, H. & Cheng, J.X. (2008). Multimodal nonliner optical microscopy and applications to central nervous system imaging. IEEE J Sel Top Quantum Electronics 14(1), 4–9.CrossRefGoogle Scholar
James, P. (2003). The Heine condenser (part 2), the operational aspects and imagery. Available at http://www.microscopy-uk.org.uk/mag/indexmag.html?http://www.microscopy-uk.org.uk/mag/artnov03/pjheine2.html.Google Scholar
James, P. (2012). Circular oblique lighting, part 1–4. Available at http://www.microscopy-uk.org.uk/mag/indexmag.html?http://www.microscopy-uk.org.uk/mag/artjan03/pjcol2.html.Google Scholar
Le, T.T., Langohr, I.M., Locker, M.J., Sturek, M. & Cheng, J-X. (2007). Label-free molecular imaging of atherosclerotic lesions using multimodal nonlinear optical microscopy. J Biomed Opt 12(5), 054007-1–10.CrossRefGoogle ScholarPubMed
Lichtscheidl, I. (2011). Interference contrast. In Light Microscopy Online—Theory and Practical Use. University of Vienna, Department of Cell Imaging and Submicroscopic Research. Available at http://www.univie.ac.at/mikroskopie/2_kontraste/interferenz/1_einleitung.htm.Google Scholar
Mann, C.J., Yu, L., Lo, C.-M. & Kim, M.K. (2005). High-resolution quantitative phase-contrast microscopy by digital holography. Opt Exp 13(22), 8693–8698.Google Scholar
Murphy, D.B. (2001). Phase contrast microscopy. In Fundamentals of Light Microscopy and Electron Imaging, Murphy, D.B. (Ed.), pp. 97–112. Chichester: Wiley.Google Scholar
Murphy, D.B., Spring, K.A., Parry-Hill, M. & Davidson, M.W. (2012). Shade-off and halo phase contrast artifacts, interactive tutorial, Nikon Comp. Available at http://www.microscopyu.com/tutorials/java/phasecontrast/shadeoff/index.html.Google Scholar
Palima, D. & Glückstad, J. (2010). Generalized phase contrast: Microscopy, manipulation and more. Contemp Phys 51(3), 249–265.Google Scholar
Piper, T. & Piper, J. (2012a). Variable bright-darkfield-contrast, a new illumination technique for improved visualizations of complex structured transparent specimen. Microsc Res Techniq 75, 537–554.Google Scholar
Piper, T. & Piper, J. (2012b). Variable phase dark-field contrast—A variant illumination technique for improved visualizations of transparent specimens. Microsc Microanal 18, 343–352.Google Scholar
Piper, T. & Piper, J. (2012c). Axial phase-darkfield-contrast (APDC), a new technique for variable optical contrasting in light microscopy. J Microsc 247(3), 259–268.Google Scholar
Popescu, G., Ikeda, T., Dasari, R.R. & Feld, M.S. (2006). Diffraction phase microscopy for quantifying cell structures and dynamics. Opt Lett 31(6), 775–777.CrossRefGoogle ScholarPubMed
Robertson, D. (1970). The phase contrast microscope. In The World beneath the Microscope, Robertson, D. (Ed.), pp. 26–30. London: Weidenfeld and Nicholson.Google Scholar
Samson, E.C. & Blanca, C.M. (2007). Dynamic contrast enhancement in widefield microscopy using projector-generated illumination patterns. New J Phys 9, 363–376.Google Scholar
Slaghter, E.M. & Slaghter, H.S. (1992). Imaging of phase objects. In Light and Electron Microscopy, Slaghter, E.M. & Slaghter, H.S. (Eds.), pp. 149–167. Cambridge UK: Cambridge University Press.Google Scholar
Warger, W.C., Laevsky, G.S., Townsend, D.J., Rajadhyasha, M. & DiMarzio, C.A. (2007). Multimodal optical microscopy for detecting viability of mouse embryos in vitro . J Biomed Opt 12(4), 044006. Google Scholar
Yelleswarapu, C.S., Tipping, M., Kothapalli, S.R., Veraska, A. & Rao, D.V. (2009). Common-path multimodal optical microscopy. Opt Lett 34(8), 1243–1245.CrossRefGoogle ScholarPubMed