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 the DeltaVision OMX SuperResolution microscope system that will allow our users to achieve sub-diffraction subcellular resolution to address a number of important biological problems. The power of this 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, the system has extended the resolution barrier to approximately 150 nm in both the lateral and axial direction. The system is also engineered to be able to acquire extremely rapid simultaneous multi-color imaging of living cells expressing fluorescent proteins. The speed and resolution of this system are unsurpassed in any other instrument. This instrumentation will allow our users to approach a wide array of questions spanning three important biomedical areas (microbial biology, chromatin biology, and mitosis) that are limited by existing microscopy methods. A major obstacle in microbial cell biology is that medically important microbes, such as Streptococcus pneumoniae are only ~0.5 5m, which is only slightly larger than the resolution of the light microscope, making imaging of the subcellular distribution of proteins virtually impossible. Asymmetry is very important in bacterial differentiation and communication in biofilm formation, highlighting the importance of being able to dynamically image the distribution of components in these organisms at high resolution. Likewise the epigenetic modifications of chromatin are critical to gene expression, and increasing evidence shows that specific DNA sites are spatially defined in chromatin structure. Identifying when and how chromatin marks are distributed provide additional examples of how super resolution imaging will make an impact on an important area of research. Finally, the dynamic organization of the cytoskeleton is critical for proper cell morphology and for mitotic progression. SuperResolution imaging has the capacity to identify when and where important dynamics regulatory proteins act, and the super speed imaging will allow us to gain novel insights into the dynamic regulation of the microtubule cytoskeleton. Overall the proposed instrumentation would have a significant impact on the research approaches and progress made by life sciences researchers at our university.
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