Biophysical Society Thematic Meeting - June 28-July 1, 2015

New Biological Frontiers Illuminated by Molecular Sensors and Actuators

Poster Abstracts

24-POS Board 24 Multi-Frequency Modulated LED for Detecting Fluorescence Lifetimes in Complex Composition Yeh Lin , Han-Tse Liu, Yi-Chun Chen. National Chiao Tung University, Tainan, Taiwan. Frequency domain fluorescence lifetime instrument (FD-FLI) is a rapid and accurate method to study biomolecular interactions and dynamics. While fluorescence lifetime represents molecular structure and molecular local environment, complex molecular composition results in multiple fluorescence lifetimes in FLI data. It is usually difficult to identify multiple fluorescence lifetime components from just one FLI measurement. In order to further apply FLI to study multiple molecular components, and to capture fast and dynamic events involving multiple fluorescence lifetimes in one measurement, we developed a novel FLI technique based on light-emitting diode (LED). We applied multi-frequency modulation to the LED, and used a lock-in detection algorithm to acquire all the lifetime components information. We were able to solve multi- lifetime cases with our FLI system. The advantages to use LED as excitation light source of FLI include: small size, high power, and low cost. Especially, LED was modulated at radio frequency directly, therefore provided nanosecond temporal resolution for fluorescence lifetime signals. Since conventional fluorescence lifetime techniques are costly due to use of laser, our LED- based FD-FLI has great potential for wider applications in complex biomolecular studies. To our knowledge, we are the first group to demonstrate applications of multi-frequency modulated LED in a FD-FLI system. We also applied this technique to Förster resonance energy transfer experiments, and resolved fluorescence lifetimes when complex donor and acceptor composition was present in the sample. With our novel FD-FLI system, we successful resolved all fluorescence lifetimes of donor alone (without acceptor), donor bound to acceptor molecules, and acceptors in the sample in one multi-frequency measurement.

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