![]() ![]() ![]() With conventional DIC, the two-dimensional distribution of optical path gradients along the shear direction generates image contrast that is not symmetrical and varies proportionally with the cosine of the angle made by the azimuth of the object and the direction of wavefront shear. ![]() Another important feature of the DIC technique is that it permits effective optical sectioning, employing high numerical aperture (NA) objectives together with high NA condenser illumination. Where there are steep gradients of optical path, image contrast is significantly increased. Those regions of the specimen where the optical paths increase along a reference direction appear brighter (or darker), while regions where the path differences decrease appear in opposite contrast. The technique produces a monochromatic shadow-cast image of optical path (dry mass) gradient with a transparent specimen. 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.ĭifferential interference contrast (DIC) microscopy is widely used to observe structure and motion in unstained living cells and isolated organelles. We present pseudo-color combined images of a crane-fly spermatocyte at diakinesis and metaphase of meiosis I. For instance, in a live dividing cell, the OI-DIC image clearly shows the detailed shape of the chromosomes while the polarization image quantitatively depicts the distribution of birefringent microtubules in the spindle, both without any need for staining or other modifications of the cell. The combined system yields two complementary phase images of thin optical sections of the specimen: distribution of refractive index and distribution of birefringence due to anisotropy of the cell structure. The OI-DIC images were obtained using optics having numerical aperture 1.4, thus achieving a level of resolution never before achieved with any phase contrast or interference microscope. Then the obtained images are used for computation of the phase gradient magnitude and azimuth distribution, and, further, the phase image. Several conventional DIC images were recorded with the specimen oriented in different directions followed by digital alignment and processing of the images. The article describes combined orientation-independent (OI-) DIC and polarization microscope and its biological applications. ![]()
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