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.
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