Development of a convenient and supersensitive high-throughput screening system for genetically encoded fluorescent probes of small molecules using a confocal microscope.

Monitoring the dynamic patterns of intracellular signaling molecules, such as inositol 1,4,5-trisphosphate (IP3 ) and Ca2+ , that control many diverse cellular processes, provides us significant information to understand the regulatory mechanism of cellular functions. For searching more sensitive and higher dynamic range probes for signaling molecules, convenient and supersensitive high throughput screening systems are required. Here we show the optimal "in Escherichia coli (E. coli) colony" screening method based on the twin-arginine translocase (Tat) pathway and introduce a novel application of a confocal microscope as a supersensitive detection system to measure changes in the fluorescence intensity of fluorescent probes in E. coli grown on an agar plate. To verify the performance of the novel detection system, we compared the changes detected in the fluorescent intensity of genetically encoded Ca2+ indicator after Ca2+ exposure to two kinds of conventional fluorescence detection systems (luminescent image analyzer and fluorescence stereomicroscope). The rate of fluorescence change between Ca2+ binding and unbinding detected by novel supersensitive detection system was almost double than those measured by conventional detection systems. We also confirmed that the Tat pathway-based screening method is applicable to the development of genetically encoded probes for IP3 . Our convenient and supersensitive screening system improves the speed of developing florescent probes for small molecules.