Anisotropic Polyethylene Nanocrystals Labeled with a Single

Article pubs.acs.org/Macromolecules

Anisotropic Polyethylene Nanocrystals Labeled with a Single Fluorescent Dye Molecule: Toward Monitoring of Nanoparticle Orientation Benjamin Scheinhardt,†,‡,∥ Justyna Trzaskowski,†,∥ Moritz C. Baier,† Beate Stempfle,† Alex Oppermann,† Dominik Wöll,*,†,‡,§ and Stefan Mecking*,†,‡ †

Department of Chemistry, University of Konstanz, Universitätsstrasse 10, 78457 Konstanz, Germany Konstanz Research School Chemical Biology, University of Konstanz, Universitätsstrasse 10, 78457 Konstanz, Germany § Zukunftskolleg, University of Konstanz, Universitätsstrasse 10, 78457 Konstanz, Germany ‡

S Supporting Information *

ABSTRACT: The three-dimensional orientation monitoring of anisotropic nanoparticles during dynamic processes is a fundamental issue. Herein we show that incorporation of a single fluorescent reporter molecule is a promising concept toward this goal. As a model system, shape anisotropic single lamella polyethylene (PE) nanocrystals bearing one single fluorescent reporter molecule were prepared via ring-opening metathesis polymerization (ROMP) of highly ring-strained trans-cyclooctene (trCOE) using a mixture of a dye-functionalized ruthenium-based initiator (1; perylene diimide (PDI) substituted Hoveyda−Grubbs second generation Ru alkylidene) and an appropriate excess of the unlabeled analogue (2; Hoveyda−Grubbs second generation Ru alkylidene) in aqueous microemulsion as a key step and subsequent exhaustive hydrogenation (>99.9%) of the main-chain unsaturated polymer in the nanoparticles to yield nanocrystals of high molecular weight, strictly linear PE (Mn = 8 × 105 g mol−1; Mw/Mn = 1.4). TEM and AFM show a particle thickness of ca. 12 nm with a lateral extension of typically 45 nm. Comparable initiation kinetics of both complexes 1 and 2, which is a key requirement for this approach, were revealed by fluorescence spectroscopy studies (ΔH‡ = 57.4 kJ mol−1, ΔS‡ = −73.0 J mol−1 K−1 for 1 vs ΔH‡ = 63.6 kJ mol−1, ΔS‡ = −80.8 J mol−1 K−1 for 2 for the initiation with n-butyl vinyl ether, respectively). The labeled nanocrystals were characterized by means of single molecule fluorescence spectroscopy. Orientational analysis via defocused wide-field fluorescence microscopy (DWFM) revealed a fixed orientation of the chromophores within the nanocrystals, with their long molecular axis predominantly oriented parallel to the polar axis of the nanoparticles.

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INTRODUCTION The morphology of colloidal nanoparticles was shown to have a crucial influence on their characteristic features, e.g., optical and plasmonic properties,1 cellular internalization behavior,2 and colloidal self-assembly,3 and consequently on the functional behavior of these particles under a wide variety of conditions. In comparison to spherical systems, an increased number of degrees of freedom exist with respect to mutual alignment of anisotropic objects. As a result, a direction dependence of different properties is added, making these systems far more complex than isotropic objects. This also applies to larger structures formed by assembly of such particles; e.g., materials generated from anisotropic particles can be expected to possess direction-dependent mechanical properties like tensile strength.4 A deeper understanding of such shape-related phenomena is desirable, as it is of high potential interest, e.g., for the design of new functional materials. In this context, concepts for a three-dimensional (3D) orientation monitoring of nanoparticles during dynamic processes via common microscopy techniques are of interest as they could deliver new insights into anisotropic interactions © 2013 American Chemical Society

in this very small size regime, such as local organization and assembly. However, so far only very few concepts which address this fundamental issue have been reported.5,6 Employing single molecule fluorescence spectroscopy techniques (SMFS) is an attractive approach toward this long-term goal. Unlike traditional microscopy techniques to resolve nanoheterogeneities, such as atomic force microscopy (AFM)7 or electron microscopy,8 they allow for highly sensitive, noninvasive online measurements and simultaneously have the power to reveal a distribution of values which can provide unique information about dynamic processes and local environments surrounding a probe molecule.9 Both translational and rotational motion of single emitters can be resolved using defocused wide-field fluorescence microscopy (DWFM),10 which exploits the characteristic transmission dipole emission patterns of individual molecules to determine their spatial molecular orientation.11,12 Its utility to detect the Received: September 2, 2013 Revised: September 17, 2013 Published: September 30, 2013 7902

