Accurately determining single molecule trajectories of molecular motion on surfaces.

This paper presents a method for simultaneously determining multiple trajectories of single molecules from sequential fluorescence images in the presence of photoblinking. The tracking algorithm is computationally nondemanding and does not assume a model for molecular motion, which allows one to determine correct trajectories even when a distribution of movement speeds is present. We applied the developed procedure to the important problem of monitoring surface motion of single molecules under ambient conditions. By limiting the laser exposure using sample scanning confocal microscopy, long-time trajectories have been extracted without the use of oxygen scavengers for single fluorescent molecules. Comparison of the experimental results to simulations showed that the smallest diffusion constants extracted from the trajectories are limited by detector shot noise giving error in locating the positions of the individual molecules. The simulations together with the single molecule trajectories and distributions of diffusion constants allowed us therefore to distinguish between mobile and immobile molecules. Because the analysis algorithm only requires a time series of images, the procedure presented here can be used in conjunction with various imaging methodologies to study a wide range of diffusion processes.

Fluorescence correlation spectroscopy: criteria for analysis in complex systems.

We have evaluated the effect of varying three key parameters for Fluorescence Correlation Spectroscopy analysis, first in the context of a one species/one environment system, and then in a complex system composed of two species, or conversely, two environments. We establish experimentally appropriate settings for the (1) minimum lag time, (2) maximum lag time, and (3) averaging times over which an autocorrelation is carried out, as a function of expected diffusion decay time for a particular solute, and show that use of appropriate settings plays a critical role in recovering accurate and reliable decay times and resulting diffusion constants. Both experimental and simulated data were used to show that for a complex binary system, to extract accurate diffusion constants for both species, decay times must be bounded by adequate minimum and maximum lag times as dictated by the fast and slow diffusing species, respectively. We also demonstrate that even when constraints on experimental conditions do not permit achieving the necessary lag time limits for both of the species in a binary system, the accuracy of the recovered diffusion constant for the one species whose autocorrelation function is fully time-resolved is unaffected by uncertainty in fitting introduced by the presence of the second species.

Chain-length dependent nematic ordering of conjugated polymers in a liquid crystal solvent.

In this communication we demonstrate the dependence of the solute order parameter on the solute molecular weight for polymer solutes dissolved in liquid crystalline solvents. Using ensemble absorption polarization spectroscopy together with single molecule fluorescence polarization measurements, we have determined the order parameter of the conjugated polymer MEH-PPV (poly[2-methoxy-5-(2'-ethyl-hexyloxy)-1,4-phenylene vinylene]) in the liquid crystal 5CB (4-cyano-4-n-pentylbiphenyl) as a function of polymer chain length. Ensemble absorption polarization measurements agree well with results obtained by single molecule fluorescence polarization spectroscopy, indicating a large-scale ordering of the MEH-PPV solute in 5CB. These results demonstrate that the increasing number of defects for larger polymer weights inherently limits the alignment of the polymer solute.