Super-resolution Microscopy Study of Molecular Transport Mechanisms

Temple Univ Of The Commonwealth, Philadelphia PA

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Abstract

PROJECT SUMMARY We are writing to request funds to acquire the Olympus FV3000 confocal microscopy system from Evident Instruments. This cutting-edge imaging system will be a valuable addition to the Yang laboratory and will greatly enhance our ability to conduct research in the field of cell biology and biophysics. The need for this high-end confocal microscope to support the NIH-funded research in our laboratory described below is both urgent and increasing. Our laboratory has developed unique tools that have enabled further progress towards understanding molecular transport mechanisms involving three sub-cellular organelles in eukaryotic cells: the nucleus, cytoplasm, and primary cilium. Macromolecular trafficking among these compartments is suggested to be gated by two unique mechanisms. One is the nuclear pore complex (NPC) embedded in the nuclear envelope that mediates bidirectional trafficking of proteins and RNAs between the cytoplasm and the nucleus; the other is the transition zone (TZ) located at the base of primary cilium that regulates the entry of membrane and cytosolic proteins into the cilium. Without access to a high-end confocal imaging system designed for researchers who require the highest image quality and imaging versatility, some research questions in our research projects cannot be answered. However, with this high-end confocal microscopy system, we can immediately obtain large-scale 3D imaging of nucleocytoplasmic transport and cytoplasm-cilium trafficking, which is urgently needed but not available in our laboratory now. Furthermore, this new confocal imaging system equipped with the two-deck configuration of Olympus IX83 inverted microscope is highly compatible with other imaging techniques currently existing in our laboratory2. For example, with the unique two-deck configuration3, we will be able to integrate epifluorescence microscopy, laser scanning confocal microscopy, total internal reflection (TIRF) microscopy, SPEED microscopy and other super-resolution microscopy into a single vibration- free platform. These advanced multifunctional features include 3D confocal imaging, single- molecule tracking, spectral imaging, high-speed imaging, super-resolution imaging, and multi- area time-lapse imaging allow us to capture dynamic biological processes in real-time and with unprecedent high precision for the MIRA-funded reach projects including nucleocytoplasmic transport and cytoplasm-cilium trafficking.