dx.doi.org/10.1021/ma401828k | Macromolecules 2013, 46, 7902−7910

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Scheme 1. Approach for the Preparation of Well-Defined Polyethylene Nanocrystals Labeled with a Single Fluorescent Reporter Molecule via ROMP in Aqueous Microemulsion and Subsequent Hydrogenationa

a Covalently attached fluorescent PDI reporter molecules are incorporated into the amorphous layers, which cover the crystalline lamella. For the sake of clarity, the adsorbed SDS molecules are not shown in the case of the PE nanocrystal.

mers in aqueous solution,21 and crystallization of precisely branched linear polyethylene in nanoscale droplets.22 An analysis of such nanoparticles composed of linear polyethylene by a combination of cryo-TEM and SAXS revealed these to be polymer single crystals composed of one lamella and with an extension of only a few tens of nanometers also in the lateral direction (cf. Scheme 1).18 However, the precise covalent labeling of the resulting anisotropic PE nanocrystals for microscopy investigations requires a different approach. Herein we report on the direct preparation of anisotropic PE nanocrystals functionalized with one single dye molecule per particle as a novel approach toward monitoring the orientation of anisotropic nanoparticles via DWFM in this challenging size regime.

anisotropic 3D orientation distribution of single molecules in thin films has recently been demonstrated.13 Thus, transferring this technique from a single molecular level to anisotropic nanoobjects by introducing a single fluorescent reporter molecule into the nanoparticles might provide deeper insights into their dynamics. Concerning an access to suitable model systems, the preparation of defined nonspherical colloidal particles with sizes smaller than ca. 50 nm is challenging. They often represent a thermodynamically unfavorable state in terms of interfacial free energy and minimization of the surface energy thus drives the particle to adopt a spherical shape.14 However, this small size regime appears relevant as e.g. in hybride materials of organic and inorganic particles the resulting very high degree of dispersion enhances interaction between the phases. While for inorganic nanoparticles, comprising metals as well as metal oxides, a broad spectrum of shapes was realized and it is well established that the shape and physical properties are controlled by the crystalline structure and the crystallization process,4,15 functionalization of such inorganic nanoparticles, e.g. with fluorescent reporter molecules, requires binding of functional organic ligands to inorganic surfaces, which generally limits this approach. Furthermore, precise control over functional ligand composition most often is difficult to achieve due to unbalanced stoichiometries between small molecules and relatively massive nanoparticles.16 By comparison, welldirected functionalization of organic materials is much more feasible and can be accomplished during the preparation process in a well-controlled manner. Thus, our concept pursued here to generate well-defined shape-anisotropic nanoparticles exploits crystalline order in polymers, which in contrast to inorganic materials results from van der Waals interactions between adjacent chain segments, in very small compartments as a structure-forming principle. In this regard polyethylene (PE) is of particular interest, since it is the prototype of a crystalline polymer and its crystallization behavior has been studied intensely in the past.17 Different methods have been developed for the synthesis of anisotropic PE single crystal nanoparticles with variable size and shape, such as insertion polymerization of ethylene in aqueous surfactant solution,18 free-radical emulsion polymerization at intermediate pressures,19 crystallization of the hydrophobic core of micelles formed from diblock copolymers with a PE and a hydrophilic block,20 self-assembly of linear ethylene−acrylic acid copoly-

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RESULTS AND DISCUSSION Fluorescence labeled polyethylene nanocrystals were prepared via a two-step reaction involving ring-opening metathesis polymerization (ROMP) of an appropriate cyclic olefin in aqueous microemulsion in the first step followed by hydrogenation of the main-chain unsaturated polymer in the nanoparticles as the second step (Scheme 1). For the introduction of a covalently bound fluorescent reporter molecule ROMP is the method of choice, since it offers variable possibilities to attach the desired fluorescence functionality23 and can be conducted in aqueous systems,24 a desirable feature for the preparation of nanoparticles directly during the polymerization reaction. trans-Cyclooctene (trCOE) was chosen as the monomer, since it combines both the potential to generate strictly linear PE25 and the high ring strain (16.7 kcal mol−1)26 essential for the achievement of high molecular weights, which is in turn crucial for the formation of crystallizable segments. Fluorescence Functionalized ROMP Initiator. The chain-end functionalization of the polymer formed was accomplished by the use of perylene diimide (PDI) labeled ruthenium-based alkylidene precursor 1 (Figure 1), which structurally resembles the well-known second generation Hoveyda−Grubbs initiator 2. This approach enables the selective introduction of the desired fluorescence functionality directly during the polymerization process and avoids further postpolymerization conversions of functional end groups. Specific anticipated features of the targeted structure of 1 were: First, tetra-bay-substituted PDI dyes as such are favored 7903


