The objective of the Section on Cellular Biophotonics is to develop new forms of microscopy and photometry to study protein-protein interactions under physiological conditions. I will outline the current status of the four major projects in the section. SCB has four Specific Aims: 1) To automate fluorescent polarization and fluctuation analysis (FPFA) microscopy, and to apply this technology to biological problems. 2) To develop a method for simultaneously monitoring two independent protein-protein interactions inside cells, and to use this approach to generate binary biosensors. 3) To investigate the mechanism of ultrafast energy transfer between fluorescent proteins. 4) To develop a methodology for monitoring protein-protein interactions using deep-brain fiber photometry.
Specific Aim 1 Under the direction of Dr. Nguyen, we have designed and built an automated microscope that is capable or collecting FPFA data from 96 samples in a glass-bottom 96-well plate overnight. This instrument has excellent repeatability across all sample wells, and the experimental variance is very low. A manuscript describing this instrumentation is being prepared. We are collaborating with Dr. Anne Kenworthys laboratory at Vanderbilt University to demonstrate the utility of this automated instrumentation.
Specific Aim 2 This project is primarily under the direction of Dr. Nguyen, with collaborative support of the Puhl. Tuan has demonstrated that homo-FRET and hetero-FRET can be measured simultaneously and that these measurements can follow independent changes in the proximity of homo-FRET and hetero-FRET pairs. Tuan has demonstrated the utility of this approach by simultaneously monitoring the binding of a CaM-Kinase-II T-site ligand using hetero-FRET while simultaneously measuring a conformational change in the kinase holoenzyme structure triggered by the ligand using homo-FRET. Dr. Puhl is currently developing binary biosensors that simultaneously monitor free calcium concentration with both low and high affinity to further demonstrate the utility of this approach.
Specific Aim 3 Dr. Kim has been using time-resolved anisotropy, FCS, antibunching, and paired-pulse correlation to develop new analysis and instrumentation to investigate ultra-fast (less than a few picoseconds) energy transfer between fluorescent proteins. His results indicate that a coherent energy transfer mechanism is responsible for this unexpected high-speed energy transfer. We speculate that such a mechanism might have utility in developing quantum computers.
Specific Aim 4 In collaboration with Dr. Lovingers laboratory (LIN), Drs. Nguyen and Kim are developing fiber optic based instrumentation to monitor FRET-based biosensors for cAMP and A-kinase activity in living mice based on monitoring changes in fluorescence lifetime.
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