This project aims to develop and apply new differential interference contrast and polarized light microscope techniques, both of whose contrast is independent of specimen orientation. During the first grant period we built an orientation-independent differential interference contrast (OI-DIC) microscope, which rapidly changes shear directions without any mechanically moved components. The OI-DIC system receives the quantitative refractive index gradient and phase (dry mass) images with the highest quality that cannot be achieved with any other optical microscope. We will design and built the improved OI-DIC with an optical stack consisting of DIC prisms and high quality liquid crystal (LC) cells. Because of the small total thickness, this stack can be placed into an existing DIC slot of a regular microscope. The new OI-DIC system will operate in both the visible and near- IR spectral regions, thus opening up the possibility of identifying structures with different wavelength- dependence of refractive indices. We will develop a new orientation-independent polarization microscope (LC- polscope), which employs only one LC cell. The DIC and LC-polscope 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. The new system will yield two complementary images of thin optical section of the specimen: distribution of refractive index gradient or dry mass and distribution of birefringence due to structural or intrinsic anisotropy. For example, in a live dividing cell, the OI-DIC image will clearly show the chromosomes while the polscope image will quantitatively depict the distribution of the birefringent microtubules in the spindle. We will develop a method for reconstruction the 3D distributions of refractive index of specimen regions. Such capabilities will be especially important for studying live cells in division. With the new approach, one will be able to determine the dry mass of chromosomes before division and observe its change during the division process. One could also count the number of condensed chromosomes without spreading or killing the cell. We will design and fabricate new video-enhanced polychromatic polscope, capable of video-rate or faster image acquisition. The polychromatic polscope will be able to provide sharp images of fast moving birefringent organelles, rapid birefringence propagation caused be nerve impulses, etc.
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 state of the cells.
