Orientation independent DIC and polarization microscopy

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DESCRIPTION (provided by applicant): This project aims to develop and apply a new, orientation-independent differential interference contrast (OI-DIC) microscope, whose contrast is independent of specimen orientation and is appropriate for investigation of live biological specimens. Extending the preliminary studies using conventional DIG coupled with specimen rotation, by which the theoretical validity of the proposed technique was validated, OI-DIC systems are proposed which require no rotation of either the specimen or the DIG prisms. With the new systems (patent applied), an OI-DIC image should be obtained in 2 seconds or less. Thus microscopes incorporating the new system will allow precise analyses of organellar morphology, motility, as well as dry mass distribution. In addition, a combination of regular DIG and orientation independent polarization (LC-Pol) microscope will be developed. This new system will yield two complementary phase images of thin optical sections of the specimen: distribution of optical path gradients and distribution of birefringence due to structural or internal anisotropy of the cell structure. The DIG and LC-Pol devices combined into one unit will allow rapid switching between the two modes without the need to move any optical components so that both images can be captured nearly simultaneously and without misalignment, yielding a combined image in ca. 0.3 seconds. The possibilities of combining the proposed technique with near-contemporaneous fluorescence images without moving any optical elements will be explored. This combination will give unique views of a specimen, where structural information can be joined with molecularly specific labels. The new devices will be used to study the architectural and molecular dynamics of live biological specimen, with emphasis on events associated with mitosis and meiosis; the OI-DIC providing enhanced chromosome images while the LC-Pol images quantitatively depicting the dynamic assembly-disassembly and distribution of microtubules in the spindle fibers and astral rays. The new microscope should generally allow advanced analyses of organellar movement and sub-organellar anisotropy that reflect the submicroscopic and ongoing molecular events, directly in living cells. RELEVANCE: In order to gain improved insight into the dynamic behavior and molecular events underlying healthy and pathologically impaired tissues, we will develop new optics and processing software for 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 concurrently reveal changes in molecular assembly and alignment, all non-invasively without perturbing the state of the cells.