Optical Microscopic Techniques for Synthetic Polymer Characterization

Review Cite This: Anal. Chem. XXXX, XXX, XXX−XXX

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Optical Microscopic Techniques for Synthetic Polymer Characterization Herman Coceancigh, Daniel A. Higgins,* and Takashi Ito*

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Anal. Chem. Downloaded from pubs.acs.org by UNIV OF LOUISIANA AT LAFAYETTE on 11/01/18. For personal use only.

Department of Chemistry, Kansas State University, 213 CBC Building, Manhattan, Kansas 66506-0401, United States

CONTENTS

Optical Microscopic Techniques for Polymer Characterization Common Instrumentation Super-Resolution Optical Microscopy Super Localization Microscopy (SLM) Particle Tracking in 3D Stimulated Emission Depletion (STED) Microscopy Stochastic Optical Fluctuation Imaging (SOFI) Super-Resolution in the Temporal Domain Spectrally-Resolved Super-Resolution Microscopy Morphological and Functional Properties of Homopolymers Morphology and Composition of Homopolymer Assemblies and Thin Films Dynamic Properties Block Copolymer-Derived Nanostructures Nanoscale Block Copolymer Aggregates Microdomains in Block Copolymer Monoliths Solution−Polymer Interfaces Interfacial Dynamics of Polymer Molecules Dynamic Molecular Behavior at Polymer Brushes Dynamic Molecular Behavior at Synthetic Polymer Surfaces Dynamic Nanoparticle Behavior in Porous Materials Conjugated Polymers Nanoscale Morphologies of Conjugated Polymer Films Polymer Chain Conformation and Aggregation Excited State and Charge Carrier Diffusion Exciton Dynamics Charge Carrier Dynamics In Situ Monitoring of Polymerization Reactions Summary and Outlook Author Information Corresponding Authors ORCID Notes Biographies Acknowledgments References

techniques can be linked to the macroscopic rheological, mechanical, optical, electrical, and/or chemical properties of synthetic polymers that are widely used in scientific research and/or in daily life. Detailed knowledge of nanoscale polymer properties is thus anticipated to facilitate designing novel polymers for various applications, including surface coating,1 chemical separations,2,3 chemical sensing,4,5 bioimaging,6 and organic electronic devices.1,5 Optical microscopic methods are widely used for in situ characterization of the structural and dynamic properties of various samples under ambient conditions. However, the spatial resolution of these methods is generally limited by the diffraction of light.7 Recent development of super-resolution optical microscopies such as superlocalization microscopy (SLM)8−11 and stimulated emission depletion (STED)12 microscopy mitigates the resolution issue, enabling the investigation of spatiotemporal heterogeneity in the properties of polymeric materials with spatial resolution of tens of nanometers.13,14 In particular, single molecule/particle tracking (SMT, SPT) techniques provide unique spatiotemporal information on the dynamic behavior of individual molecules/particles rather than ensemble-averaged information.15 Information obtained using these methods is complementary to that obtained with other characterization methods such as electron microscopy, scanning probe microscopy, and X-ray/ neutron scattering.16 Electron microscopy can offer 3D morphological information at much higher spatial resolution but, basically, can only measure dried samples under vacuum. Scanning probe microscopy is applicable for in situ nanoscale characterization of polymer surfaces under various conditions but cannot access the inside of a sample. X-ray and neutron scattering provide ensemble averaged information on periodic nanostructures under ambient conditions but not detailed information on materials heterogeneity such as single-point defects. Herein, we highlight optical microscopic studies on synthetic polymers published between January 2015 and June 2018. These papers employed optical microscopic techniques based on irradiation of samples by UV or visible light and revealed the detailed morphological, dynamic, and electronic properties of monolithic polymer films or isolated polymer molecules/ aggregates. We focus on works associated with synthetic “covalent” polymers (Figure 1) and do not discuss biopolymers,17 noncovalent polymers, such as metal organic frameworks or supramolecular polymers,18 polymer-nanoma-

B C D D E E E E E F F G H H I I I K K L M N N O O P Q Q Q Q Q Q Q Q Q

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n this review, we discuss how state-of-the-art optical microscopic techniques enhance our understanding of the morphological and dynamic properties of synthetic polymers. Microscopic/nanoscopic properties obtained using these © XXXX American Chemical Society

Special Issue: Fundamental and Applied Reviews in Analytical Chemistry 2019

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DOI: 10.1021/acs.analchem.8b04694 Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry