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(transparent) o/w microemulsions of the cyclic monomer and of the initiator 1 were prepared separately. A suitable composition which is in the microemulsion regime was found to be oil phase (monomer or a toluene solution of the initiator, respectively):SDS:pentanol:water in a mass ratio of 4:10:5:81. Initiator 1 was stable under these aqueous conditions, as concluded from the finding that no change of the fluorescence intensity was observed over more than 1 h. Note in this context that fluorescence of the dye in the precursor is quenched by the ruthenium metal center and is restored upon cleavage from the metal (vide infra). Upon mixing appropriate amounts of both microemulsions fast initiation occurred, as evidenced by a rapid increase of the fluorescence intensity. Nearly complete monomer consumption (>90%) was observed after stirring for 20 min for all entries of Table 1 (cf. Supporting Information). Addition of ethyl vinyl ether after full monomer consumption in order to terminate all metathesis-active species, including possibly noninitiated 1, and subsequent precipitation in acetone yielded light red polymer. Upon several cycles of dissolving the polymer in CH2Cl2 and precipitating in acetone, neither discoloration of the polymer nor coloration of the supernatant by free dye was observed. This confirms the absence of noninitiated 1 and full incorporation of the monofunctional dye as polymer end group. Molecular weights as determined by GPC (in THF vs PS standards) typically amount to Mn = (2−3) × 106 g mol−1, independent of the monomer-to-initiator ratio within experimental error, with a narrow molecular weight distribution of Mw/Mn = 1.2−1.5. Similar results were obtained with the unlabeled analogue 2 under otherwise identical conditions (Table 1, entries 4−6), showing that the fluorescence functionalization in initiator 1 has no undesirable impact on its catalytic properties and, thus, on the polymer properties. Polymer molecular weight distributions (Tables 1 and 2) and the finding that molecular

Figure 1. Ruthenium-based second generation alkylidene complexes used in this study. Perylene diimide (PDI) substituted Hoveyda− Grubbs (1), Hoveyda−Grubbs (2), and Grubbs (3) ruthenium alkylidenes.

chromophores in SMFS investigations due to their high quantum yields and excellent photostabilities.27 Second, Hoveyda−Grubbs type ruthenium alkylidenes have been shown to be extremely tolerant toward functional reagents, making this catalyst system an ideal candidate for our envisioned application in aqueous systems.28 Initiator 1 was synthesized via a carbene exchange reaction by gently warming a mixture of a vinyl isopropoxyphenyl substituted PDI dye and Grubbs second generation ruthenium alkylidene 3 in the presence of CuCl as a phosphine scavenger in CH2Cl2. After purification by column chromatography 1 was obtained in 60% yield. Characteristic 1H NMR resonances comprise a signal for the carbene proton at δ = 16.6 ppm. Aqueous Microemulsion Polymerizations. As a first step, polymerizations of trCOE in aqueous microemulsion with variable monomer-to-initiator ratios were performed using labeled initiator 1 (Table 1, entries 1−3). For this purpose, two

Table 2. Hydrogenation of Unsaturated Polycyclooctene Dispersionsa

initiator

equivb

Mn (106 g mol−1)c

Mw/Mnc

particle sized (nm)

1 2 3 4 5 6 7

1 1 1 2 2 2 2:1 = 150

5 000 10 000 20 000 5 000 10 000 20 000 20 000

2.5 3.6 2.5 1.7 2.4 3.0 3.8

1.19 1.17 1.34 1.49 1.33 1.20 1.26

21 23 29 22 26 31 29

functional chain endb

Mn (105 g mol−1)c

Mw/Mnc

particle sized (nm)

1 2 3

R = PDI (from 1) R = H (from 2) R = H/PDI

6.0 8.4 8.1

1.37 1.40 1.40

34 31 29

a

Monomer/initiator = 20 000 (Table 1, entries 3, 6, and 7); hydrogenation conditions: 100 bar H2, 65 °C, 65 h, addition of an aqueous microemulsion of 3 (quenched with ethyl vinyl ether), monomer/3 = 1500. bDetermined by the initiator used during the microemulsion polymerization process. cDetermined by GPC at 160 °C in TCB vs polyethylene standards. dNumber-average particle size determined by DLS.

