Project Summary A major goal of biologists is to ?see? inside cells to discover where and when each individual protein acts. Conventional light microscopy approaches limit our spatial resolution to ~250 nm, which in several instances is only slightly smaller than the organism itself. This classic physical limitation has been broken through the development of Super-Resolution microscopy. Our proposal is to acquire a new DeltaVision OMX-SR imaging system to replace an aging/ailing system, which is a flagship piece of equipment at the IU Light Microscopy Imaging Center. The OMX 3D-Structured Illumination Microscope (SIM) has enabled our users to make breakthrough discoveries by achieving sub-diffraction subcellular resolution to address a number of important biological problems. This has been most notable in the area of microbial cell biology in which the small size of the organism precludes subcellular resolution using conventional widefield or confocal technologies. The power of the OMX system is that the super resolution is achieved with conventional lasers and computer technology, the microscope system is a standard high-end widefield microscope, conventional fluorophores can be used obviating the need to develop new reagents, up to 4 color labeling can be achieved by the use of multiple laser lines, and the system can be maintained and operated by an experienced cell biologist. Practically speaking, these features are what have allowed our users to implement this technology to answer a wide array of cell biological questions and have led to a number of significant new discoveries by the user group. Our current system is no longer serviceable due to a lack of replacement parts, and there has been an escalation in the amount of down-time of the current instrument, impacting the work of our NIH-funded user group. After assessing a number of competing technologies and instruments, the DeltaVision OMX-SR emerges as a clear leader of the 3D-SIM field due to its sensitivity, its resolution, its speed of data acquisition, its robustness in maintaining alignment, and in the potential for users to carry out live SIM with suitable temporal resolution.