Single molecule fluorescence microscopy investigations on heterogeneity of translational diffusion in thin polymer films

Translational diffusion of single perylene diimide molecules in 25 nm thin polymer films was investigated by single molecule widefield fluorescence microscopy. Spatial heterogeneities in single molecule motion were detected and analyzed by a new, quantitative method which draws a comparison of log-Gaussian fits of experimentally determined diffusion coefficient-distributions and diffusion coefficient-distributions from Monte Carlo random walk simulations. Heterogeneities could be observed close to the glass transition temperature, but disappear at ca. 1.1 × T(g). At higher temperatures, heterogeneities do not exist or they average out on the time and length scales of observation. The observed heterogeneities also explain why the dependency of diffusion coefficients on temperature does not follow Vogel-Fulcher-Tammann behavior.     

Multi-beam single-molecule defocused fluorescence imaging reveals local anisotropic nature of polymer thin films

We developed a method to determine full three-dimensional orientation distribution of individual molecules based on wide-field defocused fluorescence imaging. Excitation efficiencies of out-of-plane oriented molecules were improved dramatically by illuminating molecules with multiple laser beams. Our high throughput approach allowed us to obtain unbiased statistical distributions of orientations of doped molecules in spin-coated polymer thin films. We found thickness- and glass transition temperature-dependent distributions of the molecular orientations which reflect local chain orientations and relaxation in the polymer thin films.     

Non-fickian diffusion in thin polymer films

Diffusion of penetrants through polymers often does not follow the standard Fickian model. Such anomalous behavior can cause difficulty when designing polymer networks for specific uses. One type of non-Fickian behavior that results is so-called case II diffusion, where Fickian-like fronts initially move like √t with a transition to a non-Fickian concentration profile and front speed for moderate time. A mathematical model is presented that replicates this behavior in thin polymer films, and an analysis is performed that yields relevant dimensionless groups for study. An unusual result is derived: In certain parameter ranges, the concentration profile can change concavity, reflecting Fickian behavior for short times and non-Fickian behavior for moderate times. Asymptotic and numerical results are then obtained to characterize the dependence of such relevant quantities as failure time, front speed, and mass transport on these dimensionless groups. This information can aid in the design of effective polymer protectant films. © 1996 John Wiley & Sons, Inc.     

Specific features of high-temperature oxidation of thin polymer films

The dependence of oxidation rate on the sample thickness for high-temperature oxidation of polyphenylene oxide and of polycarbonate has been studied. Sharp maxima in curves of film thickness vs oxidation rate were observed for both polymers. These maxima can be explained assuming that at least two types of mobile low-molecular-weight radicals take part in the high-temperature oxidation reactions.     

Rh-V alloy formation in Rh-VOx thin films after high-temperature reduction studied by electron microscopy

Rh nanoparticles (mean size 10 and 15 nm), prepared by epitaxial growth on NaCl surfaces, were covered with layers of crystalline vanadium oxide (mean thickness 1.5 and 25 nm) by reactive deposition in 10(-2) mbar O2. The 1.5 nm film was further stabilized with a coating layer of 25 nm amorphous alumina. The so-obtained Rh/vanadia films, containing vanadium in the V3+ and V2+ state, were treated in 1 bar O2 at 673 K for 1 h and thereafter reduced in 1 bar H2 at increased temperatures, particularly between 723 and 873 K. The structural and morphological changes were followed by (high-resolution) transmission electron microscopy and selected area diffraction. Oxidation at 673 K transforms the purely vanadia-supported samples into Rh/V2O5, while in the alumina-supported films containing only small amounts of VOx, the formation of topotactic V2O3 is observed. The formation of Rh-V alloys during the subsequent reduction is strongly determined by the intimate contact and the structural and orientational relationship between Rh particles and the surrounding VOx phase. Reduction above 473 K transforms the support into substoichiometric vanadium oxides of composition VO and V2O. Analysis of high-resolution images and diffraction patterns reveals the presence of different alloy phases after reduction with increasing T (from 573 up to 823 K). In the alumina-supported film (low V/Rh ratio) the epitaxial alignment between the Rh particles and the surrounding V2O3 phase apparently favours the primary formation of defined alloys of type V3Rh and VRh3, followed by VRh at higher temperature. On the contrary, mainly V3Rh5 is formed in the purely VOx-supported Rh/films, due to different epitaxial relations in the initial state. Possible pathways of alloy formation are discussed.     

Circular dichroism probed by two-photon fluorescence microscopy in enantiopure chiral polyfluorene thin films

Two-photon fluorescence scanning confocal microscopy sensitive to circular dichroism with a diffraction-limited resolution well below 500 nm is demonstrated. With this method, the spatial variation of the circular dichroism of thermally annealed chiral polyfluorene thin films has been imaged. We observed circular dichroism associated with submicrometer-sized domains showing helicoidally twisted macromolecular organization. Domains with opposite chiroptical properties, corresponding to left- or right-handed molecular organization, coexist in the film. Our results are consistent with those obtained by one-photon imaging and illustrate the potential of two-photon imaging for use in studying helical macromolecular organization.     

