Recent developments of imaging techniques have enabled fluorescence microscopy to investigate the localization and dynamics of intracellular substances of interest even at the single-molecule level. However, such sensitive detection is often hampered by autofluorescence arising from endogenous molecules. Those unwanted signals are generally reduced by utilizing differences in either wavelength or fluorescence lifetime; nevertheless, extraction of the signal of interest is often insufficient, particularly for in vivo imaging. Here, we describe a potential method for the selective imaging of nitrogen-vacancy centers (NVCs) in nanodiamonds. This method is based on the property of NVCs that the fluorescence intensity sensitively depends on the ground state spin configuration which can be regulated by electron spin magnetic resonance. Because the NVC fluorescence exhibits neither photobleaching nor photoblinking, this protocol allowed us to conduct long-term tracking of a single nanodiamond in both Caenorhabditis elegans and mice, with excellent imaging contrast even in the presence of strong background autofluorescence.

Actin filament dynamics are critical in cell motility. The structure of actin filament changes spontaneously and can also be regulated by actin-binding proteins, allowing actin to readily function in response to external stimuli. The interaction with the motor protein myosin changes the dynamic nature of actin filaments. However, the molecular bases for the dynamic processes of actin filaments are not well understood. Here, we observed the dynamics of rabbit skeletal-muscle actin conformation by monitoring individual molecules in the actin filaments using single-molecule fluorescence resonance energy transfer (FRET) imaging with total internal reflection fluorescence microscopy (TIRFM). The time trajectories of FRET show that actin switches between low- and high-FRET efficiency states on a timescale of seconds. If actin filaments are chemically cross-linked, a state that inhibits myosin motility, the equilibrium shifts to the low-FRET conformation, whereas when the actin filament is interacting with myosin, the high-FRET conformation is favored. This dynamic equilibrium suggests that actin can switch between active and inactive conformations in response to external signals.

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We report the first real time imaging of single fluorophores attached to protein molecules on metal surfaces in aqueous solution using surface plasmon resonance fluorescence microscopy. The fluorescence was enhanced by the surface plasmons as theoretically predicted for gold and silver. Active movement of single molecules of the fluorescently labeled motor protein, coupled to the ATPase reaction, was observed on the surfaces of gold and aluminum. This microscopy should prove a powerful tool to directly detect single molecule processes in biomolecule systems organized on a metal surface.[ S0031-9007(98)

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Superfamily I helicases are nonhexameric helicases responsible for the unwinding of nucleic acids. However, whether they unwind DNA in the form of monomers or oligomers remains a controversy. In this study, we addressed this question using direct single-molecule fluorescence visualization of Escherichia coli UvrD, a superfamily I DNA helicase. We performed a photobleaching-step analysis of dye-labeled helicases and determined that the helicase is bound to 18-basepair (bp) double-stranded DNA (dsDNA) with a 3' single-stranded DNA (ssDNA) tail (12, 20, or 40 nt) in a dimeric or trimeric form in the absence of ATP. We also discovered through simultaneous visualization of association/dissociation of the helicase with/from DNA and the DNA unwinding dynamics of the helicase in the presence of ATP that these dimeric and trimeric forms are responsible for the unwinding of DNA. We can therefore propose a new kinetic scheme for the helicase-DNA interaction in which not only a dimeric helicase but also a trimeric helicase can unwind DNA. This is, to our knowledge, the first direct single-molecule nonhexameric helicase quantification study, and it strongly supports a model in which an oligomer is the active form of the helicase, which carries important implications for the DNA unwinding mechanism of all superfamily I helicases.

We have monitored the reaction dynamics of the DNA hybridization process on a liquid/solid interface at the single-molecule level by using a hairpin-type molecular beacon DNA probe. Fluorescence images of single DNA probes were recorded by using total internal reflection fluorescence microscopy. The fluorescence signal of single DNA probes during the hybridization to individual complementary DNA probes was monitored over time. Among 400 molecular beacon DNA probes that we tracked, 349 molecular beacons (87.5 %) were hybridized quickly and showed an abrupt fluorescence increase, while 51 probes (12.5 %) reacted slowly, resulting in a gradual fluorescence increase. This ratio stayed about the same when varying the concentrations of cDNA in MB hybridization on the liquid/surface interface. Statistical data of the 51 single-molecule hybridization images showed that there was a multistep hybridization process. Our results also showed that photostability for the dye molecules associated with the double-stranded hybrids was better than that for those with the single-stranded molecular beacon DNA probes. Our results demonstrate the ability to obtain a better understanding of DNA hybridization processes using single-molecule techniques, which will improve biosensor and biochip development where surface-immobilized molecular beacon DNA probes provide unique advantages in signal transduction.

Objectives: This randomized controlled clinical trial evaluated the 3-year clinical performance of a hybrid (Clearfil AP-X; AP) and a flowable (Clearfil Flow FX; FX) resin composite in 98 non-carious cervical lesions. Methods:Twenty-two patients, 11 male and 11 female (mean age: 61.9 years, range: 29-78 years) regularly visiting the Nagasaki University Hospital, participated in the study. Each patient received both materials randomly. All restorations (48 restorations for AP and 50 restorations for FX) were placed in conjunction with an all-in-one system (Clearfil S 3 Bond) by one dentist. The restorations were blindly evaluated by two examiners at baseline, 6 months, 1, 2 and 3 years using modified USPHS criteria. The data were statistically analyzed using the Cochran's Q test and Fisher's exact test.Results: All the patients were examined at each recall. However, five restorations could not be evaluated at 3-year recall as two teeth had been extracted and three restorations had been lost. The only minor problem was the integrity of the enamel margin. The incidence and extent of marginal staining increased with time, but it was still superficial. Marginal staining occurred adjacent to 11 restorations for AP and 12 restorations for FX after 3 years. Neither lesion size nor depth had influence on marginal staining adjacent to each type of resin composite. There were no significant differences in the clinical performances between AP and FX for each variable. Conclusions:Under the protocol used in this study, both types of resin composite in conjunction with S 3 Bond demonstrated an acceptable clinical performance up to 3 years.