This award supports the acquisition of the first commercially available 4Pi-Confocal Laser Scanning Microscope (4Pi-CLSM). The 4Pi-CLSM offers immediate three-dimensional optical resolution in the 100 nm range. This instrument will be housed at the Institute for Molecular Biophysics (IMB), a research unit of the University of Maine (UM), established through a consortium between UM, The Jackson Laboratory (TJL) and Maine Medical Center Research Institute (MMCRI). The 4Pi-CLSM will enable axial optical resolving power that is five to seven fold higher than that offered by conventional confocal microscopy. This unprecedented progress in the potential to acquire quantitative structural information from fluorescence-labelled small regions in individual cells will enable new understanding of dynamic processes within cellular structures. The projects have been selected to test the capacity of the 4Pi-CLSM to resolve a diverse set of cellular structures at sufficient power to address longstanding questions of gene expression and gene product structure and function. Several major users propose studies that will examine the relationship of genome architecture and control of gene expression during embryonic development in mouse and zebrafish. The 4Pi-CLSM will enable entirely new kinds of investigation into the relationship between chromatin structure and transcription at the level of individual genes, permitting investigators to see how small differences in structure and epigenetic modification control the timing and sequence of gene activation. The relationship between the structures of individual chromosome regions, their positioning within the nuclei of different cell types and coordinated gene expression can be examined. Other users will apply the instrument for the analysis of cytoplasmic and membrane structures. These studies will examine the processes that guide protein migration and multi-protein complex assembly, the mechanisms by which nuclear trafficking of proteins is associated with cell cycle control and cell fate determination. A third group of users tests the capabilities of 4Pi-CLSM to optically resolve membrane structures and transmembrane molecules, a longstanding challenge for optical microscopy. In consequence, the 4Pi-CLSM will allow a wealth of biological and biophysical research applications that could not previously be addressed with light microscopy. The participating investigators collaborate with faculty at the three participating institutes and train students at several educational levels.
