This award supports a collaborative effort between the PI and investigators at the University of Colorado to develop and evaluate an enhanced differential interference contrast (DIC) microscope that will permit the visualization and measurement of dynamic processes in living cells. A wealth of potentially useful information can be provided by subtle variations in the concentrations of the molecular components of living cells. The microscope will detect and record minute changes in light phase resulting from the effect of the variation on the optical path length of light passing through the specimen. The information will be visualized as precisely altered image intensities. The microscope will be able to separate changes in intensity due to altered phase properties from those due to absorption, which existing DIC instruments are unable to do. The new instrument is based on phase-shifting interferometry principles that require careful modeling of the DIC image formation process in order to design appropriate digital image reconstruction routines. The result will be a fully automated combination of optical and digital processing technologies that include novel extended-depth-of-field imaging capabilities recently developed for fluorescence microscopy. Biologists will be able to view, within a single image, three-dimensional features that would otherwise appear out of focus. Potential applications of the new quantitative DIC microscope are plentiful in live-cell biology. Thus, initial tests of the instrument will determine sensitivity for detecting very subtle stages of the cell cycle (e.g. what stage of S phase, or whether in early onset of mitosis) simply by measuring refractive index. Other applications that will be used to develop and refine the instrument include comparison of pre- and post-synaptic neurons and measurement of the rate of change due to protein localization. The new system will be implemented within standard commercial DIC microscopes using a novel liquid crystal device design, making the instrument fully automated and easily accessible to biologists. The microscope is expected to minimize or eliminate the need for fluorescent probes, which can alter normal cell function. The investigators' relation with local companies is expected to facilitate transfer of new technology to commercial development. The project will provide training opportunities for graduate and undergraduate students, and the investigators will incorporate knowledge gained in the project into graduate and undergraduate courses and a public website.
