The long-term project goal is to create small, highly fluorescent, and optically modulatable nanomaterials to enable a whole new array of high sensitivity imaging and detection capabilities in extremely high background biological environments. Beautifully adept at probing mechanistic heterogeneity, single molecule (SM) experiments hold great potential to unravel the complex steps leading to biological activity. Unfortunately, all SM experiments are at some level limited by the disadvantageous properties of available fluorescent labels, with at least 10-fold improvements in brightness and photostability being necessary for facile observation in the high background cellular milieu. Selective modulation of the probe of interest has long been utilized in spectroscopy to extract very weak signals from high background environments, but no fluorophores exhibit modulatable emission without also simultaneously modulating background emission, precluding similar sensitivity gains in biological imaging. Through two Specific Aims, we will demonstrate intracellular observability of individual fluorophores through brightness gains and selective optical modulation possible with our unique Ag nanodot emitters.
In Aim I, we will elucidate the unique photophysics of our Ag nanodots that enable long wavelength, secondary laser-induced optical depletion of a photoaccessible dark state to significantly increase overall emission rate. Since this secondary laser is of lower energy than both the primary laser excitation and the fluorescence it enhances, we can selectively modulate the bright Ag nanodot emission independent of the background through modulation of the secondary laser intensity. Detailed photophysical characterizations of all nanodots created to date are expected to identify at least 5 spectrally pure nanodots exhibiting modulation-based sensitivity gains.
In Aim II we will employ these 5 different color modulatable nanodots for extraction of true intracellular SM fluorescence signals through whole image modulation. Limits of permissible background for SM signal extraction will be directly probed for each emitter in well- designed control experiments. Modulation will also be utilized for signal extraction in live cell fluorescence correlation spectroscopy-based observations of single molecules. This selective optical modulation should enable entire images to be modulated and synchronized with detection for potential >10-fold sensitivity increases in SM or bulk nanodot imaging. With a high probability of success, we will develop these ultrabright, highly photostable emitters with the goal of optical modulation-based intracellular SM observation.
Studies of single molecule imaging and dynamics, as well as fluorescence-based medical imaging are limitedby the disadvantageous properties of available fluorophores. This proposal outlines the creation and utilization of Ag nanodots for >10-fold sensitivity gains, making them ideal for imaging in high background cellular environments. These materials may also be directly extendable to minimally invasive medical imaging and diagnostics applications.
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|Richards, Chris I; Hsiang, Jung-Cheng; Dickson, Robert M (2010) Synchronously amplified fluorescence image recovery (SAFIRe). J Phys Chem B 114:660-5|
|Richards, Chris I; Hsiang, Jung-Cheng; Khalil, Andrew M et al. (2010) FRET-enabled optical modulation for high sensitivity fluorescence imaging. J Am Chem Soc 132:6318-23|
|Patel, Sandeep A; Cozzuol, Matteo; Hales, Joel M et al. (2009) Electron transfer-induced blinking in Ag nanodot fluorescence. J Phys Chem C Nanomater Interfaces 113:20264-20270|
|Yu, Junhua; Choi, Sungmoon; Dickson, Robert M (2009) Shuttle-based fluorogenic silver-cluster biolabels. Angew Chem Int Ed Engl 48:318-20|
|Jung, Soonkyo; Dickson, Robert M (2009) Hidden markov analysis of short single molecule intensity trajectories. J Phys Chem B 113:13886-90|
|Patel, Sandeep A; Richards, Chris I; Hsiang, Jung-Cheng et al. (2008) Water-soluble Ag nanoclusters exhibit strong two-photon-induced fluorescence. J Am Chem Soc 130:11602-3|
|Richards, Chris I; Choi, Sungmoon; Hsiang, Jung-Cheng et al. (2008) Oligonucleotide-stabilized Ag nanocluster fluorophores. J Am Chem Soc 130:5038-9|
|Yu, Junhua; Choi, Sungmoon; Richards, Chris I et al. (2008) Live cell surface labeling with fluorescent Ag nanocluster conjugates. Photochem Photobiol 84:1435-9|
|Zheng, Jie; Nicovich, Philip R; Dickson, Robert M (2007) Highly fluorescent noble-metal quantum dots. Annu Rev Phys Chem 58:409-31|
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