The microscope we propose in this application is the new Zeiss Elyra 7 system, which combines multiple modes of super-resolution imaging. The new Lattice SIM technology of the Elyra 7 enables structured illumination super-resolution imaging of common fluorescent proteins and labels, even in tissue samples, due to the increased signal to noise imaging with the lattice pattern. Increased light efficiency allows 2x diffraction limited imaging at high speed with less phototoxicity and bleaching ? up to 255 fps. One can examine the fastest processes in living samples ? in large fields of view, in 3D, over long time periods, and with multiple colors. The Elyra 7 also has single molecule localization microscopy (SMLM) capabilities for techniques such as photoactivation localization microscopy (PALM), direct stochastic optical reconstruction microscopy (dSTORM) and points accumulation for imaging in nanoscale topography (PAINT) with high power laser lines and dual camera functionality. The new Apotome mode gives superfast optical sectioning of 3D samples with minimal photobleaching. We note PALM/STORM and lattice-structured illumination of the Elyra 7 includes Zeiss software solutions for the analysis of images acquired in each of these modes and thus adds capabilities in super-resolution imaging that currently do not exist on Duke campus. The flexibility and new technologies of the ELYRA 7 will significantly contribute to new research capabilities related to NIH funded developmental biology, neuroscience, and cell biology questions relevant to human disorders as outlined in the research statement. The instrument would be housed in a large shared resource serving the entire research base at Duke University and Duke School of Medicine where the impact and use would be widespread. The LCMF Director has experience with these forms of imaging and has general high-levels of imaging expertise and so is well placed to immediately and effectively help implement these methods for an important array of basic research. Additionally, as part of our broad long-term objectives with institutional support, the ELYRA 7 works seamlessly with ZEISS SEMs in a correlative light-EM imaging workflow via integrated software solutions that enable the precise landmarking of light imaging regions of interest to be automatically recalled from the same sample for electron microscopy. This added benefit will be paradigm shifting for many research programs in our user group.
STATEMENT This research is relevant to the mission of NIH because it examines biology underlying developmental and neurobiological mechanisms that are implicated in many diseases and that are germane to basic mechanisms to modulate cellular behavior. Thus, important advances in understanding these mechanisms, down to nanometer detail, are expected. Super-resolution microscopy of lattice SIM and point localization techniques are essential to advance these projects. It is also expected that knowledge gained in these studies will shed light on normal and abnormal processes important to priorities of NIH research initiatives.
