Phase I We propose to develop an ultra-sensitive real-time polarized light microscope (""""""""Real-Time PolScope"""""""") for analyzing the dynamics of the molecular architecture in living cells, in whole organisms and in cell-free model systems through birefringence imaging, a powerful and quantitative new imaging contrast technique. The instrumentation builds on the PolScope platform previously developed through a partnership between CRI, Inc and Dr. Oldenbourg at the Marine Biological Laboratory While the spatial resolution and analytical capabilities of the original PolScope system are still state-of-the-art, the temporal resolution of the imaging system is insufficient for resolving and analyzing many potentially interesting cellular events. In Phase I of this application we will speed up image acquisition and thereby greatly improve the fidelity of birefringence measurements. The major innovation we are planning is the development of a four-sector liquid crystal universal compensator that is added to an imaging beam splitter that projects in parallel four polarization images onto a high-resolution digital camera. The simultaneous recording of four images for polarization analysis is expected to increase by more then 10-fold the number of images that we can acquire per minute. Furthermore, the parallel recording of polarization-analyzed images will eliminate most of the artifacts that arise from the internal motion of sample components. Ideally, the presentation of calculated birefringence images will occur at a rate commensurate with the imaging speed. Overall, the goal of the proposed work in Phase I and II is a Real-Time PolScope with a temporal resolution of at least 10 times better than that of the current model with little or no sacrifice in the sensitivity of the current instrument. In Phase II we will add software and hardware components that will create a highly sophisticated polarization and fluorescence analysis tool This instrument will provide much-needed information complementary to other microscope modes used for live-cell imaging. To test the utility of the proposed microscope on biological material, we will continue our studies of the architectural dynamics within living cells, with specific emphasis on the microtubule-containing spindle apparatus during mitosis and meiosis, and mechanical links to biochemical signal transduction pathways. Phase II Phase I should have accomplished implementation of a near-real-time birefringence retardance imaging system by exploiting existing advanced technology of the following components: high-speed, sensitive digital camera; a four-way beam splitter to allow collection of up to four polarization images simultaneously; and an ultra-bright pulsed light source. These components will be complemented by a new four-sector universal compensator that is employed inside the beam splitter and by new polarimetric algorithms that can calculate and display retardances images as fast as they are collected. In Phase II we will extend these capabilities and add engineering and software refinements to create a commercially acceptable, affordable and useful instrument platform. New capabilities will include near simultaneous fluorescence imaging; optical modifications to provide new imaging options and to reduce polarization aberrations in the imaging path; development of a new pulsed, multi-spectral light source, additional algorithms that add new modes of polarization analysis; and 3-D optical sectioning. Other tasks will be directed towards replacing, where necessary, off-the-shelf items used in Phase I when either improved function or a dramatic decrease in cost can be achieved, along with systems engineering to create a beta version suitable for commercialization. This includes advances in the user interface and other software functionalities. These technical advances will be deployed in collaborations.
