Orientation-independent DIC and orientation-independent polarization techniques 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). For example, in a live dividing cell, the DIC image will clearly show detailed shape of the chromosomes while the polarization image will quantitatively depict the distribution of the birefringent microtubules in the spindle, both without any need for staining or other modifications of the cell.
Here pseudo-color examples of OI-DIC and OI-Pol images of spermatocytes from the crane-fly, Nephrotoma suturalis, during meiosis I. are presented. The set-up was a Nikon Microphot-SA microscope equipped 60x/1.4 NA oil immersion objective and a Universal Achromatic-Aplanat condenser with the same NA at wavelength is 546 nm. The changing of bias and rotating the shear direction during DIC image acquisition were done manually. Also, in order to switch between polarization and DIC imaging modes, the pair of liquid crystal waveplates was replaced with DIC prisms (see ). Unfortunately, these mechanical manipulations took some time, thus resulting in significant sacrifice of temporal resolution during the test, as discussed below.
Summary of protocol: 4 raw polarization mode images were acquired with intervals of 0.03
sec. (total acquisition time 0.12 second). Switching from the polarization mode to the DIC mode and adjustment of the bias took 2min. 30 sec, then 3 DIC mode images with inverse biases and with zero bias were made with intervals of 5 sec., followed by rotation of the microscope stage for 28 sec, then another set of 3 DIC mode images with intervals of 5 seconds. Total time of DIC image acquisition was 10+28+10= 48 seconds.
and are images of the same spermatocyte during meiosis I first recorded during diakinesis () and then later at metaphase (). They contain computed phase (dry mass) mode (left top); computed retardance mode (left bottom); and color combination of the dry mass and retardance modes (right), in which red and green colors correspond to dry mass distribution and retardance, respectively.
Diakinesis of meiosis I in a crane-fly spermatocyte.
Metaphase of meiosis I in a crane-fly spermatocyte.
Those figures illustrate morphological structures that are especially prominent in the phase mode image, such as the chromosomes. Other features, such as the birefringent spindle fibers (actually bundles of microtubles) exhibit much better contrast in the retardance mode.
The phase image () reveals a dry mass difference between the kinetochore (K-) fibers and the domains of the spindle that surround them. Most notable are the K-fibers extending toward the bottom pole. In the retardance image they are clearly resolved as birefringent structures, whereas in the gradient image, they appear as weakly refractile structures, just slightly brighter than the surround. The image is very black, but the K-fibers are clearly evident as slightly brighter (whiter) than their surround. Thus, those metaphase K-fibers provide a good test object for gradient and phase mode imaging, due to their slightly greater optical density than their surround.
But the K-fibers also raise a problem. They show visible structures in conventional DIC mode (), which are almost absent in the gradient and phase images. This aberration likely is caused by movement(s) occurring during the time interval between subsequent regular DIC images. This explanation is further supported by D. Biggs’ observation that when he deconvolved the DIC images, the first set appeared to be very different from the second set, likely a consequence of a longer time interval between the sets than the time between frames within a set, as described above. When the proposed setup diagrammed in our patent application13
will be implemented, the time interval for raw DIC image acquisition is expected to be 0.3 sec, and thus such artifacts due to movement of (or within) the specimen will be minimized, if not fully abolished.
and provide clear evidence of our notion that the proposed technique can reveal architecture (morphology) of live cells without staining and fluorescent labeling. The phase image acquired in this fashion yields the true distribution of optical path differences using 1.4 NA optics, a feat never before achieved with any interference microscope.