Table 1. Aqueous Microemulsion Polymerizations of trCOE Using Initiators 1 and 2a entry

entry

weights do not vary strongly with the monomer-to-initiator ratio suggest that a certain extent of backbiting occurs during polymerization at the high substrate concentration present in the microemulsion droplets.29 Note in this context that in terms of absolute values the molecular weights determined on the hydrogenated polymers (Table 2) more accurately reflect true values, as they are determined vs polyethylene standards by comparison to apparent molecular weights of the unsaturated polycyclooctene vs polystyrene standards. Number-average particle sizes of 21−33 nm, slightly increasing with a higher monomer-to-initiator ratio, with a narrow particle size distribution of typically 99.9%) PE nanocrystals, as concluded from the absence of olefinic resonances at δ = 5.35 ppm in 1H NMR spectra and from the absence of =CH vibrations at 667 cm−1 (cis) and 965 cm−1 (trans) of isolated bulk material (Figures S7 and S8). As a consequence of the hydrogenation process, a lamella forms by crystallization of saturated PE segments in the nanoscale droplets to yield anisotropic PE nanocrystals. Differential scanning calorimetry (DSC) typically revealed a peak melting point of Tm = 137 °C, associated with a melt enthalpy of ΔHm = 160 J g−1, and thus confirmed the crystalline nature of the as-obtained PE nanoparticles. Particle sizes remained unaltered during the hydrogenation procedure, indicating that the reaction occurs in individual particles which preserve their identity (Figure 2a). Fluorescence spectroscopy on multiple labeled (initiated with 1, without added 2) nanoparticle dispersions before (Table 1, entry 3) and after (Table 2, entry 1) hydrogenation showed no alteration in absorption and emission spectra (Figure 2b). This confirms the retained intact nature of the dye throughout the synthesis process. The fluorescence quantum yield after hydrogenation slightly decreased to 74%, probably due to residual ruthenium in the particles. Molecular weights as determined by GPC (in TCB vs PE standards) amount to Mn = (6−8) × 105 g mol−1 with a narrow molecular weight distribution of Mw/Mn = 1.4. Transmission electron microscopy (TEM) on the single labeled PE nanocrystals NP-1 (Table 2, entry 3) confirmed an anisotropic oblate-like shape, with a shorter extension along the z-direction (Figure 2c,d). The particles possess an 7905


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Figure 3. (a) Wide-field fluorescence image of NP-1 spin-coated on a glass substrate. (b) Time-resolved fluorescence intensities of the particles indicated in (a).

Figure 4. (a) Schematic representation of the angular coordinates: the out-of-plane angle θ is defined as the inclination angle between the emission dipole (red arrow) and the optical axis (z-axis), and ϕ indicates the in-plane (xy image plane) angle. (b) Calculated defocused patterns of a single emitter for 10 different out-of-plane orientations (constant in-plane angle ϕ). (c) Typical defocused widefield image of NP-1 spin-coated on a glass substrate. (d) Frequency histogram of the out-of-plane angle θ determined for single labeled PE nanocrystals NP-1 spin-coated on a glass substrate (analysis of 209 individual particles). The black stars represent an orientation distribution expected for randomly oriented molecules. (e) Typical defocused wide-field image of NP-1 embedded in a 50−300 nm thick PVA matrix. (f) Frequency histogram of the out-of-plane angle θ determined for single labeled PE nanocrystals NP-1 embedded in a 50−300 nm thick PVA matrix (analysis of 109 individual particles). The black stars represent an orientation distribution expected for randomly oriented molecules.