Segment dynamics in thin polystyrene films probed by single-molecule optics

Single fluorescent molecules (represented by spheres with a volume equal to the actual van der Waals volume of the molecule) has been embedded in a polystyrene matrix (left). Such molecules act as probes for the study of polymer nanoscale (segmental scale) dynamics in thin films deposited on a glass cover slide (right).     

An ensemble and single-molecule fluorescence microscopy investigation of phase-separated monolayer films stained with Nile Red

Phase-separated Langmuir-Blodgett monolayer films prepared from mixtures of arachidic acid (C19H39COOH) and perfluorotetradecanoic acid (C13F27COOH) were stained via spin-casting with the polarity sensitive phenoxazine dye Nile Red, and characterized using a combination of ensemble and single-molecule fluorescence microscopy measurements. Ensemble fluorescence microscopy and spectromicroscopy showed that Nile Red preferentially associated with the hydrogenated domains of the phase-separated films, and was strongly fluorescent in these areas of the film. These measurements, in conjunction with single-molecule fluorescence imaging experiments, also indicated that a small sub-population of dye molecules localizes on the perfluorinated regions of the sample, but that this sub-population is spectroscopically indistinguishable from that associated with the hydrogenated domains. The relative importance of selective dye adsorption and local polarity sensitivity of Nile Red for staining applications in phase-separated LB films as well as in cellular environments is discussed in context of the experimental results.     

Analysis of high-temperature superconducting films by X-ray fluorescence analysis

Radionuclide X-ray fluorescence analysis was used for the determination of Cu, Y and Ba in very thin high-temperature superconducting films. The precision of the method is better than 3% for about 1 m thick films. The atomic emission ICP spectrometry was used to testify results of XRF analysis. An acceptable agreement of both methods was obtained.     

Segmental dynamics near the chain end of polystyrene in its ultrathin films: A study by single-molecule fluorescence de-focus microscopy

Rotational motion of fluorophores chemically attached to polystyrene chain-ends in ultra-thin films on solid substrates was studied by single-molecule fluorescence de-focus microscopy. The collective feature of the rotational motion was found and evidenced by the sharp change of the population of fluorophores undergoing rotational motion within a very narrow temperature range (named as the changing temperature, T _c). The T _c value was found to depend on film thickness and interfacial chemistry and the variation of the T _c value is also dependent on the molecular weight of the polymer. The results demonstrate that the spatial confinement effect enhances the segmental mobility near the polymer chain-ends while the interfacial attraction restricts the segmental motion inside the thin film.     

Nanostructure formation in polymer thin films influenced by humidity

The influence of relative humidity (RH) during the film preparation on the surface morphology and on the material distribution of the resulting technical polymer blend films consisting of poly (methyl methacrylate) (PMMA) and poly (vinyl butyral) (PVB) is investigated by atomic force microscopy. Both pure polymers and polymer blends with different compositions of PVB/PMMA dissolved in tetrahydrofuran (THF) were used. Polymer films prepared under dry conditions (RH < 20%) are compared with those that have the same polymer composition but were prepared under increased humidity conditions (RH > 80%). The films consisting of the pure polymers showed a nonporous surface morphology for low‐humidity preparation conditions, whereas high‐humidity preparation conditions lead to porous PVB and PMMA films, respectively. These pores are explained as the result of a breath figure formation. In the case of the polymer blend films containing both polymers, porous or phase‐separated surface structures were observed even at low‐humidity conditions. A superposition of the effects of phase separation and breath figure formation is observed in the case of polymer blend films prepared under high‐humidity conditions. Atomic force microscopy (AFM) images taken before and after the treatment with ethanol as a selective solvent for PVB indicate that PMMA is deposited on top of a PVB layer in the case of the low‐humidity preparation process whereas for high‐humidity conditions the silicon substrate is covered with a PMMA film. Copyright © 2006 John Wiley & Sons, Ltd.     

Study of bulk morphology of polymer latex films using laser confocal fluorescence microscopy

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Molecular probe techniques for studying diffusion and relaxation in thin and ultrathin polymer films

Two optically based, molecular probe techniques are employed to study relaxation and small-molecule translational diffusion in thin and ultrathin (thicknesses < ∼200 nm) polymer films. Second harmonic generation (SHG) is used to study the reorientational dynamics of a nonlinear optical chromophore, Disperse Red 1 (DR1) (previously shown to be an effective probe of α-relaxation dynamics) either covalently attached or freely doped in polymer films. Our studies on films ranging in thickness from 7 nm to 1 μm show little change in T_g with film thickness; however, a substantial broadening of the relaxation distribution is observed as film thickness decreases below approximately 150 nm. Experimental guidelines are given for using fluorescence nonradiative energy transfer (NRET) to study translational diffusion in ultrathin polymer films. Appropriate choice of a fluorescence donor species is important along with ensuring that diffusion is slow enough to be measured appropriately. Initial results on the diffusion of a small-molecule probe, lophine, in poly(isobutyl methacrylate) indicates that there is little change in probe diffusion coefficients in films as thin as 90 nm as compared to bulk films. © 1997 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 35: 2795–2802, 1997     

