Type Ib diamonds emit bright fluorescence at 550 -800 nm from nitrogen-vacancy point defects, (N-V) 0 and (N-V) ؊ , produced by high-energy ion beam irradiation and subsequent thermal annealing. The emission, together with noncytotoxicity and easiness of surface functionalization, makes nano-sized diamonds a promising fluorescent probe for single-particle tracking in heterogeneous environments. We present the result of our characterization and application of single fluorescent nanodiamonds as cellular biomarkers. We found that, under the same excitation conditions, the fluorescence of a single 35-nm diamond is significantly brighter than that of a single dye molecule such as Alexa Fluor 546. The latter photobleached in the range of 10 s at a laser power density of 10 4 W/cm 2 , whereas the nanodiamond particle showed no sign of photobleaching even after 5 min of continuous excitation. Furthermore, no fluorescence blinking was detected within a time resolution of 1 ms. The photophysical properties of the particles do not deteriorate even after surface functionalization with carboxyl groups, which form covalent bonding with polyL-lysines that interact with DNA molecules through electrostatic forces. The feasibility of using surface-functionalized fluorescent nanodiamonds as single-particle biomarkers is demonstrated with both fixed and live HeLa cells.blinking ͉ photobleaching ͉ single-molecule detection ͉ single-particle tracking ͉ live cell O ne of the key avenues to understanding how biological systems function at the molecular level is to probe biomolecules individually and observe how they interact with each other directly in vivo. Laser-induced fluorescence is a technique widely adopted for this purpose owing to its ultrahigh sensitivity and capabilities of performing multiple-probe detection (1-3). However, in applying this technique to imaging and tracking a single molecule or particle in a biological cell, progress is often hampered by the presence of ubiquitous endogenous components such as flavins, nicotinamide adenine dinucleotides, collagens, and porphyrins that produce high fluorescence background signals (4-6). These biomolecules typically absorb light at wavelengths in the range of 300-500 nm and fluoresce at 400-550 nm (Fig. 1). To avoid such interference, a good biological fluorescent probe should absorb light at a wavelength longer than 500 nm and emit light at a wavelength longer than 600 nm, at which the emission has a long penetration depth through cells and tissues (5, 7). Organic dyes and fluorescent proteins are two types of molecules often used to meet such a requirement (1,8,9); however, the detrimental photophysical properties of these molecules, such as photobleaching and blinking, inevitably restrict their applications for long-term in vitro or in vivo observations. Fluorescent semiconductor nanocrystals (or quantum dots), on the other hand, have gained considerable attention in recent years because they hold a number of advantageous features including high photobleaching thresholds a...

Fluorescent nanodiamond (FND) contains nitrogen-vacancy defect centers as fluorophores. The intensity of its fluorescence can be significantly enhanced after deposition of the particle (35 or 140 nm in size) on a nanocrystalline Ag film without a buffer layer. The excellent photostability (i.e. neither photobleaching nor photoblinking) of the material is preserved even on the Ag film. Concurrent decrease of excited state lifetimes and increase of fluorescence intensities indicate that the enhancement results from surface plasmon resonance. Such a fluorescence enhancement effect is diminished when the individual FND particle is wrapped around by DNA molecules, as a result of an increase in the distance between the color-center emitters inside the FND and the nearby Ag nanoparticles. A fluorescence intensity enhancement up to 10-fold is observed for 35 nm FNDs, confirmed by fluorescence lifetime imaging microscopy.

To investigate the local environment's effect on the lifetime and quantum yield of extended polymer chains in the absence of intra-and interchain aggregation, short, rodlike polymers of poly(2,5-di-n-octyloxy-1,4-phenylenevinylene) (DO-PPV) were dissolved in chloroform and then embedded in a polystyrene matrix. The fluorescence lifetime was found to increase by 45% in moving from the solution to the matrix form. By using the absorption and emission spectra of the chloroform solution to estimate the radiative and nonradiative rate constants for the polymer in solution, along with calculations based on an exciton model, the corresponding decay rate constants for the polymer embedded in the matrix were obtained. The close agreement between the calculated and experimental values of fluorescent lifetime in the matrix proved the applicability of the exciton model used. On the basis of the model, the average quantum yield of isolated polymers in the matrix was calculated to be a factor of 2 higher than in solutionsan effect arising from a 59% decrease in the nonradiative rate constant and, to a smaller extent, from a 20% increase in the radiative decay rate due to the different dielectric constants of the environments. These results suggest that by extending and isolating single luminescent polymers, high quantum yield devices are possible.