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.

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.

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.

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.

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.