equatorial diameter of ca. 45 nm and a height of ca. 12−16 nm, which is in good agreement with atomic force microscopy (AFM) analysis of individual particles (Figure 2e and Figure S13), indicating a single crystalline habitus, where the crystalline lamella is sandwiched between two amorphous layers (cf. Scheme 1).32 Three-Dimensional Orientation Analysis of Incorporated Chromophore Molecules. For wide-field fluorescence microscopy, the particles NP-1 (Table 2, entry 3) were spincoated on a glass substrate and their fluorescence was imaged on a CCD camera. The majority of fluorescent particles exhibited a binary blinking and one-step photobleaching behavior typical for a single emitter (Figure 3). Only a minor fraction of nanoparticles showed two-step bleaching in the fluorescence time traces indicating the presence of two

chromophores within one particle. This confirms the number of incorporated PDI dye molecules per nanoparticle to follow Poisson statistics, as anticipated (vide supra). For the analysis this small number of double-labeled nanoparticles was discarded. The 3D orientation of the incorporated chromophores could be visualized directly using DWFM, since the transition dipole moment of the S0−S1 excitation of the PDI dye, described by the azimuthal angle ϕ and the polar angle θ (Figure 4a), is aligned parallel to its long molecular axis. This technique images the anisotropic fluorescence pattern of single fluorophores which depends on the orientation of the transition dipole moment with respect to the optical axis of the microscope. Defocused emission patterns were calculated for different polar angles θ (Figure 4b). The patterns for different 7906


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azimuthal angles ϕ can be obtained by turning each of these patterns by the corresponding angle. For DWFM measurements, a quasi total internal reflection (qTIRF) excitation was used to ensure efficient excitation of molecules with low polar angles (out-of-plane oriented molecules),33 and the sample was positioned ∼1 μm toward the microscope objective from the focus. The single labeled nanocrystals NP-1 were spin-coated on a glass substrate, adjusting the particle density in such a way that the minimal distance between two adjacent particles was ∼1 μm. This distance assures that the patterns are well separated for an accurate analysis. AFM studies on individual particles revealed a uniform thickness of the nanocrystals of 12−16 nm, indicating that under these conditions the crystals orient exclusively with their lamellae lying flat on the substrate, i.e., with their polar axis perpendicular to the substrate (Figures S13 and S14). Although the defocused images obtained showed various emission patterns, a high fraction of asymmetric ringed patterns, typical for substantially out-of-plane oriented transition dipole moments, was observed (Figure 4c and Figure S16). Reorientation of the incorporated reporter molecules, i.e., translational or rotational mobility, was not observed, indicating that their orientation is fixed within the nanocrystal and consequently is steadily linked to the nanoparticle orientation.34 In terms of monitoring absolute nanoparticle orientations, a uniform orientation of the incorporated reporter molecules with respect to the nanoparticle symmetry axes is essential in order to enable a correlation between both orientations. Hence, for a detailed orientation analysis, the chromophore orientations in 209 independent particles were determined by visual comparison of the observed patterns with the calculated patterns. Note that this mode of analysis was found to be more reliable and less prone to errors for our system than a fully automated pattern recognition. In this context it is worth mentioning that the most pronounced alteration of defocused patterns under the conditions applied here occurs in a range of 10° ≤ θ ≤ 60°, where the accuracy of angle determination is higher than at polar angles of >60°, where it becomes progressively harder to distinguish the patterns (Figure 4b). The resulting distribution of polar angles θ clearly shows a distinct nonstatistical behavior (Figure 4d): Approximately 60% of the reporter molecules possess an orientation of their transition dipole moment with a polar angle of ≤30°, which represents a 4-fold increase compared to randomly oriented molecules (Figure S18). Thus, correlating this finding with the nanocrystal orientation under the measurement conditions (vide supra), the long molecular axis of the chromophore is preferentially oriented parallel to the polar axis of the nanoparticles. Since the particles lie flat on the substrate with an arbitrary in-plane orientation, they exhibit an equal distribution of azimuthal angles of the embedded reporter molecules which, thus, were not further considered. In order to underline this finding, the single labeled particles NP-1 were embedded in a 50−300 nm thick poly(vinyl alcohol) (PVA) matrix since under these conditions a statistical orientation distribution of the nanocrystals and consequently of the incorporated chromophores was expected. DWFM on these PVA films demonstrated that the majority of obtained patterns show a clear two-lobe structure, as expected for a statistical orientation distribution (Figure 4e and Figure S17). Analysis of 109 individual embedded particles revealed a random distribution (Figure 4f), demonstrating the suitability of this approach for nanoparticle orientation analyses.

