DESCRIPTION (from the application): In TRD1, we propose three subprojects that will result in substantial additions to our hardware and software. Collectively, these subprojects will enhance techniques and methods developed at the LFD. TRD1 with its three subprojects is not a mere update or maintenance of instrumentation. On the contrary, each of the subprojects is novel and has its own merits and they are necessary for our DBP's and collaborations. Specific Aims: During the last five years, fluorescence microscopy has experienced a revolution due to several factors. These factors include better understanding of the physics of optics for achieving super resolution, availability of new types of sensitive detectors, use of computers as an integral part of the microscope, development of light sheet techniques, multi foci multiphoton excitation and the availability of new fluorescent probes, both synthetically produced (quantum dots) and genetically encoded, that offer better brightness and photochemical stability. Consequently, fluorescence microscopy has evolved into a sophisticated technique. Today, there is a major effort to exploit these new capabilities to address urgent problems in cell biology. Imaging methods have become advanced to the point that single molecule detection inside the cell is feasible. One major challenge is the development of methodologies to extract information from the natural systems where all of the biological actions occur simultaneously. The three subprojects address different needs that are not generally found in current efforts for increasing the microscope performance but rather are targeting specific applications that are important for the LFD. These subprojects form a cohesive plan formulated to achieve new quantitative measurements in microscopy.