At the core of biological function lays the ability of proteins to interact and associate with each other and the imaging methods capable of reporting on molecular events and interactions with high sensitivity and high resolution become indispensible. Fluorescence methods have a potential for highly sensitive detection and become essential for studying molecular processes with high specificity and sensitivity through a variety of signaling mechanisms. Tens of fluorescence probes are developed every year to be used for proteins/DNA/RNA labeling and to study molecular pathways and interactions as well as tissue imaging. However after many years of significant effort we are still missing "perfect" probes. There are many obstacles for markers that can be used for studying biological processes in cells and tissue. Two fundamental common problems for biomedical imaging, from single molecule studies and cellular imaging to whole body imaging are the background signal and availability of highly bright probes with suitable fluorescence lifetimes. The background signal (sample autofluorescence, scattering, and non- specific probe binding) always compromise sensitivity and specificity. The need for imaging kinetics and dynamics of molecular interactions/processes (like protein-protein interactions) requires probes with fluorescence lifetimes comparable to the mobility of interacting molecular partners. Many membrane and cellular proteins are large with molecular masses from 20 kDa to much over 100 kDa for which tumbling time and conformational changes are within tens and hundreds of nanoseconds. Within the large arsenal of dyes available today we have many bright fluorophores with fluorescence lifetimes of few nanoseconds or less and some luminophores like lanthanides with exited state lifetimes in microseconds. At present we lack fluorophores in red spectral range with fluorescence lifetimes over 10 ns. In this application we propose to utilize and further develop new group of small organic compounds [1,2]. The new group of azaoxa-triangulenium dyes offers excellent physico-chemical properties that will have unprecedented impact on molecular imaging. The rigid and small triangular frame of this organic compound has very favorable spectral properties including high photostability and most importantly unprecedented long single exponential fluorescence lifetime (~20 ns). We now propose to develop active and enhanced forms of these compounds to be used for studying molecular processes and interactions on a single molecule level, cellular level, and tissue imaging. In parallel to dyes development and tuning their spectral properties we will develop novel methodologies based on time gated detection to eliminate background signal and study dynamics of molecular processes and interactions by fluorescence polarization and FRET. This will enable: (1) precise time-resolved imaging that brings dynamic information about observed processes in large molecular complexes not available from steady-state measurements;(2) use of time-gated detection that will dramatically decrease background and improve imaging sensitivity over 100 folds;(3) new molecular beacon-type probes based on FRET, long lived donor, and time-gated detection that will have enormous signal gain of 105. In addition, 10-30 ns fluorescence lifetimes are much longer from the lifetime of typical background signal and in the same time easy for gating. Importantly time-resolved measurements for such lifetimes can be comfortably made with a pulsed laser source with a repetition rate of 1-5 MHz, in contrast it will require significantly longer time to collect enough photons in case of the lanthanides where the repetition rates are only in kHz.

Public Health Relevance

In order to understand many complex biological processes, it is important to characterize complex interactions that occur in living subject by developing strategies, technologies, and probes that can monitor intracellular communication pathways, including protein-protein interactions. Our immediate goal is to develop probes and technologies that will allow for noninvasive imaging of molecular events openings new ways for studying complex interactions, as well as testing of drugs in cell cultures and in living subjects. The long term goal is to develop methods to image arrays of diverse molecular processes simultaneously to provide adequate in vivo characterization of diseases and allow accurate prognosis and rational disease treatment.

National Institute of Health (NIH)
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
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Special Emphasis Panel (ZRG1-SBIB-A (55))
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Conroy, Richard
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University of North Texas
Other Domestic Higher Education
Fort Worth
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Raut, Sangram; Rich, Ryan; Fudala, Rafal et al. (2014) Resonance energy transfer between fluorescent BSA protected Au nanoclusters and organic fluorophores. Nanoscale 6:385-91
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Xia, Guiquan; Li, Yuqi; Li, Yu et al. (2014) Optimize the Separation of Fluorinated Amphiles Using High-Performance Liquid Chromatography. J Fluor Chem 164:39-42
Raut, Sangram L; Rich, Ryan; Fudala, Rafal et al. (2014) BSA Au clusters as a probe for enhanced fluorescence detection using multipulse excitation scheme. Curr Pharm Biotechnol 14:1139-44
Shtoyko, Tanya; Raut, Sangram; Rich, Ryan M et al. (2014) Preparation of plasmonic platforms of silver wires on gold mirrors and their application to surface enhanced fluorescence. ACS Appl Mater Interfaces 6:18780-7
Raut, S L; Fudala, R; Rich, R et al. (2014) Long lived BSA Au clusters as a time gated intensity imaging probe. Nanoscale 6:2594-7
Rich, Ryan M; Mummert, Mark; Gryczynski, Zygmunt et al. (2013) Elimination of autofluorescence in fluorescence correlation spectroscopy using the AzaDiOxaTriAngulenium (ADOTA) fluorophore in combination with time-correlated single-photon counting (TCSPC). Anal Bioanal Chem 405:4887-94
Thyrhaug, Erling; Sorensen, Thomas Just; Gryczynski, Ignacy et al. (2013) Polarization and symmetry of electronic transitions in long fluorescence lifetime triangulenium dyes. J Phys Chem A 117:2160-8
Sorensen, Thomas Just; Thyrhaug, Erling; Szabelski, Mariusz et al. (2013) Azadioxatriangulenium (ADOTA(+)): A long fluorescence lifetime fluorophore for large biomolecule binding assay. Method Appl Fluoresc 1:25001
Maliwal, Badri P; Fudala, Rafal; Raut, Sangram et al. (2013) Long-lived bright red emitting azaoxa-triangulenium fluorophores. PLoS One 8:e63043

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