Because of the pronounced sterical demand and hydrophobicity of the PDI reporter molecule, excluding its localization in the polymer crystalline phase and the surrounding aqueous phase, respectively, it is anticipated to be incorporated into the amorphous region surrounding the nanocrystal (cf. Scheme 1). The orientation of PDI dyes bearing coordinating substituents, e.g. pyridyl groups, on inorganic (nanoparticle) surfaces recently was found to show a preferred perpendicular orientation of the long molecular axis with respect to the inorganic surface.35 Thus, our current understanding for the finding that the incorporated PDI dye molecules are preferentially oriented parallel to the polar axis of the nanoparticles is that this concept also applies to the system reported herein, in which the polymer chain covalently attached to the PDI molecule functions as a linker to the polymer crystalline lamella to result in a directed orientation of the reporter molecule within the amorphous layers covering the nanocrystal. Thus, this preferred chromophore orientation enables an estimation of nanoparticle orientations. Incorporation of an alternative fluorescence marker potentially could enhance this effect to result in an even more uniform chromophore orientation with respect to the nanoparticle symmetry axes.

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SUMMARY AND CONCLUSIONS In conclusion, we demonstrated a novel approach toward monitoring the orientation of anisotropic nanoparticles. To date, only very few concepts have been reported which address this relevant but challenging issue. Our concept pursued here to generate well-defined, anisotropic nanoparticles as model system exploits polymer crystallinity in very small compartments as a structure-forming principle. Single fluorescent reporter molecules were incorporated into the nanoparticles via a perylene-functionalized ruthenium alkylidene initiator in combination with microemulsion polymerization of transcyclooctene as a strained reactive monomer and exhaustive postpolymerization hydrogenation in the nanoparticles. The orientation of the incorporated reporter molecule was found to be fixed within the nanocrystal. Thus, our system reported herein is thought to be suited for following both the translational and rotational motion of individual anisotropic nanoparticles in this challenging size regime. A combination of DWFM and AFM revealed the long molecular axis of the incorporated chromophore to be predominantly oriented parallel to the polar axis of the nanocrystal. Incorporation of an alternative fluorescence marker which potentially enhances this effect, thus, could even allow for the detection of absolute nanoparticle orientations. Overall, the findings reported provide an approach toward gaining insights into nanoparticle dynamics, e.g., assembly processes of nonspherical building blocks in this challenging size regime.

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EXPERIMENTAL SECTION

General Methods and Materials. All manipulations of air and moisture sensitive compounds were carried out under an inert gas atmosphere using standard glovebox or Schlenk techniques. Toluene was distilled from sodium under argon prior to use. All other solvents were utilized in technical grade as received unless noted otherwise. Distilled water was degassed by saturation with nitrogen. Hydrogen 5.0 grade (99.999% purity) was purchased from Air Liquide; pentanol (99%) supplied by Sigma-Aldrich was degassed via freeze−pump− thaw cycles and used without further purification. Sodium dodecyl sulfate (SDS), ethyl vinyl ether, Grubbs second generation catalyst, Hoveyda−Grubbs second generation catalyst, and poly(vinyl alcohol) 7907


