Dynamic dark state depletion: A path to high sensitivity imaging

Silver nanoclusters encapsulated in oligonucleotide scaffolds exhibit remarkably bright emission at both the bulk and single molecule level. With emissive species across the visible and into the near infrared, these clusters offer the potential to create a new class of biological labels with vast improvements over existing dyes.;Here we evaluate and optimize many of the characteristics necessary for extending these fluorophores toward eventual applications in biological imaging. Through spectral purification, photophysical characterization at the bulk and single molecule level was achieved for several fluorescent species. Studies reveal the presence of a microsecond blinking component resulting from a short-lived dark state. The lack of intermittency on a longer time scale leads to much higher emission rates than common organic dyes.;This dark state was found to be photo-accessible with a very efficient depopulation transition leading to repopulation of the emissive state. Secondary excitation on resonance with this transition significantly shortens the residence time in the dark state giving rise to as much as 5-fold fluorescence enhancement. Manipulation of the secondary laser can be used to impose a specific waveform onto the fluorescent signal resulting in a regular modulation of the emission. Signal processing techniques can be employed to extract the modulated signal from large backgrounds, leading to drastically improved sensitivity.;This new imaging concept can be extended, beyond Ag nanoclusters, to common organic fluorophores that demonstrate large dark state quantum yields. These fluorophores simultaneously illustrate the utility of this technique and help to define a general set of parameters for engineering ideal dyes for modulated signal extraction. Ideally suited for fluorescence enhancement, FRET pairs can be used to engineer a wide range of modulatable systems, based on detecting donor emission in the presence of a laser directly exciting the acceptor. The utility of Ag nanoclusters, organic dyes, and FRET systems for improved sensitivity are thoroughly investigated in this work.