Review

Figure 1. Chemical structures of synthetic polymers discussed in this review: (a) homopolymers (except conjugated polymers), (b) block copolymers, and (c) conjugated polymers.

commonly used to visualize polymer films and nanostructures, and to reveal materials morphology and heterogeneity. Discussion of these methods can be found in ubiquitous online and print materials and are not included here. Rather, the discussion below emphasizes existing and emerging stateof-the-art imaging methods having spatial resolution that exceeds the fundamental diffraction limit of light, or that simultaneously provide multiple forms of data by coupling temporal or spectral resolution with spatial resolution. After introducing some of the common instrumental components and configurations, the discussion goes on to highlight a few representative and unique optical imaging and spectroscopic methods that have been implemented for the characterization of polymer films, nanostructures, and single molecules. Some methods and experiments have been excluded because they

terial composites,18 or polymer solutions.15 In this review, we first summarize the principles and instrumentations of superresolution optical microscopic techniques and then discuss their applications for investigations of the structural and dynamic properties of synthetic homopolymers and block copolymers. Finally, we will provide outlooks for possible future directions in this research field.

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OPTICAL MICROSCOPIC TECHNIQUES FOR POLYMER CHARACTERIZATION A diverse and ever expanding toolbox of optical microscopic methods exists for the characterization of polymeric materials.13−15,18−20 Many conventional methods such as standard bright field and dark field imaging, differential interference contrast, phase contrast, and fluorescence imaging are B



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Figure 2. (a) Common microscope configurations for confocal (left) and wide-field (right) imaging. In the former, a collimated laser beam is delivered to the objective lens, producing a small spot of light of diffraction-limited size in the sample. In the latter, the laser is focused into the back aperture of the objective, yielding broad illumination of the sample. Only the epi-illumination mode is shown. Transmission mode would require delivery of the incident light from the opposite side of the sample. (b) Optical path in conventional wide-field illumination (left) and total internal reflection mode illumination (right). In the former, the sample is uniformly illuminated along its depth dimension while in the latter, only the region exposed to the evanescent field is exposed to the incident light to a depth of ∼100−200 nm.

In confocal imaging experiments (Figure 2a, left), the sample is illuminated with a single point of light, produced by delivering a collimated laser beam to the microscope objective. To produce an image, either the sample position is scanned in relation to the illuminated point, or the incident beam is scanned across the surface, while the transmitted, reflected, scattered, or emitted light is detected. In wide-field imaging (Figure 2a, right), a broad region of the sample is illuminated all at once. This requires focusing the incoming laser into the back aperture of the objective so that its focal point appears in the back focal plane of the objective. Conventional wide-field illumination involves focusing the incident light along the optical axis of the objective (Figure 2b, left). In this case, little depth resolution is achieved and relatively large background often results when it is used for imaging optically thick samples. Background from sample luminescence or light scattering can be greatly reduced by the implementation of total internal reflection (TIR) mode illumination (Figure 2b, right). In this case, the incoming light is focused well off-axis into a high numerical aperture (NA) objective. To achieve TIR, the light emerging from the front side of the objective must be incident on the interface between a sample having high refractive index and a neighboring medium of low refractive index. The incident angle must exceed the critical angle of the interface, producing an evanescent field in the low index medium that decays over ∼100−200 nm. This limits the sample volume exposed to the incident light and, hence, greatly reduces the background produced. Any of a wide variety of microscope objectives may be employed in imaging experiments. Commonly, these are high

have been addressed in recent reviews. For example, correlative imaging by optical and physical methods has been extensively discussed by Hauser et al.,17 and recent advances in superresolved imaging by tip-enhanced Raman scattering (TERS) and near-field scanning optical microscopy (NSOM) methods have been reviewed by Xiao and Schultz.21 Common Instrumentation. Most optical microscopy is performed using home-built or commercial light microscopes of a standard design.13,22 Figure 2 provides schematics of common microscope configurations. Lasers are often used as the light source because many produce optical beams having Gaussian intensity profiles (i.e., TEM00) that are easily focused. Light from lasers with different mode profiles can be converted to TEM00 by passing the light through a single mode optical fiber. Single mode fibers also serve as a convenient tool to deliver light to the microscope. Prior to entry into the microscope, the laser light is usually directed through appropriate polarizers and wave plates to set the power and to manipulate the optical polarization state. The laser light is subsequently directed into the microscope and can be used to illuminate the sample in a number of different configurations. In transmission experiments, the light is sent directly into the objective used for sample illumination. For experiments where inelastically scattered light or emission from the sample is to be collected in reflection, the incoming light is reflected from a dichroic mirror and then directed into the back aperture of the objective lens. This constitutes the socalled epi-illumination mode. The dichroic mirror must be properly chosen to reflect the incident laser light while transmitting the collected signal. C



