Single-stranded oligonucleotides stabilize highly fluorescent Ag nanoclusters, with emission colors tunable via DNA sequence. We utilized DNA microarrays to optimize these scaffold sequences for creating nearly spectrally pure Ag nanocluster fluorophores that are highly photostable and exhibit great buffer stability. Five different nanocluster emitters have been created with tunable emission from the blue to the near-IR and excellent photophysical properties. Ensemble and single molecule fluorescence studies show that oligonucleotide encapsulated Ag nanoclusters exhibit significantly greater photostability and higher emission rates than commonly used cyanine dyes.

The water-soluble, near-IR-emitting DNA-encapsulated silver nanocluster presented herein exhibits extremely bright and photostable emission on the single-molecule and bulk levels. The photophysics have been elucidated by intensity-dependent correlation analysis and suggest a heavy atom effect of silver that rapidly depopulates an excited dark level before quenching by oxygen, thereby conferring great photostability, very high singlemolecule emission rates, and essentially no blinking on experimentally relevant time scales (0.1 to >1,000 ms). Strong antibunching is observed from these biocompatible species, which emit >10 9 photons before photobleaching. The significant dark-state quantum yield even enables bunching from the emissive state to be observed as a dip in the autocorrelation curve with only a single detector as the dark state precludes emission from the emissive level. These species represent significant improvements over existing dyes, and the nonpower law blinking kinetics suggest that these very small species may be alternatives to much larger and strongly intermittent semiconductor quantum dots.correlation ͉ photophysics ͉ silver nanoclusters ͉ single-molecule spectroscopy ͉ fluorescence intermittency W hile myriad dyes exist with varying photophysical properties (1, 2), organic dye-based single-molecule and even bulk in vivo imaging dynamics studies suffer from low probe brightness, poor photostability (3), and oxygen sensitivity (4). Advances in nanotechnology such as the use of quantum dots (5, 6) have ameliorated some of these issues but at the cost of toxicity (7), broad excitation (8, 9), power-law blinking (10-12), and large probe size (13,14). While quantum dots are readily excited with low-intensity sources, their fluorescence exhibits intermittency on all time scales (10-12), thereby causing problems when used for tracking or imaging studies. Arising from Auger processes (15), these photophysical dynamics are apparent at all excitation intensities and appear without characteristic times. While functionalization, large size (Ϸ10-20 nm in diameter), and cellular uptake are potential problems, the strong nonmolecular power-law fluorescence intermittency is a major drawback of these materials as single-molecule reporters (10-12). Recently, Ϸ35-nm-sized fluorescent nanodiamonds have also been reported as single-molecule emitters, but these also raise concerns about label size (16). Consequently, for both in vitro and in vivo single-molecule studies, fluorophores with high emission rates and excellent photostability must be identified that are completely devoid of blinking on all relevant time scales, while maintaining small overall sizes.By combining the virtues of chemistry and nanotechnology, we have developed few-atom, molecular-scale noble metal nanoclusters as a class of emitters that simultaneously exhibit bright, highly polarizable discrete transitions, good photostability, and small size, all within biocompatible scaffolds (17-20). Recent observations that DNA encapsulates Ag nanoclust...

Fluorescence imaging in biological sciences is hindered by significant depth-dependent signal attenuation and high fluorescent backgrounds. We have developed optically modulated near-IR-emitting few-atom Ag nanodots that are selectively and dynamically photobrightened upon simultaneous excitation with a secondary laser enabling high sensitivity image extraction to reveal only the demodulated fluorophores. Image demodulation is demonstrated in high background environments to extract weak signals from completely obscuring background emission.

Various single-standed DNA-encapsulated Ag nanoclusters (nanodots) exhibit strong, discrete fluorescence with solvent polarity-dependent absorption and emission throughout the visible and near-IR. All species examined, regardless of their excitation and emission energies, show similar µs single-molecule blinking dynamics and near IR transient absorptions. The polarity dependence, µsec blinking, and indistinguishable µsec-decaying transient absorption spectra for multiple nanodots suggest a common charge transfer-based mechanism that gives rise to nanodot fluorescence intermittency. Photoinduced charge transfer that is common to all nanodot emitters is proposed to occur from the Ag cluster into the nearby DNA bases to yield a long-lived charge-separated trap state that results in blinking on the single molecule level.

A pilot cross sectional study was conducted to investigate the role of red blood cells (RBC) deformability in type 2 diabetes mellitus (T2DM) without and with diabetic retinopathy (DR) using a dual optical tweezers stretching technique. A dual optical tweezers was made by splitting and recombining a single Nd:YAG laser beam. RBCs were trapped directly (i.e., without microbead handles) in the dual optical tweezers where they were observed to adopt a “side-on” orientation. RBC initial and final lengths after stretching were measured by digital video microscopy, and a Deformability index (DI) calculated. Blood from 8 healthy controls, 5 T2DM and 7 DR patients with respective mean age of 52.4yrs, 51.6 yrs and 52 yrs was analysed. Initial average length of RBCs for control group was 8.45 ± 0.25 μm, 8.68 ± 0.49 μm for DM RBCs and 8.82 ± 0.32 μm for DR RBCs (p < 0.001). The DI for control group was 0.0698 ± 0.0224, and that for DM RBCs was 0.0645 ± 0.03 and 0.0635 ± 0.028 (p < 0.001) for DR group. DI was inversely related to basal length of RBCs (p = 0.02). DI of RBC from DM and DR patients was significantly lower in comparison with normal healthy controls. A dual optical tweezers method can hence be reliably used to assess RBC deformability.