Automation of quantitative DIC microscopy for 3D live-cell imaging using programm

DESCRIPTION (provided by applicant): The primary objective proposed is to utilize Boulder Nonlinear System's (BNS) spatial light modulator (SLM) technology to automate the Cogswell group's recently-developed quantitative DIC microscope (Q-DIC) and make it accessible to biologists in the marketplace. Q-DIC is a full-field (non-scanning) phase imaging technique which provides optical path length measurements indicative of either thickness, density, or chemical variations. The present limitations of the University of Colorado (CU) lab Q-DIC system can be overcome by adapting BNS spatial light modulators for the microscope in such a way that it will be possible to dynamically vary the three requisite DIC optical design parameters (i.e. phase bias, shear distance and shear direction). A SLM in this context is an electrically driven, anisotropic liquid crystal device with the ability to dynamically change the complex phase of an incident optical wavefront. Precise control of DIC parameter variability using SLM technology will enable automated acquisition of Q-DIC images for non-invasive, real-time quantitative phase (optical path length) imaging. This new capability will allow evaluation of the advantages of Q-DIC for high-resolution biological imaging and will also strengthen the utility of DIC comparisons to fluorescence imaging. Its ability to produce contrast in live-cell images without introducing dyes (which potentially interfere with cellular dynamics) will be of special interest to the cellular biology community. After first proving the feasibility of automating DIC image acquisition with existing SLM technology, BNS proposes to design and fabricate two new and distinctly different SLM configurations in order to compare and contrast their performance and determine which (if either) is best-suited for further development in Phase II. A marketable SLM design must meet the technical challenges of retro-fitting to existing commercial microscopes without limiting their multimode functionality or their high-resolution optical quality. These new configurations will be tested and evaluated through in vitro imaging studies of developing Drosophila embryos, provided by our collaborating biologist (Dr. T. Su). The success of this Phase I research will be measured by the ability to demonstrate accurate quantification of a known cellular development process through a live-cell Q-DIC imaging experiment. In addition to automating the CU lab Q-DIC microscope, this proposal includes initial investigation into whether the new microscope-compatible SLM configurations can also be used more generally for enhancing optical microscope design and performance in the future. Directly coupling new methods of active optical wavefront manipulation to novel methods for digital image processing has the potential to inspire pioneering methods of optimized optical and digital imaging processing. Examples include extending the depth of field of high resolution objectives, active aberration correction, and super-resolution. Such innovations could aid understanding of basic biological problems and biomedical research. PUBLIC HEALTH RELEVANCE: Quantitative differential interference contrast (Q-DIC) microscopy is a full-field-of-view (non-scanning) phase imaging technique which extracts optical path length measurements indicative of either thickness, density, or chemical variations from contrast in images of non-absorbing (or partially absorbing) objects without introducing dyes (which potentially interfere with cellular dynamics). Automated Q-DIC microscopy will enable 3D live-cell Q-DIC imaging and the non-invasive study of dynamic properties during cell development which may reveal new insights into the processes through which the onset of disease takes place.