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For emission or scattering experiments when single point illumination is employed, detection of the collected light is most commonly accomplished by use of an avalanche photodiode or a photomultiplier tube. For wide-field imaging experiments performed at very low light levels (i.e., single molecule detection), electron-multiplying charge coupled device (EM-CCD) cameras are widely employed.22 These have an advantage over conventional CCD cameras in that they have built-in gain. The gain allows for the read noise of the CCD to be virtually eliminated as a factor in determining the signal-to-noise ratio at high readout rates. When higher and more variable noise levels can be tolerated and higher frame rate videos are needed, modern scientific-grade complementary metal oxide semiconductor (sCMOS) cameras are commonly used.23 Super-Resolution Optical Microscopy. Ernst Abbe first described how the spatial resolution of optical imaging methods is limited to about half the wavelength of light by diffraction effects in 1873.7 Soon thereafter, scientists began to take on the challenge of overcoming this limit. Some of the most important recent developments in the characterization of organic polymers have come as a result of the successful development of methods for breaking the diffraction limit. Super Localization Microscopy (SLM). Perhaps the most successful means for achieving subdiffraction-limited spatial resolution in optical imaging has come from the realization that a single particle or single molecule (both referred to as single particles in this section) can be located within an image to very high precision in scattering and emission microscopy experiments.24 The precision of particle localization in the sample plane scales as the diffraction limited width of the particle image, or point-spread function, divided by the square root of the number of photons detected from it.25 Background and dark counts must also be included in the calculation.25 The many forms of single-particle or single-molecule super localization microscopy (SLM) based on this realization have relied on either natural fluctuations in the signal24 or on the ability to switch particle emission on and off in different frames of a video to obtain high-resolution images.8,9 These signal fluctuations may be due to particle motion, bleaching, blinking (stochastic optical reconstruction microscopy, STORM), or photoinduced transitions to dark states (photoactivated localization microscopy, PALM).26 After determining the location of each particle in each video frame, a high-resolution image of the sample is reconstructed by compiling the locations of all the individual particles. Previous reviews cover the full details of these methods and their applications in the imaging of materials.3,24,26 Recently developed SLM methods used for the characterization of polymers include one reported by Park et al., in which the bleaching of conjugated single MEH-PPV molecules was used to limit the number of emitters in each video frame, allowing for the locations of the individual emitters along each polymer chain to be determined.27 Advances were made in this study by subtracting all possible combinations of image pairs, affording reduced uncertainty in the location of each emitter. Jiang and co-workers developed PFBT nanoparticles doped with a fullerene derivative, PCBM, that produced bursts of luminescence and could be readily implemented in SLM.28 The PCBM dopant led to production of hole polarons when the nanoparticles were illuminated with light, and these charge carriers efficiently quenched the nanoparticle luminescence. Fluctuations in the number of charge carriers led to occasional

Figure 3. (a) STReM experimental setup. Fluorescently tagged molecules at the water−polymer interface are excited with a TIR geometry. Fluorescence is transferred into the Fourier domain after the first image plane, and subframe dynamics are encoded in the resulting point spread functions by a rotating phase mask. The final image is captured by an sCMOS camera positioned at the second image plane. (b) (A) Representative STReM single frame containing subframe (blue and green) and longer (red) adsorption events. (B− D) Zoomed PSFs of part A. The surface residence time is determined from the arc length of the point spread function, shown with the blue arrows in parts B and C. The green arrows denote one camera exposure of 100 ms. For events longer than one frame, such as part D, the surface residence time is determined by cumulating its arc lengths in all consecutive frames. Reproduced from Wang, W.; Shen, H.; Moringo, N. A.; Carrejo, N. C.; Ye, F.; Robinson, J. T.; Landes, C. F. Langmuir 2018, 34, 6697−6702 (ref 36). Copyright 2018 American Chemical Society.

