ShEEP Request for Nikon TIRF STORM microscope

点击翻译按钮获取中文摘要

Super-resolution microscopy has opened unprecedented opportunities to image single molecules or groups of molecules with resolution below 50 nm. However microscopes with these capabilities, if purchased from a commercial vendor as ready to assemble, are beyond the budgets of most individual investigators. And cutting expenses by building one's own requires an understanding of optics which is beyond the knowledge of most biologists. Multiple investigators associated with the Iowa City VA Medical Center have reached a point in their investigations where applying super-resolution microscopy to living cells is required to move their research forward. This research is targeted towards diseases which are relevant to US military veterans: pulmonary emphysema, alterations in host defense observed in chronic infections such as hepatitis C, cytomegalovirus, or Leishmaniasis, glaucoma and obesity. There is currently no microscope on the VA or University of Iowa campuses which will enable these types of studies. Therefore, we are requesting a Nikon Eclipse Ti-e2 inverted microscope equipped with total internal reflection (TIRF) and stochastic optical reconstruction microscopy (STORM) modules. TIRF optics will benefit those studying receptors which signal by assembling adapter proteins at the plasma membrane (T-cell receptor, Drs. Bishop and Stapleton) or retain active signaling after entering endosomes (Dr. McGowan). Combining TIRF and STORM will enable Drs. McGowan, Allen and Anderson to study the assembly of podosomes or remodeling of the sub-cortical cytoskeleton. STORM will enable Drs. Wilson, Meier, and Stapleton to observe the assembly of proteins from pathogens with those from host cells to learn how viruses or parasites evade host defenses. Dr. Meier will use STORM to study targeted degradation of viral or mammalian proteins or the interaction of viral proteins with chromatin. All of these projects require multicolor fluorescent imaging, at high resolution, to detect and localize photons, at rapid frame rates and with high directional and temporal precision. We have chosen the Nikon H-TIRF/N- STORM system because it meets all of these requirements in a package which provides these features in a largely automated format, which is highly desirable for a multi-user instrument. To enable live cell imaging the microscope will be equipped with a stage-top incubator to control the environment and allow reagents to be introduce without altering the field of view. We are also requesting powerful PC workstations and Nikon software which is required to acquire and analyze images. A more complete description of the instrument may be found in the Specific Aims section. Because this package involves emerging state-of the art technology, we hope that it will also enhance the research programs of new investigators as they join the VA research program and pursue novel projects tailored to the future health needs of US veterans.