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(Mw = 89 000−98 000 g mol−1, 99% hydrolyzed) were purchased from Sigma-Aldrich. trans-Cyclooctene (trCOE) was prepared according to a reported procedure36 and was purified by vacuum transfer before each use. NMR spectra were recorded on a Varian Unity INOVA 400 and a Bruker AVANCE III 400. 1H and 13C chemical shifts were referenced to the solvent signal. High-temperature NMR measurements of polyethylenes were performed in 1,1,2,2-tetrachloroethane-d2 at 130 °C. IR spectra were acquired on a PerkinElmer Spectrum 100 instrument with an ATR unit. Differential scanning calorimetry (DSC) was performed on a Netzsch DSC 204 F1 instrument at a heating rate of 10 K min−1. Crystallinities were determined assuming a melt enthalpy of 293 J g−1 for 100% crystalline polyethylene.37 Gel permeation chromatography (GPC) of unsaturated polymers was carried out on a Polymer Laboratories PL-GPC 50 with two PLgel 5 μm MIXED-C columns and a RI detector in THF at 40 °C against polystyrene standards. GPC of polyethylenes was carried out on a Polymer Laboratories 220 instrument equipped with Olexis columns with differential refractive index, viscosity, and light scattering (15° and 90°) detectors in 1,2,4-trichlorobenzene at 160 °C against polyethylene standards. Dynamic light scattering (DLS) was performed on a Malvern Nano-ZS ZEN 3600 particle sizer (173° backscattering). The autocorrelation function was analyzed using the Malvern dispersion technology software 5.10 algorithm to obtain volume and number-averaged particle size distributions. For the determination of particle sizes, a few drops of latex sample were diluted with approximately 3 mL of water. Polymer nanoparticle fluorescence quantum yields and absorptions and emission spectra were measured on a Hamamatsu Absolute PL Quantum Yield Measurement System C9920-02 CCD spectrometer. Temperature-dependent fluorescence spectra for initiation kinetics were recorded on the same system equipped with a temperature controlled cuvette holder (qpod 2e, Quantum Northwest) at an excitation wavelength of 550 nm. All measurements were conducted under inert gas atmosphere in quartz cuvettes closed with a PTFE-coated silicone septum. Transmission electron microscopy was performed on a Zeiss Libra 120 with an accelerating voltage of 120 keV. The dispersions were dialyzed extensively to remove free surfactant. Samples for TEM were prepared by applying a drop of the dialyzed dispersion to a carbon-coated grid. Atomic force microscopy (AFM) was performed on a JPK Nano Wizard instrument in the intermittent contact mode using a silicon tip with a force constant of 40 N m−1 and a resonant frequency of about 300 kHz. Synthesis of the Fluorescence-Functionalized Initiator 1. The vinyl isopropoxyphenyl substituted PDI dye N-(2,6-diisopropylphenyl)-N′-(3-vinyl-4-isopropoxyphenyl)-1,6,7,12-tetra[4-(1,1,3,3tetramethylbutyl)phenoxy]perylene-3,4,9,10-tetracarboxylic diimide was synthesized in six steps from 3,4,9,10-perylenetetracarboxylic dianhydride (cf. Supporting Information for experimental details). For the synthesis of 1, N-(2,6-diisopropylphenyl)-N′-(3-vinyl-4isopropoxyphenyl)-1,6,7,12-tetra[4-(1,1,3,3-tetramethylbutyl)phenoxy]perylene-3,4,9,10-tetracarboxylic diimide (137 mg, 90 μmol), CuCl (9 mg, 91 μmol), and Grubbs second generation Ru alkylidene 3 (76 mg, 90 μmol) were dissolved in 6 mL of dry CH2Cl2 under an argon atmosphere. Upon stirring at 40 °C for 1 h the reaction mixture was filtered through a syringe filter and purified by column chromatography on silica gel with CH2Cl2 as eluent to yield 102 mg of initiator 1 (57%). 1H NMR (400 MHz, CD2Cl2, 25 °C): δ 16.59 (s, 1H, carbene H), 8.12 (s, 4H, perylene H), 7.43 (m, 2H, phenylic H para to N, HAr), 7.35 (d, 3JHH = 8.8 Hz, 4H, phenylic H meta to O), 7.35 (d, 3JHH = 8.8 Hz, 4H, phenylic H meta to O), 7.30 (d, 3JHH = 7.8 Hz, 2H, phenylic H meta to N), 7.03 (s, 4H, phenylic H of the NHC ligand), 6.93 (m, 9H, phenylic H ortho to O, HAr), 6.83 (d, 3JHH = 2.2 Hz, 1H, HAr), 4.92 (sept, 3JHH = 6.6 Hz, 1H, PhOCHMe2), 4.13 (s, 4H, MesNC2H4NMes), 2.69 (sept, 3JHH = 6.8 Hz, 2H, NPhCHMe2), 2.43 (s, 12H, PhCH3 in the NHC ligand ortho to N), 2.31 (s, 6H, PhCH3 in the NHC ligand para to N), 1.74 (s, 8H, CH2CMe3), 1.36 (s, 24H, OPhC(CH3)2), 1.28 (d, 3JHH = 6.6 Hz, 6H, PhOCH(CH3)2), 1.09 (d, 3JHH = 6.8 Hz, 12H, NPhCH(CH3)2), 0.76 (s, 18H, C(CH3)3), 0.75 (s, 18H, C(CH3)3).