NA oil- or water-immersion lenses, but air objectives can also be used. High NA is important for achieving the highest spatial resolution and for most efficient collection of light from the sample. High NA is also required to achieve TIR illumination in the through-objective configuration shown in Figure 2b. In the epi-illumination mode, only a single objective is required to illuminate the sample and to collect the reflected, scattered, or emitted light. In transmission mode, a second objective must be positioned opposite the illumination objective to collect light passing through the sample. Once the light has been collected from the sample, it is commonly passed through additional optics, prior to detection. In experiments where emission or inelastic scattering from the sample is being collected and detected, the light must be passed through appropriate filters to remove residual laser light. It may also be sent through a polarization analyzer for polarization-dependent imaging experiments. In experiments where two or more signals are recorded simultaneously using the same camera, it may also be passed through an image splitter. D



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Stochastic Optical Fluctuation Imaging (SOFI). Another method for achieving subdiffraction-limited spatial resolution in imaging experiments involves recording the fluctuating emission signals from a small ensemble of single particles. A camera is again used to record video data. The signal fluctuations may result from particle motions or from particle blinking. In any case, the number of particles must be sufficiently small that the signal fluctuations due to single particle events can be detected. The fluctuating signal detected for each pixel is subsequently autocorrelated and the amplitude of the autocorrelation function at each pixel used to construct an image of the sample. Equation 1 gives the second-order autocorrelation function at position r:

bursts of strong luminescence from the individual particles that allowed for their superlocalized detection with ∼0.6 nm precision. These nanoparticles were tested for imaging applications in single cells, where ∼5 nm localization precision was achieved. The same nanoparticles could be used for imaging of nanostructured organic polymer materials. Particle Tracking in 3D. Achieving diffraction-limited optical resolution in the longitudinal direction (along the zdimension) is accomplished in a confocal microscope by inserting an appropriately sized pinhole into the primary image plane. In a wide-field microscope, z-resolution is more challenging to achieve, but recent advances in single particle detection have now afforded subdiffraction-limited resolution in this dimension as well. Superlocalization of particles along the z-dimension could be achieved by simply inserting a cylindrical lens in the detection path, imparting astigmatism on the particle image.29 Alternatively, a phase mask could be inserted into the optical path of the microscope to access phase information related to the z-position of the particle.30 In both cases, the z-position was encoded in 2D images of the particle. Determining the z-position of the particle required fitting of the image to a Gaussian or an elliptical Gaussian, in cases where astigmatism was employed to encode the z-position. In cases where phase masks were used, more sophisticated algorithms were required to extract the position of the particle in 3D. Recent work in this area by Shuang et al.31 described a generalized algorithm that worked with several different phase masks. Their method employed an alternating direction method of multipliers to deconvolute particle positions in 3D. Machine learning was used to refine the output. Stimulated Emission Depletion (STED) Microscopy. An alternative method for subdiffraction-limited fluorescence imaging involves the spatially patterned stimulated depletion of a population of excited fluorescent molecules or nanoparticles. In stimulated emission depletion (STED) microscopy,12 a local region within the sample is initially excited by a diffraction-limited focused laser spot of Gaussian mode profile. The excitation wavelength is chosen to fall within the absorption spectrum of the chromophore employed. A second laser, having a toroidal mode profile, is subsequently used for depletion of the excited state population. The toroidal beam is centered over the Gaussian excitation beam. Its wavelength is chosen to fall near the red edge of the Stokes-shifted emission spectrum of the chromophore so that stimulated emission is induced, depleting the population of excited molecules in this region. Saturation of the depletion transition at high laser powers produces an excited region of subdiffraction-limited size, affording the desired high resolution. Images are usually acquired by raster scanning the sample while it is exposed to both laser beams. Either pulsed or continuous wave lasers can be employed. A modified form of STED microscopy recently developed by Penwell and co-workers was employed for subdiffractionlimited imaging of conducting polymer nanoparticles (CPNs).32 STED imaging with CPNs and some dyes is made difficult by two-photon absorption of the stimulated emission beam by the ground-state chromophores. These authors overcame the associated challenges by modulating the excitation beam. Synchronous detection of the fluorescence allowed for the two-photon-excited fluorescence background to be discriminated against and for clusters of the CPNs to be imaged at high resolution.

