This project aims to develop and apply new quantitative differential interference contrast and polarized light microscope techniques, both of whose contrast is independent of specimen orientation. During the previous grant period we built the orientation-independent differential interference contrast (OI-DIC) microscope, which rapidly changes shear directions without any mechanically moved components. The OI-DIC technique, which we have developed, provides the highest quality of optical path length (dry mass) map of thin optical section of unstained and stained specimens with lateral resolution ~250-300nm, axial discrimination depth ~100nm, and optical path length sensitivity ~0.5nm at wavelength 546nm. We believe that images with such high level of resolution cannot be produced by any other currently available interference and phase microscopy techniques. We also built the polychromatic polarized light microscope (polscope), which employs new principle of generating interference color and produces a color image of the birefringent structures with retardances of several nm, which was not possible before. The hue of the structure indicates its slow axis orientation, and the brightness of the structure is proportional to its retardance. We will engineer the improved high-resolution OI- DIC with and exploit two principally new OI-DIC approaches. The new OI-DIC will be combined with the orientation-independent polarization (LC-polscope) and confocal fluorescence techniques. The improved OI- DIC and the combined setup will be used to study the architectural dynamics of live biological specimens, with emphasis on events associated with mitosis and meiosis. We will build an instantaneous OI-DIC, which will simultaneously capture 4 raw DIC images with the orthogonal shear directions. The instantaneous OI-DIC techniques will provide the best temporal resolution and allow the elimination of artifacts caused by movements of the cytosol and organelles. We will develop OI-DIC technique to restore the 3D distribution of both dry mass (phase-related information) and refractive index. With this technique, one will be able to determine the dry mass of chromosomes before division and observe its change during the division process. The new high- sensitive polychromatic polscope will visualize birefringent structures with retardances less than 1nm. The polychromatic polscope will be combined with phase contrast and dark field techniques, so that image brightness displays dry mass distribution and the color depicts molecular orientation. These combined techniques will be used to study metaphase of meiosis I, and analyze the correspondence between structural signatures in different live organisms, as well as their genetic background. We will create a quantitative polychromatic polscope, which will provide a two-dimensional distribution both the specimen?s retardance and slow axis orientation. The quantitative polychromatic polscope will be employed to study collagen fibers in cancer tissues.
In order to gain improved insight into the dynamic behavior and molecular events underlying healthy and pathologically impaired tissues, we will develop new light microscopes that allow speedy capture of informative images without the need to destroy or stain the cells. The new systems will make cell organelles, including their changes and movement more visible, allow measurement of their dry mass and anisotropy, and concurrently reveal changes in molecular assembly and alignment, all non-invasively without perturbing state of the cells.
