New Frontiers in Polarized Light Microscopy for Live Cell Imaging

Polarized light microscopy provides unique opportunities for analyzing the architectural dynamics of living cells, tissues, and whole organisms. To effectively harness this analytic power, we are developing polarized light imaging techniques, including fluorescence polarization, that employ new optical configurations, and acquisition and processing algorithms for generating time-lapse images that reveal the dynamics of single molecules and molecular assemblies in organelles, cells, and tissues. In the Cellular Dynamics Imaging Group at the MBL, we have developed several configurations of the LC-PolScope, a new type of polarizing microscope that reveals the birefringence of cellular components and measures their alignment over the whole field of view at once. New optical configurations have led to comprehensive polarization and phase analysis for imaging living cells. We developed the orientation independent differential interference contrast (OI-DIC) microscope for quantitative imaging of dry mass distributions, complementing the birefringence data. In recent years we expanded the LC-PolScope technique to include the measurement of polarized fluorescence of GFP and other molecules, and applied it to record the remarkable choreography of septin proteins during cell division, displayed in yeast to mammalian cells. Using a carefully designed beam splitting arrangement, we are able to follow the orientation dynamics of single fluorescent molecules attached to proteins that assemble into membrane-bound higher order structures in vitro and in vivo. Polarization analysis in microscopy combines the exquisite morphological detail available in modern microscope images with the submicroscopic resolution available with polarization analysis that reveals the alignment of molecular bonds, of fine structural form, and of fluorescent dipoles. Fluorescence polarization further combines the molecular specificity available with fluorescent labeling with the structural specificity afforded by polarization analysis. In fact, most if not all contrast methods in microscopy, when coupled with polarization analysis, can reveal new, vital information about the architectural dynamics in living cells, tissues, and whole organisms.