G2(r , τ ) = ⟨δF(r , τ ) ·δF(r , t + τ )⟩

(1)

where δF represents the signal fluctuation from its average value, τ is the time basis for the autocorrelation, and the brackets (⟨ ⟩) indicate that the average over video time t is calculated. As shown by Dertinger et al., enhancement to the spatial resolution scales inversely as the square root of the autocorrelation order.33 That is, second-order autocorrelation leads to a 2 improvement in spatial resolution over the diffraction limit. Recent work reported by Kisley and co-workers demonstrated the implementation of SOFI for high-resolution imaging of nanostructured polymer hydrogels and other materials.34 In these experiments, the diffusion of fluorescent dye molecules produced the signal fluctuations. Their work confirmed that enhanced resolution could be achieved by this method and demonstrated its utility in the imaging of nanostructured materials such as synthetic polymers, hydrogels, and organized block copolymer mesophases. Super-Resolution in the Temporal Domain. Wide-field imaging experiments frequently suffer from the relatively low time resolution with which videos can be acquired. Although fast sCMOS-based cameras continue to be developed, the time resolution that can be achieved is still limited by the rate at which the detector array can be read out. This limitation restricts the range of time scales that can be probed in dynamics studies. Researchers are now working to develop novel new methods for overcoming the temporal resolution limits of camera-based imaging. Recent work published by Wang et al. has provided representative examples of one such method (STReM, Figure 3).35,36 In this case, the authors employed a rotating phase mask placed in the microscope detection path to encode additional temporal information in images of fluorescent beads and a dye-labeled protein adsorbing to a surface (Figure 3a). The orientation of the particle image for rapidly adsorbing and desorbing species represented a snapshot of the point-spread function produced when each particle adsorbed to the surface. Knowing the position and speed of the rotating phase mask allowed for the exact time of adsorption to be determined (Figure 3b). For particles that remain adsorbed for long periods of time, arcshaped point-spread functions were obtained in which the arc length provided a measure of the surface residence time. The authors achieved ∼20-fold improvement in the time resolution, demonstrating the utility of this method for following the interfacial exchange of particles on time scales that are short compared to the camera exposure time. Spectrally-Resolved Super-Resolution Microscopy. Single molecule tracking and super-resolution methods provide a wealth of information on the details of materials structure, but E



Review

Figure 4. (a) Schematic illustration of the experimental setup. A free-standing PMA film doped with PDI molecules was uniaxially elongated using a microtensile testing setup and observed with wide-field fluorescence polarization microscopy. The fluorescence signal was divided into two polarization components that were parallel and perpendicular to the elongation direction (Ip and Is, respectively) and recorded using a CCD camera. (b) Single PDI molecules were recognized as individual spots with intensity of (Ip + Is). The orientation of the in-plane projection of the transition dipole, φ, was calculated using φ = arctan( Is/Ip ). Local elongation was calculated from changes in Δx and Δy. (c) Individual PDI molecules and their in-plane orientation before and after elongation in the arrow direction (upper) and the distribution of orientations (bottom). Reproduced from Krause, S.; Neumann, M.; Frobe, M.; Magerle, R.; von Borczyskowski, C. ACS Nano 2016, 10, 1908−1917 (ref 63). Copyright 2016 American Chemical Society.

reported.41,42 Here, the authors employed an achromatic beam splitter to separate the fluorescence collected from the individual molecules into two different detection pathways. One was directly sent to the CCD camera for the superlocalized detection of the fluorescent spots produced. The other was passed through a prism and then on to the CCD camera to produce wavelength-dispersed streaks, representing the emission spectrum of each molecule. An alternative geometry in which two objectives and two separate detection pathways were employed was also reported.42 The latter configuration afforded improved collection efficiency in light-starved applications. While these demonstrations described applications to studies of biological samples, these methods could easily be implemented in studies of polymer films and particles. Such studies are certain to be reported soon.

usually very little information on local materials composition is obtained. The implementation of solvatochromic dyes in these experiments provides a viable route to obtaining such chemical information. Solvatochromic dyes exhibit dramatic shifts in their absorption and emission spectra when exposed to environments of different polarity, as defined by the local dielectric constant. By simultaneously locating single molecules of these dyes while also acquiring information on their spectral emission, much can be learned about materials composition on super-resolved length scales. A particularly useful probe molecule for such studies is Nile Red.37 Early experiments in which single molecule spectra were obtained using a sample scanning confocal microscope demonstrated the utility of this method for characterizing local environments in polymer films.38 Quasi-single molecule imaging methods have since been used by Hess et al. to obtain the fluorescence emission wavelength of Nile Red doped into 30 μm-thick PVDF films.39 These data allowed them to visualize spatial variations in the local dielectric constant of the polymorphic polymer films. More recently, Nile Red single molecule spectra were obtained in a wide-field imaging modality by inserting a transmission grating in the detection path of the microscope to obtain both the position of each molecule and its spectrum.40 A method for simultaneously acquiring subdiffractionlimited spatially resolved images and the full emission spectrum of individual Nile Red molecules was also recently

