Probing Lipid Vesicles by Bimolecular Association and Dissociation Trajectories of Single Molecules

Abstract

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.

Wide-field subdiffraction imaging by accumulated binding of diffusing probes

“…Nile red does not fluoresce in water but exhibits strong fluorescence in a hydrophobic environment, such as the lipid core. The mean residence time that was reported previously for Nile Red associated with liquid-phase LUVs is Ϸ6 msec when the vesicle radius was chosen as 50 nm (12). With the present conditions of Nile red concentration (10 Ϫ9 M or N ϭ 6⅐10 11 cm Ϫ3 ) and laser-power density (1 kW/cm 2 ), we found that the probability of observing two fluorescent molecules on a single LUV is very low.…”

“…These vesicles have been well characterized (15). The probe was chosen to be Nile red because the collisional kinetics of Nile red with LUVs has been studied in detail (12). Nile red does not fluoresce in water but exhibits strong fluorescence in a hydrophobic environment, such as the lipid core.…”

“…The collision rate of probes with a single object is easily controlled, because it depends linearly on the concentration of the probes. The rate of appearance of fluorescent spikes is then approximated by the diffusion-controlled bimolecular reaction rate constant (11) times the probability that a collision will lead to a binding configuration that generates a signal from the probe (12). Further discrimination between locations in an image can be obtained if the probe fluorescence is sensitive to the chemical composition of the object.…”

Characterization of the Surface Contribution to Fluorescence Correlation Spectroscopy Measurements

“…A threshold should be set where the on-and off-time histogram does not change significantly with a slight change in the threshold. 25,44 For our data, 0.1 ms was the smallest possible binning time for an analyzable signal-to-noise ratio and a threshold of 1 was the suitable value. The on-and off-time distribution is well described by an exponential function, the characteristic time of which is the average distribution.…”

“…Recently, FCS has been widely used to study the kinetics of biocells near the surface to understand the nature of membranes. 4,6,9,24,25 When investigating the dynamics of living cells or studying the properties of cell membranes, one cannot avoid taking into account the surface contribution; this is referred to as surface artifacts. [26][27][28] The surface contribution needs to be characterized to get an actual picture of the dynamical properties at the cell membrane.…”