Concentric-ringed structures in polymer thin films

Novel polymer crystalline structures containing micrometer-sized concentric rings (or bands) were observed in thin poly(bisphenol A hexane ether) (BA-C6) films. The origin of the banded structures was found to be different from that of traditional banded spherulites in polymer systems. Analyses based on optical microscopy (OM) and atomic force microscopy (AFM) revealed that the banded structures contained alternating ridge and valley bands of polymer crystals in the flat-on orientation. No lamellar twisting was observed within the concentric-ringed structures, which were developed as a result of the formation of a depletion zone during crystallization. The formation of a depletion zone was determined to be caused by the specific volume decrement between the crystal and the melt and by the diffusion of polymer chains to the fold surfaces of the flat-on lamellae. The height of the ridges and the interband widths could be adjusted by controlling the diffusion rate. Time-of-flight secondary ion mass spectrometry ion images showed higher concentrations of low-molecular-weight polymer chains on the surfaces of the ridges than in the valleys.     

Detection of single-molecule DNA hybridization by using dual-color total internal reflection fluorescence microscopy

We examined the use of prism-type simultaneous dual-color total internal reflection fluorescence microscopy (TIRFM) to probe DNA molecules at the single-molecule level. The system allowed the direct detection of the complementary interactions between single-stranded probe DNA molecules (16-mer) and various lengths of single-stranded target DNA molecules (16-mer and 55-mer) that had been labeled with different fluorescent dyes (Cy3, Cy5, and fluorescein). The polymer-modified glass substrate and the extent of DNA probe immobilization were easily characterized either with standard TIRFM or with atomic force microscopy. However, only dual-color TIRFM could provide unambiguous images of individual single-stranded target DNA molecules hybridized with the correct sequence in the range of fM–aM. Succinic anhydride showed low RMS roughness and was found to be an optimal blocking reagent against non-specific adsorption, with an efficiency of 92%. This study provides a benchmark for directly monitoring the interactions and the detection of co-localization of two different DNA molecules and can be applied to the development of a nanoarray biochip at the single-molecule level.     

应用全内反射单分子荧光成像研究蛋白复合物亚基组成

细胞的生化过程大都是由蛋白复合物完成的,研究蛋白复合物亚基的组成对于了解蛋白质的结构和生物学功能具有重要的意义,然而如何准确确定蛋白复合物中蛋白质亚基的数量（stoichiometry）仍然是一个挑战.近年来,活细胞体系单分子荧光成像技术的不断发展为原位实时动态地研究蛋白质的结构和性质提供了新的手段.本文主要介绍了应用活细胞全内反射单分子荧光成像技术表征细胞膜区蛋白复合物组成的3种方法,包括单分子漂白步数分析、荧光强度统计分布以及蛋白运动分析,并结合其基本原理介绍了这几种方法在活细胞体系膜蛋白研究中的应用.     

Preparation of combinatorial arrays of polymer thin films for transmission electron microscopy analysis

We present a new method for harvesting multiple thin film specimens from polymer combinatorial libraries for transmission electron microscopy (TEM) analysis. Such methods are of interest to researchers who wish to integrate TEM measurements into a combinatorial or high-throughput experimental workflow. Our technique employs poly(acrylic acid) plugs, sequestered in an elastomer gasket, to extract a series of film patches from gradient combinatorial libraries. A strategy for simultaneous deposition of the array of film specimens onto TEM grids also is described. We demonstrate our technique using nanostructured polymer thin film libraries as test cases in which the nanoscale details can be successfully imaged. Microscopy of test case specimens demonstrates that these samples are of sufficient quality for morphology screening via TEM, and in some cases are sufficient for more detailed morphological studies.     

Structure of water incorporated in amphoteric polymer thin films as revealed by FT-IR spectroscopy

The structure and hydrogen bonding of water incorporated in a thin film of amphoteric terpolymers composed of various ratios of MA, DMAPMA, and BMA were analyzed using the band shapes of the O--H stretching in the IR spectra. At an early stage of sorption of water, the IR spectrum for the water incorporated in the film with comparative contents of MA and DMAPMA residues was similar to that for free water. This is consistent with the tendency for zwitterionic polymers, but is in contrast with the drastic change in the IR spectrum of water incorporated in non-ionic polymer films such as polyBMA. These results suggest a correlation between the mildness of the charge-balanced polymers to the structure of incorporated water and their blood compatibilities.     

Facile measurement of surface heat loss from polymer thin films via fluorescence thermometry

Quantitative determination of heat loss and transport within complex systems having inhomogeneous temperatures and several different components is important for applications ranging from electronics to solar cells. An approach and material system to study heat transport within and heat loss from polymer thin films is presented. In a thin film configuration with a cylindrical heating source, the theoretical solution for temperature as a function of radial position can be determined from fundamental principles. Use of embedded fluorescent molecules as temperature probes and manipulation of the relative location of heating and thermometry light sources allows experimental measurements of temperature versus position within the plane of the film. For a large range of practical cases, the exact theoretical solution can be well‐approximated by a single term, which enables a fit to experimental data, and subsequent determination of either the heat loss coefficient at the film's surface or the material's effective thermal conductivity. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2018 , 56, 643–651