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MORPHOLOGICAL AND FUNCTIONAL PROPERTIES OF HOMOPOLYMERS In this section, we discuss recent optical microscopic works aiming to reveal the nanoscale morphology and dynamic properties of assemblies and thin films based on synthetic homopolymers (Figure 1a). Morphology and Composition of Homopolymer Assemblies and Thin Films. SLM has been used to gain time-averaged morphologies of hydrogel-based nanostructures F



Review

crystal microbalance.55 The inhomogeneous mobility observed for the quantum dots in the film reflected spatial variations in the local viscoelasticity. Wursch and co-workers compared the orientations and perpendicular positions of single shapepersistent wheel molecules in a thin PMMA film before and after the film was exposed to toluene vapor.56 The orientations of individual molecules could be determined from excitation polarization fluorescence anisotropy data reflecting isotropic and anisotropic absorption by molecules oriented flat in the 2D film plane and those with perpendicular orientation, respectively. In addition, the positions of single molecules along the film normal could be estimated from their fluorescence intensity. Probe molecules within the film gave weaker fluorescence, reflecting the involvement of the triplet state due to the lack of oxygen access. They showed that solvent vapor exposure to the thin film (250 nm thick) led to the diffusion and accumulation of the probe molecules at the polymer−air interface, where they were found to orient horizontally with respect to the film surface. Over the last 2 decades, considerable efforts have directed toward a thorough understanding of the dynamic properties of polymer segments near their Tg.51,57 SMT revealed the spatiotemporally heterogeneous evolution of changes in dynamic properties for thin PVAc and PMA films around their Tg.58 More recently, Dev Verma and co-workers employed multidimensional time correlation functions to obtain quantitative results on the heterogeneity of rotational behavior for single rhodamine 6G molecules in and on PCA films.59 Paeng and Kaufman investigated segmental dynamics in thin PS films (200 nm thick) near Tg using single-molecule fluorescence polarization measurements.60 They observed spatially inhomogeneous rotational dynamics for individual PDI molecules in a film, reflecting the presence of distinct environments in terms of PS segmental mobility that were distributed in space and evolved in time. Characteristic time scales associated with dynamic exchange were widely dispersed and were significantly longer than the structural relaxation of the PS segmental dynamics. Zhang et al. used defocused singlemolecule fluorescence microscopy to show the slower rotational dynamics of PDI probes having larger sizes61 or those involving stronger probe−polymer interactions62 in thin PVAc films at temperatures below and above Tg. Single-molecule fluorescence microscopy was used to gain real-time nanoscale information on the viscoelastic properties of a mechanically elongated polymer film. Krause and coworkers monitored the nanoscale deformation of a freestanding polymer film induced by mechanical elongation and subsequent stress relaxation using single-molecule fluorescence polarization microscopy.63 The deformation was assessed from the position and orientation of individual PDI molecules doped into a free-standing PMA film, the latter of which was estimated from fluorescence intensities recorded in two orthogonal polarizations (Figure 4a). The measurements were carried out at a temperature slightly higher than Tg to minimize the translational and rotational diffusion of the probe molecules. Film elongation was shown to induce the reorientation of probe molecules in the stretching direction (Figure 4b), as supported by the close temporal correlation between probe reorientation and local matrix elongation. During stress relaxation, the relaxation of probe orientation was much slower than polymer segment relaxation, suggesting that the probe relaxation reflected the shape deformation of a polymer coil in the film.

