Single molecule tracking studies of lower critical solution temperature transition behavior in poly(N-isopropylacrylamide).

Spatial and temporal heterogeneities in expanded and collapsed surface bound poly(N-isopropylacrylamide), pNIPAAm, films are studied by single molecule tracking (SMT) experiments. Tracking data are analyzed using both radius of gyration (R(g)) evolution and confinement level calculations to elucidate the range of behaviors displayed by single Rhodamine6G (R6G) molecules. Confined diffusion that is dictated by the free volume within surface tethered chains is observed with considerable dispersion among individual R6G molecules. Thus, the distribution of probe behavior reflects nanometer-scale information about the behavior of the probe-polymer system at temperatures above (T > T(LCST)) and below (T < T(LCST)) the lower critical solution temperature (LCST). In this context, confinement-level analysis and R(g) evolution both show a larger degree of confinement of the probe in pNIPAAm at T > T(LCST). Temperature-dependent changes in confinement are evidenced at T > T(LCST) by a higher percentage of confined steps, longer periods of confined events, and smaller area of confined zones, as well as a shift in the overall distribution of R(g) evolution paths and final R(g) distributions.

Quantitative fluorescence microscopy to determine molecular occupancy of phospholipid vesicles.

Encapsulation of molecules in phospholipid vesicles provides unique opportunities to study chemical reactions in small volumes as well as the behavior of individual proteins, enzymes, and ribozymes in a confined region without requiring a tether to immobilize the molecule to a surface. These experiments generally depend on generating a predictable loading of vesicles with small numbers of target molecules and thus raise a significant measurement challenge, namely, to quantify molecular occupancy of vesicles at the single-molecule level. In this work, we describe an imaging experiment to measure the time-dependent fluorescence from individual dye molecules encapsulated in ~130 nm vesicles that are adhered to a glass surface. For determining a fit of the molecular occupancy data to a Poisson model, it is critical to count empty vesicles in the population since these dominate the sample when the mean occupancy is small, λ ≤ ~1. Counting empty vesicles was accomplished by subsequently labeling all the vesicles with a lipophilic dye and reimaging the sample. By counting both the empty vesicles and those containing fluors, and quantifying the number of fluors present, we demonstrate a self-consistent Poisson distribution of molecular occupancy for well-solvated molecules, as well as anomalies due to aggregation of dye, which can arise even at very low solution concentrations. By observation of many vesicles in parallel in an image, this approach provides quantitative information about the distribution of molecular occupancy in a population of vesicles.

Structural characterization of individual vesicles using fluorescence microscopy.

Extrusion of hydrated lipid suspensions is frequently employed to produce vesicles of uniform size, and the resulting vesicles are often reported to be unilamellar. We describe a method for the quantitative fluorescence image analysis of individual vesicles to obtain information on the size, lamellarity, and structure of vesicles produced by extrusion. In contrast to methods for bulk analysis, fluorescence microscopy provides information about individual vesicles, rather than averaged results, and heterogeneities in vesicle populations can be characterized. Phosphatidylcholine vesicles containing small fractions of biotin-modified phospholipid and fluorescently labeled 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD) phospholipid were immobilized through biotin-avidin-biotin binding to the surface of a biotin-modified glass coverslip. Biotin was attached to the surface in a mixed cyano-terminated silane monolayer. Initial fluorescence intensities for each immobilized vesicle were recorded, and a solution of membrane impermeable quencher was passed through the flow cell to quench the fluorescence of the outer layer. Fluorescence from individual vesicles was measured by fitting the spots to 2-dimensional Gaussian functions. The integrated signals under the peaks yielded a pre- and postquench intensity. From the fractional loss of intensity, the number and structure of the bilayers in individual vesicles could be quantified; the results showed that extruded vesicles exhibit a distribution of size, lamellarity, and structure.

Trajectory analysis of single molecules exhibiting non-brownian motion.

Four techniques for analyzing single molecule tracking data--confinement level analysis, time series analysis and statistical analysis of lateral diffusion, multistate kinetics, and a newly developed method, radius of gyration evolution analysis--are compared using a set of sample fluorophore trajectories obtained from the lipophilic carbocyanine dye 1,1'-dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine, DiIC(18), partitioned into surface tethered poly(n-isopropylacrylamide). The purpose here is two-fold: first to test that these techniques can be applied to single molecules trajectories, which typically contain a smaller total number of frames than those obtained from other particles, e.g. quantum dots or gold nanoparticles; and second to critically compare the information obtained from each method against the others. A set of five SMT trajectories, ranging in length from 41 to 273 steps with a 30 ms frame transfer exposure, were all successfully analyzed by all four techniques, provided two important criteria were met: enough steps to define the motion were acquired in the trajectory, generally on the order of 50 steps, and the fast and slow diffusion coefficients differ by at least a factor of 5. Beyond that the four trajectory analysis methods studied provide partially confirmatory and partially complementary information. SMT data resulting from more complex physical behavior may well benefit from using these techniques in succession to identify and sort populations.

Identification of single fluorescent labels using spectroscopic microscopy.

Detection of single, fluorescently labeled biomolecules is providing a powerful approach to measuring molecular transport, biomolecular interactions, and localization in biological systems. Because the biological molecules of interest rarely exhibit sufficient intrinsic fluorescence to allow observation of individual molecules, they are usually labeled with fluorescent dye molecules, fluorescent proteins, semiconductor nanocrystals or quantum dots, or fluorescently doped silica or polymer nanospheres to allow their detection. Differences in the photophysical and spectral properties of different labels allow one to identify individual molecules by distinguishing their corresponding labels. A simple approach to measuring fluorescence spectra of individual fluorescent labels can be implemented in a standard wide-field fluorescence microscope, where a grating or prism is incorporated into the path from the microscope to an imaging detector to disperse the emission spectrum. In this work, principal components and cluster analysis are applied to the identification of fluorescence spectra from single fluorescent labels, with statistical tests of the classification results. Spectra are determined from diffracted images of fluorescent nanospheres labels, where emission maxima are separated by less than 20 nm, and of single dye-molecule labels with 30 nm separation. Clusters of points in an eigenvector representation of the spectra correctly classify known labels (both nanospheres and single molecules) and unambiguously identify unknown labels in mixtures.