Orientation-independent differential interference contrast microscopy and its combination with an orientation-independent polarization system

Abstract

The article describes combined orientation-independent (OI-) DIC and polarization microscope and its biological applications. Several conventional DIC images were recorded with the specimen oriented in different directions followed by digital alignment and processing of the images. Then the obtained images are used for computation of the phase gradient magnitude and azimuth distribution, and, further, the phase image. 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. 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. 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. We present pseudo-color combined images of a crane-fly 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.

Differential Interference Contrast Microscopy (DIC)

“…Both methods, however, have the same shortcomings: they require the proper orientation of a specimen in relation to the optical system in order to achieve high quality results, and the images are not quantitative. Hence, it would be beneficial to combine the OI-DIC microscope with an orientation-independent differential polarization (OI-Pol) system [45].…”

“…For instance, in a live dividing cell, the OI-DIC image clearly shows 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. [45]. The changing of bias and rotating the shear direction during DIC image acquisition were done manually.…”

Peering Beneath the Surface: Novel Imaging Techniques to Noninvasively Select Gametes and Embryos for ART

“…In another example of combining imaging approaches, DIC and polarization microscopy have been used in tandem, and through the use of algorithms and computer-recombined images, a quantitative and orientation-independent approach has been used to visualize crane fly spermatocytes [116,117] (Fig. 8).…”

Video-rate imaging of microcirculation with single-exposure oblique back-illumination microscopy

“…Strategies based on noncollimated illumination have the advantage that they can be used with extended sources. Examples are quantitative DIC, 12,13 lateral shearing, 14 and oblique illumination, 15,16 or oblique detection. [17][18][19] Alternatively, numerical techniques based on the solution of the transport of intensity equation have been used.…”