Aqueous Microemulsion Polymerization Procedure. In a 25 mL Schlenk flask 750 mg of SDS and 375 mg of degassed pentanol were dissolved in 6.1 mL of degassed water under an argon atmosphere. To this solution 300 mg of freshly vacuum transferred trans-cyclooctene was added, and the mixture was stirred until a transparent microemulsion had formed (∼5 min). In a separate Schlenk flask 6.0 g of SDS and 3.0 g of degassed pentanol were dissolved in 48.6 mL of degassed water under an argon atmosphere, and a solution of a mixture of 0.2 mg of initiator 1 and 9.4 mg of the unlabeled analogue Hoveyda−Grubbs second generation Ru alkylidene 2 in 2.4 g of degassed toluene was added. After stirring for ∼10 min a clear microemulsion was obtained. An appropriate amount of this catalyst microemulsion (depending on the desired monomer/ initiator ratio; e.g., 0.54 mL for a monomer/initiator ratio of 20 000) was injected into the monomer microemulsion via syringe, and the mixture was stirred for 20 min. Aqueous microemulsion polymerizations with neat initiators 1 and 2, respectively, for comparative studies were carried out in the same fashion by applying an appropriate higher amount of the respective initiator. Hydrogenation of Unsaturated Aqueous Polymer Dispersion. In a 10 mL Schlenk flask 6.9 mg of Grubbs second generation Ru alkylidene 3 was dissolved in 540 mg of degassed toluene, and 0.1 mL of ethyl vinyl ether was added. Upon heating the mixture to 50 °C for 30 min the color changed from red to yellowish brown. This solution was added to a solution of 755 mg of SDS and 316 mg of degassed pentanol in 6.5 mL of degassed water. After stirring for ∼1 h a transparent yellow microemulsion was obtained. 1.5 mL of this microemulsion was added to the polymer latex, and the dispersion was hydrogenated at 100 bar of H2 and 65 °C for 65 h. Hydrogenations were carried out in a 280 mL or a 22 mL stainless steel pressure reactor equipped with a heating/cooling jacket controlled by a thermocouple. The reactor was evacuated and purged with nitrogen multiple times before the unsaturated aqueous polymer dispersion was transferred to the reactor against a nitrogen flow. The reactor was pressurized with hydrogen and heated to the reaction temperature. After the desired reaction time the reactor was cooled to room temperature and vented. For polymer analysis, an aliquot of the dispersion was precipitated in 200 mL of methanol, filtered, washed with methanol, and dried in vacuo. Defocused Wide-Field Fluorescence Microscopy. The object based total internal reflection fluorescence microscopy (TIRFM) setup was constructed according to literature (cf. Supporting Information for details).38 The defocused patterns were calculated with a MatLab routine developed by J. Enderlein (University of Göttingen) and written by H. Uji-i (K. U. Leuven). Sample Preparation. A dispersion of single labeled PE nanocrystals (NP-1) was dialyzed extensively (Spectra/Por membrane, MWCO = 6−8000) against deionized water to remove excess surfactant originating from the microemulsion synthesis process. The dialyzed dispersion was diluted with Milli-Q water in a 1:80 ratio. The particles were deposited on a cleaned glass substrate by spin-coating (65 s, 1500 rpm) and were analyzed via defocused wide-field fluorescence microscopy. Single labeled PE nanocrystals NP-1 embedded in a PVA matrix for comparative studies were prepared by diluting the dialyzed particle dispesion with Milli-Q water in a 1:80 ratio and addition of one drop of this diluted dispersion to a 2.5 wt % aqueous solution of poly(vinyl alcohol) (PVA). A PVA film with embedded nanocrystals NP-1 was obtained by spin-coating this solution on a cleaned glass substrate and subsequent drying of the film at 50 °C under vacuum.

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ASSOCIATED CONTENT

S Supporting Information *

Additional experimental details and analytical data. This material is available free of charge via the Internet at http:// pubs.acs.org. 7908


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AUTHOR INFORMATION

Corresponding Authors

*E-mail [email protected] (D.W.). *E-mail [email protected] (S.M.). Author Contributions ∥

These authors contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support by the Konstanz Research School Chemical Biology and the Zukunftskolleg of the University of Konstanz is gratefully acknowledged. M.C.B. thanks the “Fonds der Chemischen Industrie” for a scholarship. We thank Prof. Dr. Jörg Enderlein (University of Göttingen) and Prof. Dr. Hiroshi Uji-i (K. U. Leuven) for providing the Matlab routine to analyze the defocused wide-field images, Lars Bolk for DSC and GPC measurements, and Marina Krumova for TEM analysis.

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Anisotropic Polyethylene Nanocrystals Labeled with a Single

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