We are building and applying an ultra-sensitive and high-resolution polarized light microscope on liquid based crystal technology (the LC-PolScope). We use the unique instrumentation to reveal and decipher the functional role of the dynamic architecture of the cytoskeleton, with a focus on the mitotic apparatus during cell division. The proposed instrumentation continues the development of the LC-PolScope that has greatly enhanced the utility of polarized light for live cell imaging. During the last grant period, we removed an important limitation by adding an aperture scanning device to the microscope optical train. Unlike any other polarizing microscopes, including the LCPolScope, the new Scanned Aperture PolScope can reveal the birefringence of objects that are inclined to the plane of focus. Therefore, this technique can finally reveal birefringent objects, such as mitotic spindles, that have any orientation, and it is not limited to objects that either by chance or by special manipulation are oriented in the plane of focus. Building on the proof of concept, we now propose to refine the aperture scanning technique by modifying the design of the aperture scanner and adding a second, similar device in the imaging path of the microscope. These changes and additions to the optical set-up will make it possible (a) to measure birefringence more accurately and sensitively, and (b) measure dichroism in specimens that exhibit polarization-dependent absorption. Furthermore, with the addition of highly discriminating interference filters to the imaging path, the instrument will be capable of revealing fluorescence polarization. Birefringence, dichroism, and fluorescence polarization are important indicators of anisotropy in local molecular order. Structural anisotropy often proves to be important in establishing biological function, such as the alignment of lipid molecules in forming membranes or the linear arrangement of spindle microtubules for the directed segregation of chromosomes during cell division. By taking advantage of the new and improved capabilities of the Scanned Aperture PolScope, we will investigate mechanisms of chromosome segregation during meiosis in insect spermatocytes. Spermatocytes of Sciara and crane flies provide the opportunity to compare the results from normal chromosome segregation with results obtained with chromosomes that show"""""""" either natural (monopolar spindle in Sciara and late segregation of sex chromosomes in crane fly) or induced (cold treatment or drug exposure) abnormalities in their segregation behavior.

Agency
National Institute of Health (NIH)
Institute
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Type
Research Project (R01)
Project #
5R01EB002583-11
Application #
6915169
Study Section
Special Emphasis Panel (ZRG1-SSS-U (10))
Program Officer
Zhang, Yantian
Project Start
1992-08-12
Project End
2009-04-30
Budget Start
2005-05-01
Budget End
2006-04-30
Support Year
11
Fiscal Year
2005
Total Cost
$493,533
Indirect Cost
Name
Marine Biological Laboratory
Department
Type
DUNS #
001933779
City
Woods Hole
State
MA
Country
United States
Zip Code
02543
Thievessen, Ingo; Fakhri, Nikta; Steinwachs, Julian et al. (2015) Vinculin is required for cell polarization, migration, and extracellular matrix remodeling in 3D collagen. FASEB J 29:4555-67
Koike-Tani, Maki; Tani, Tomomi; Mehta, Shalin B et al. (2015) Polarized light microscopy in reproductive and developmental biology. Mol Reprod Dev 82:548-62
Henson, John H; Yeterian, Mesrob; Weeks, Richard M et al. (2015) Arp2/3 complex inhibition radically alters lamellipodial actin architecture, suspended cell shape, and the cell spreading process. Mol Biol Cell 26:887-900
McQuilken, Molly; Mehta, Shalin B; Verma, Amitabh et al. (2015) Polarized Fluorescence Microscopy to Study Cytoskeleton Assembly and Organization in Live Cells. Curr Protoc Cell Biol 67:4.29.1-13
Abrahamsson, Sara; McQuilken, Molly; Mehta, Shalin B et al. (2015) MultiFocus Polarization Microscope (MF-PolScope) for 3D polarization imaging of up to 25 focal planes simultaneously. Opt Express 23:7734-54
Noda, Naoki; Tamm, Sidney L (2014) Lithocytes are transported along the ciliary surface to build the statolith of ctenophores. Curr Biol 24:R951-2
Henson, John H; Gianakas, Anastasia D; Henson, Lauren H et al. (2014) Broadening the spectrum of actin-based protrusive activity mediated by Arp2/3 complex-facilitated polymerization: motility of cytoplasmic ridges and tubular projections. Cytoskeleton (Hoboken) 71:484-500
Mehta, Shalin B; Shribak, Michael; Oldenbourg, Rudolf (2013) Polarized light imaging of birefringence and diattenuation at high resolution and high sensitivity. J Opt 15:
Gibaud, Thomas; Barry, Edward; Zakhary, Mark J et al. (2012) Reconfigurable self-assembly through chiral control of interfacial tension. Nature 481:348-51
DeMay, Bradley S; Noda, Naoki; Gladfelter, Amy S et al. (2011) Rapid and quantitative imaging of excitation polarized fluorescence reveals ordered septin dynamics in live yeast. Biophys J 101:985-94

Showing the most recent 10 out of 21 publications