An essential component of this research will include neurodevelopmental disorders and analysis of stem cell fate via manipulation of signalling pathways that are involved in altering fate specification and/or transformation of these cells leading to brain diseases.This will be accomplished via live imaging and tracing the fate of fluorescence tagged proteins that are altered in diseased conditions. In another major line of studies, the research team will dissociate the role of the key oncogenic complex, in which thecytokine receptor for Oncostatin M (OSMR) serves as a required co-receptor of the oncogenic protein EGFRvIII. Proteins will be specifically designed and tagged for localized expression in different cell compartments, followed by in vivo live imaging. Finally, the laboratory will conduct drug screening assays to inhibit the interaction of OSMR/EGFRvIII complex via Förster Resonance Energy Transfer (FRET ) microscopy. To achieve the successful completion of these three objectives, the use of a multiplex mode inverted microscope is required on a daily basis for the experimental procedures. The requested multiplex mode inverted microscope will be equipped with fluorescence for GFP, Cy3, mCherry, Cy5 and Cy7 in order to trace tagged protein in live cells via in vivo live imaging as well as the translocation of these proteins in response to external ligands. In addition, the FRET assay in drug counter-screening requires mCherry and GFP tracing to establish small molecules that inhibit the interactionof tagged m-Cherry-OSMR and tagged GFP-EGFRvIII. In other experiments, the laboratory will routinely perform proximity ligation assays to analyze protein-protein interaction required to induce oncogenic signalling pathways in brain cancer models and organoids. Furthermore, the microscope is required on a daily basis to confirm the efficiency of electroporation in live cells and spatial and temporal expression of proteins of interest prior to processing of the cells. In addition, the microscope will be heavily used for analysis of tissues from patient derived brain tumors and rodent brain that harbor patient mutations. For example, the microscope will be able to scan the entire mouse brain with super resolution to understand spatial and temporal expression and localization of multiple proteins of interest at the same time. The microscope will also be equipped for fast scanning of large areas using 2.5, 10x, 20x, & 40x objectives. The microscope will be able to remove blur areas when imaging in thick samples. A full enclosure for live cell (with temperature and CO2 controls) and light-proofing will be mounted on this microscope. In addition, the microscope will be well equipped with high resolution and sensitivity digital cameras for detailed color imaging. A definite focus hardware monitoring and correction system for focus stability will be used during all image acquisitions. Finally, the microscope will be well equipped with software and powerful computer capabilities of extended depth of focus, software autofocus, multiposition experiments, large area high resolution imaging, colocalization, machine learning image analysis, and intelligent acquisition.