immobilized on substrate surfaces. Conley et al. demonstrated in situ measurements of the internal density profile of fluorescently labeled PNIPAM microgels during water-based swelling and methanol-induced collapse.43 Gelissen et al. demonstrated high-resolution 3D imaging of a microgel consisting of a PNIPAM core and a PNIPMAM shell via selective fluorescent labeling of the core or shell.44 Gilbert et al. visualized the internal morphology of PNIPAM-PVP-based interpenetrating network hydrogels that consisted of PNIPAMrich domains entrapped in a clustered PVP-rich phase using two-color SLM.45 SLM was also used for chemical characterization of polymer surfaces. ONeil et al. visualized the distribution of −COOH groups on two types of thermoplastic surfaces generated via UV/O3 or O2-plasma treatment by covalently labeling them with photoswitchable fluorescent probes.46 SLM images revealed the heterogeneous distribution of surface −COOH groups, which could not be observed using conventional fluorescence microscopy. The resulting nonuniform charge distribution could be related to the recirculation of electroosmotic flow, which led to the degradation of separation efficiency in microchip electrophoresis using a nanochannel. On the other hand, Raman spectroscopy was explored for in situ, label-free measurements of layered polymer films. For example, spatially offset Raman spectroscopy (SORS) was used to measure Raman spectra from a turbid surface PTFE layer and a buried PP layer.47 SORS is based on the acquisition of a Raman signal from a region displaced from where the laser initially enters the material and can give a Raman spectrum for deeper regions by increasing the lateral offset between the initial laser entry point and the detection region.48 Scanning angle Raman spectroscopy, which collected Raman spectra as a function of the incident angle of laser excitation, was applied to determine the total thickness and interface locations of stacked polymer layers based on PS/PC49 and PS/PMMA.50 Dynamic Properties. Polymer dynamic properties such as molecular permeability near polymer−substrate and polymer− air interfaces are known to be different from those of the bulk.51 Detailed information on spatiotemporal heterogeneity in these properties was investigated using SMT. Ito and coworkers used the astigmatism imaging method to record 3D SMT data for hydrophobic perylenediimide (PDI) molecules diffusing in glass-supported thin polymer films (100∼1000 nm thick) of polyHEA at a temperature significantly higher than its Tg.52 They observed negligible diffusion in the perpendicular direction and faster lateral diffusion in the thicker films. These results suggested PDI molecules were confined to a horizontal layer defined by polymer−substrate interactions propagated over a fairly long distance (>100 nm). Subsequently, the same group employed 2D SMT to show the temporal fluctuation of the local permeability of a polyHEA film.53 In this study, they employed a diarylethene dye that could be switched on and off and could be photoexcited using a single wavelength laser. Meanwhile, Piwonski et al. correlated the lower oxygen permeability of a polymer matrix to the higher photostability for terrylene diimide (TDI) molecules in single-molecule fluorescence experiments.54 The permeability of a polymer can be tuned by solventinduced swelling. SLM and related techniques were used to gain nanoscopic detail on polymer swelling. Hoang et al. measured the translational diffusion of single quantum dots (3 nm in diameter) in a toluene-swollen PMMA film using SPT while the extent of swelling was monitored using a quartz G



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Figure 5. (a,b) Experimental setups for FRAP and SMT measurements. (a) For FRAP, a circular region in a PS-b-PEO film was first photobleached using intense laser light (left). Then, fluorescence images were recorded by irradiating the sample with attenuated laser light from the top (right). (b) Subsequently, the wider observation area was further photobleached by more intense laser light (left), followed by recording SMT data under broad laser illumination (right). (c) In the analysis of FRAP data, x and y were defined as the directions parallel and perpendicular to the direction of solvent vapor penetration (SVP), which was used to align elongated cylindrical PEO microdomains (top). L|| and L⊥ were defined to be parallel and perpendicular to the long-axis of the elliptical photobleached region. The fluorescence intensity was fitted to a 2D Gaussian function to measure the widths of the intensity profile (bottom). Here, θFRAP represents the recovery direction (L||) with respect to the SVP direction (−90° ≤ θFRAP ≤ 90°). (d) A set of FRAP data obtained just after photobleaching (t = 0 s) and at longer t. An arrow at t = 0 s indicates the SVA direction. (e) A typical 1D single molecule trajectory (red) and its best-fit line using orthogonal regression methods. Here, θSMT represents the tilt angle (red) of the single trajectory with respect to the SVP direction (−90° ≤ θSMT ≤ 90°) while θ̅SMTdepicts the average trajectory orientation from all 1D trajectories found in each set of SMT data (green). (f) Single molecule trajectories measured at the identical area used for the FRAP measurement across the film thickness (z = 0 (near the polymer-glass interface) and 2 μm). 1D trajectories are shown in red, whereas 2D and immobile trajectories are depicted in gray and black, respectively. Reproduced from Tran-Ba, K.-H.; Higgins, D. A.; Ito, T. Anal. Chem. 2015, 87, 5802−5809 (ref 83). Copyright 2015 American Chemical Society.

