Vesicles prepared by DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) and SOPC (1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine) lipid molecules having sizes smaller than the diffraction-limited focused laser beam have been used to confine single molecules in the laser focus. The confinement of single molecules in a volume smaller than the focused laser beam leads to a Gaussian distribution of single molecule fluorescence intensity. The interactions of single Nile Red molecules with DMPC and SOPC lipid bilayers were studied by single molecule fluorescence confocal microscopy. Nile Red molecules were observed to associate with and dissociate from individual DMPC and SOPC vesicles adsorbed on a glass surface, generating on-and-off fluctuations in a fluorescence signal representing a very low noise two-state trajectory. Off-time statistics were used to investigate the mean radius of the vesicles and the size distribution functions. The means of the on-time distributions of Nile Red in DMPC and SOPC vesicles were significantly different. The association and dissociation reactions of single Nile Red molecules with a vesicle have been studied. Features of the bimolecular interaction between the probe Nile Red and the vesicle were evaluated from the uncorrelated mean on-time and vesicle radius distributions, and the linear Nile Red concentration dependence of the mean off-time. Nile Red is shown to be a useful probe of the structural fluctuations and heterogeneity of these membrane structures, and it is a useful model with which to directly study a diffusion-influenced reversible bimolecular reaction.

The long and the short of it: Luminol chemiluminesces with a beautiful blue color; however, to be useful for biotechnological applications, the emission must be shifted to much longer wavelengths. Energy‐transfer cassettes like that shown in the picture provide one solution.

The fluorescence intensity-time records of individual metal-free porphyrin cytochrome-c and Zn porphyrin cytochrome-c molecules whose translational motions are restricted by encapsulation in trehalose are examined by single-molecule spectroscopy by means of a two-channel confocal microscope that records transient fluorescence signals in two orthogonal polarization directions. Large angular motions often occur on time scales ranging to many seconds. Measurements of the photobleaching time distributions indicate that the trehalose glass restricts the accessibility of the fluorescent molecules to oxygen.

We previously demonstrated using noninvasive technologies that the interferon-gamma (IFN-γ) receptor complex is preassembled [1]. In this report we determined how the receptor complex is preassembled and how the ligand-mediated conformational changes occur. The interaction of Stat1 with IFN-γR1 results in a conformational change localized to IFN-γR1. Jak1 but not Jak2 is required for the two chains of the IFN-γ receptor complex (IFN-γR1 and IFN-γR2) to interact; however, the presence of both Jak1 and Jak2 is required to see any ligand-dependant conformational change. Two IFN-γR2 chains interact through species-specific determinants in their extracellular domains. Finally, these determinants also participate in the interaction of IFN-γR2 with IFN-γR1. These results agree with a detailed model of the IFN-γ receptor that requires the receptor chains to be pre-associated constitutively for the receptor to be active.

Single-molecule spectroscopy is used to characterize the microenvironments found in silicate thin films dried under different conditions. Local film properties are assigned on the basis of the fluorescence emission characteristics of individual dopant (rhodamine B) molecules. The samples studied include those characterized immediately after being spin cast onto a glass substrate (fresh samples) and after drying at ≈80°C in a vacuum oven for at least 12 h (dried samples). The single-molecule fluorescence spectra shift to the red for films dried under more rigorous conditions, reflecting increased average film polarity. The distribution of fluorescence emission maxima also broadens slightly with drying, reflecting an increase in film heterogeneity. Bimodal distributions in the widths of the emission maxima are observed. These distributions exhibit a narrowing of the single-molecule emission with drying, pointing to greater microenvironmental rigidity. Studies of the time-dependent emission characteristics of the single molecules show the total number of photons emitted (prior to bleaching) by the molecules in the dried films is four (3.6 ( 0.6) times greater than in the fresh films. A 4-fold (4.3 ( 0.7) increase in the average survival time of the molecules is also observed, proving that increased dye emission from the dried films results primarily from an increase in dye stability, rather than an increase in fluorescence quantum yield. It is also shown that the single-molecule emission fluctuates more rapidly in the dried films, possibly due to an increase in the rate of triplet formation and/or an increase in the triplet lifetime. Increased dopant stability is attributed to reduced oxygen and dye mobility within the more dense, highly cross-linked silicate network of the dried films. FTIR studies of the thin films provide additional support for these conclusions.