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Proceedings Paper

Quantitative orientation-independent differential interference contrast (DIC) microscopy
Author(s): Michael Shribak; James LaFountain; David Biggs; Shinya Inoué
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Paper Abstract

We describe a new DIC technique, which records phase gradients within microscopic specimens independently of their orientation. The proposed system allows the generation of images representing the distribution of dry mass (optical path difference) in the specimen. Unlike in other forms of interference microscopes, this approach does not require a narrow illuminating cone. The orientation-independent differential interference contrast (OI-DIC) system can also be combined with orientation-independent polarization (OI-Pol) measurements to yield two complementary images: one showing dry mass distribution (which is proportional to refractive index) and the other showing distribution of birefringence (due to structural or internal anisotropy). With a model specimen used for this work -- living spermatocytes from the crane fly, Nephrotoma suturalis --- the OI-DIC image clearly reveals the detailed shape of the chromosomes while the polarization image quantitatively depicts the distribution of the birefringent microtubules in the spindle, both without any need for staining or other modifications of the cell. We present examples of a pseudo-color combined image incorporating both orientation-independent DIC and polarization images of a spermatocyte at diakinesis and metaphase of meiosis I. Those images provide clear evidence that the proposed technique can reveal fine architecture and molecular organization in live cells without perturbation associated with staining or fluorescent labeling. The phase image was obtained using optics having a numerical aperture 1.4, thus achieving a level of resolution never before achieved with any interference microscope.

Paper Details

Date Published: 19 February 2007
PDF: 12 pages
Proc. SPIE 6441, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues V, 64411L (19 February 2007); doi: 10.1117/12.709901
Show Author Affiliations
Michael Shribak, Marine Biological Lab. (United States)
James LaFountain, Univ. at Buffalo (United States)
David Biggs, AutoQuant Imaging Inc. (United States)
Shinya Inoué, Marine Biological Lab. (United States)

Published in SPIE Proceedings Vol. 6441:
Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues V
Daniel L. Farkas; Robert C. Leif; Dan V. Nicolau, Editor(s)

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