The Analytical and Surface Chemistry (ASC) program of the Division of Chemistry will support the research program of Prof. Alexander Li of Washington State University. The essence of this project is to design and synthesize novel polymer nanoparticles, whose red-fluorescence signal can be modulated using external light pulses so that it has amplitude and a phase. When the nanoparticle recognizes the analyte associated with a green-fluorescence donor, fluorescence resonance energy transfer (FRET) will propagate the modulation from the nanoparticle to the green fluorescence donor, whose signal will be in turn modulated at the same frequency, unambiguously confirming interactions. This study will provide a new analytical method that dramatically improves the statistical confidence of fluorescence detection, including fluorescence imaging, fluorescence sensors and fluorescence-based quantitative analyses. It will provide excellent educational training opportunities for graduate students and postdoctoral trainees in the use of advanced spectroscopic methods in quantitative bioassays.
Fluorescence imaging has provided a wealth of information about biological processes and mechanisms. Optical microscopy is particularly insightful because it can be used to study live cells and those events having living characters. However, the wave-like property of light restricts the optical resolution of current microscopes to about 300 nm—the diffraction limit. This project developed a technique that had circumvented the diffraction limit by stochastically photoswitching molecules on and off. The beyond-diffraction-limit optical imaging of cells reveals biological mechanisms, cellular structures, and physiological processes in nanometer scale. Harnessing the photoswitching properties, high-resolution fluorescence nanoscopy successfully resolved nanostructures and subcellular organelles when photoswitchable particles containing optically active molecules were used as the fluorescent probes. Fluorescence imaging has become an indispensible tool for direct visualization of dynamic processes in biological systems. Another challenge in applying fluorescence-based technologies is to solve the ubiquitous fluorescent interferences, which have rendered low reliability in fluorescence detections and limited the wide usage of fluorescence-based chemical and biological sensors. The fundamental barrier is that a single probe, especially a single molecule, has limited brightness in time-domain imaging or detections. Such limitations frequently render individual molecules, toxins, or pathogens undetectable in the presence of interference or complex environments. However, a single photoswitchable probe or molecule produces a frequency-locked signal, which can be separated from none frequency-dependent interference or noise using photoswitching-enabled Fourier transformation. As a result, the light-modulated probes can be made super bright in the frequency domain simply by acquiring more cycles in the time-domain. This project has demonstrated such an innovative method using frequency-domain imaging or detection, which is expected to make a giant stride forward in the area of fluorescence-based technologies. Additionally, this program has enriched the graduate education at WSU in both research and teaching by bringing cut-edge research into classrooms. The broader impacts include training graduate students in chemical synthesis, spectroscopic detections, and microscopic imaging as well as educating high school and middle school students in the Eastern Washington and Idaho regions.
