Optically modulated, sequence- and color-tunable single Ag nanodot biolabels

Georgia Institute Of Technology, Atlanta GA

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Abstract

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.