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...

We investigate experimentally the effects of electrostatic interactions and topological constraints on DNA dynamics in nanoslit confinement by studying the equilibrium shape and dynamics of single linear and circular λ-DNA confined in a silicon/glass nanoslit. Having examined the dependence of chain radius of gyration R ∥, shape asphericity A, and relaxation time τ on chain topology, slit height h (20–782 nm), and solvent ionic strength I (8.2–268.8 mM), it is found that the chain shape becomes more aspherical as h and I decrease. Moreover, in strong sub-Kuhn length confinement, the DNA relaxation time increases with decreasing h in a smooth and broad transition. Our results provide experimental evidence to confirm that the scaling exponents of radius of gyration and of relaxation time are the same for linear and circular DNA and help resolve conflicting observations of the qualitative dependencies of chain radius of gyration and relaxation time in sub-Kuhn length slits.

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Nearly thirty years ago, Daoud and de Gennes derived the scaling predictions for the linear polymer chains trapped in a slit with dimension close to the Kuhn length; however, these predictions have yet to be compared with experiments. We have fabricated nanoslits with vertical dimension similar to the Kuhn length of ds-DNA (110nm) using standard photolithography techniques. Fluorescently labeled single DNA molecules with contour lengths L ranging from 4 to 75 microm were successfully injected into the slits and the chain molecules undergoing Brownian motions were imaged by fluorescence microscopy. The distributions of the chain radius of gyration and the two-dimensional asphericity were measured. It is found that the DNA molecules exhibit highly anisotropic shape and the mean asphericity is chain length independence. The shape anisotropy of DNA in our measurements is between two and three dimensions (2D and 3D). The static scaling law of the chain extension and the radius of gyration , approximately L(nu) were observed with nuR(parallel)=0.65+/-0.02 and nu(Rg)=0.68+/-0.05. These results are close to the average value between two (nuR parallel,Rg=0.75) and three (nuR parallel,Rg=0.6) -dimensional theoretical value. The scaling of the extensional and rotational relaxation time are between Rouse model in nanoslits and Zimm model in the bulk solution, respectively. We show that the conformation and chain relaxation of DNA confined in a slit close to Kuhn length exhibit the quasi-two-dimensional behavior.

We investigate the conformation and relaxation dynamics of single DNA molecules in strong confinement (smaller than persistence length) with coarse-grained semiflexible chain (SFC) models using overdamped Langevin dynamics simulations. DNA properties in nanochannels and nanoslits are studied in confinement with height (H) ranging from the DNA radius of gyration (R g) to smaller than the persistence length (P). Qualitatively different dependences of chain conformation and relaxation time on H in moderate (P < H < R g) and strong (H < P) confinement are observed for very stiff SFC in the nanochannel but not in the nanoslit. The chain relaxation time (t relax) exhibits strong power-law dependence in H < P nanochannels, verified with and without including hydrodynamic interactions (HI). The inclusion of hydrodynamic interactions affects chain relaxation dynamics even in strong confinement, indicating the intersegmental hydrodynamic interactions affect dominant segmental relaxation mechanisms of strongly confined polymers.