AAAS Annual Meeting, Boston, February 2013

15 January 2013

AAAS  Annual  Meeting,  Boston,  February  2013   Symposium  #5913,  Innovations  in  Imaging,  Saturday,  Feb.  16 New Frontiers in Polarized Light Microscopy for Live Cell Imaging Rudolf Oldenbourg, Michael Shribak, Tomomi Tani, and Shinya Inoué Cellular Dynamics Program, Marine Biological Laboratory, Woods Hole MA 02543 Lay-language summary: Polarization is a basic property of light that is often overlooked, because the human eye is not sensitive to polarization. Therefore, we don’t have an intuitive understanding of it and optical phenomena that are based on polarization either elude us or we find them difficult to comprehend. Meanwhile, polarization plays an important role in nature and can be used to enhance our understanding of the architectural dynamics of living cells, tissues and whole organisms through the use of the polarized light microscope. Like most scientific instruments, the polarized light microscope translates polarization effects so they can be perceived by our senses, in this case by our eyes, and makes them amenable to quantitative and analytical analysis. At the Marine Biological Laboratory, we are developing polarized light imaging techniques, including fluorescence polarization, that employ new optical components and configurations, and acquisition and processing algorithms for generating timelapse images that clearly reveal the otherwise invisible dynamics of single molecules and molecular assemblies in organelles, cells, and tissues. We created the LC-PolScope, a modern polarized light microscope, by enhancing the traditional microscope with liquid crystal devices, electronic imaging and digital image processing techniques to reveal and measure the alignment of molecules in living cells over the whole field of view at once [1]. We developed the orientation independent differential interference contrast (OI-DIC) microscope for quantitative imaging of mass density in cells [2]. In recent years we expanded the LC-PolScope technique to include the measurement of polarized fluorescence of GFP and other fluorescent molecules, and applied it to record the remarkable choreography of septin proteins during cell division, displayed in yeast to mammalian cells [3]. Using a custom 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 [4]. 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 and tissues.

15 January 2013 References 1. Oldenbourg, R., A new view on polarization microscopy. Nature, 1996. 381(6585): p. 811-2. 2. Shribak, M. and S. Inoue, Orientation-independent differential interference contrast microscopy. Appl Opt, 2006. 45(3): p. 460-9. 3. DeMay, B.S., et al., Septin filaments exhibit a dynamic, paired organization that is conserved from yeast to mammals. J Cell Biol, 2011. 193(6): p. 1065-81. 4. Tani, T., et al., Trafficking of a ligand-receptor complex on the growth cones as an essential step for the uptake of nerve growth factor at the distal end of the axon: a single-molecule analysis. J Neurosci, 2005. 25(9): p. 2181-91.