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BLOCK COPOLYMER-DERIVED NANOSTRUCTURES

fragments can form nanoscale micellar aggregates in appropriate solutions. Such materials are being explored for applications as drug carriers,66 templates for nanomaterial synthesis,67 and precursors of isoporous membranes.68 Advanced fluorescence microscopic techniques were used to measure the sizes and shapes of individual nanoscale aggregates.18 For example, SLM was used to visualize the PS cores of sub-100 nm cylindrical PS-b-PEO micelles and vesicles using an optically switchable spiropyran69 or diarylethene70 dye. In these studies, the use of the hydrophobic photoswitchable dyes enabled the visualization of hydrophobic PS cores at high spatial resolution (∼50 nm). Boott and co-workers employed STED and SLM to discuss the growth of red-green-red triblock cylindrical PFS-b-PDMS

Block copolymers (Figure 1b), which consist of distinct homopolymer chains that are linked covalently, have attracted considerable interest owing to their capability to form various nanostructures.64,65 The sizes and shapes of these nanostructures can be controlled by adjusting the properties and lengths of the fragments. State-of-the-art microscopic techniques have been used to quantitatively assess the sizes, shapes, and molecular permeability of single nanostructures under ambient conditions at a spatial resolution beyond the diffraction limit. Nanoscale Block Copolymer Aggregates. Amphiphilic block copolymers based on hydrophobic and hydrophilic H



Review

microdomains (Figure 5b,e,f). The ensemble diffusion coefficient was similar to the average of the single-molecule diffusion coefficients, indicating the presence of defect-free microdomains on length scales >100 μm. Sapkota and co-workers employed SMT to reveal a relationship between the permeability and transverse width of individual cylindrical PEO microdomains swollen by an organic solvent to different extents.84 The measurements were carried out in thin PS-b-PEO films comprising sulforhodamine B-doped PEO microdomains elongated by the shear flow of concentrated PS-b-PEO solutions.82 The analysis of individual single molecule trajectories revealed a wide distribution of microdomain permeabilities and widths. These parameters showed no evidence of correlation on a single molecule basis, reflecting the different extents to which individual microdomains were swollen. On average, SMT data showed more significant deswelling-induced decreases in microdomain permeability and width for THF than benzene due to its higher affinity for PEO, providing insights into the roles of solvents in microdomain alignment via solvent vapor annealing.85 Baier and co-workers employed SMT to investigate the effects of temperature on molecular diffusion in thin films of PS-b-PB-b-PS, which is used as a thermoplastic elastomer based on the low and high Tg of PB and PS, respectively.86 Either a small perylenediimide (PDI) molecule or a PDIlabeled polymer (MW = 13 kg/mol) was doped into a thin PSb-PB-b-PS film comprising horizontally oriented cylindrical PB or PS microdomains. The temperature of the thin film was controlled by an ITO-based heating stage during SMT measurements, which were performed under a nitrogen atmosphere.87 Small PDI molecules diffused in the PB microdomains below the Tg of PS (Tg(PS) ∼ 100 °C) and exhibited penetration across the PS microdomains above Tg(PS). In contrast, PDI-labeled polymer molecules seemed to be primarily distributed into the PS microdomains and diffused only after heating above Tg(PS). The temperature dependences of the diffusion coefficients of these fluorescence probes permitted the estimation of formal diffusion coefficients at infinite temperature and activation energies for diffusion using the Arrhenius function.

comicelles via living crystallization-driven self-assembly in native organic media.71 The PFS-b-PDMS was labeled by caged red or green dyes for the super-resolution fluorescence measurements. Micelle length distributions obtained with STED and SLM were in good agreement with those obtained by TEM. More importantly, SLM revealed that the lengths of the two micelle termini were equivalent, indicating that the red micelles symmetrically grew from the termini of cylindrical seed (green) micelles at the same rate. Gong et al. visualized the PS regions of PS-b-PEO micelles at high spatial resolution using SLM and studied the morphological changes of the micelles induced by toluene vapor exposure.72 They employed a fluorescent probe tagged with a photoswitchable quencher that could be reversibly activated by irradiation with light at the excitation wavelength of the fluorophore. The authors demonstrated SLM measurements using a single-wavelength laser both for the photoswitching of probe fluorescence and for probe photoexcitation. Meanwhile, Handschuh-Wang et al. employed FLIM to estimate the wall thickness of water-filled vesicles formed from PS-b-PAA and PEG-b-PLA.73 FLIM data of calcein-doped vesicles gave three fluorescence lifetime components originating from water-dissolved dyes, wall-incorporated dyes, and dried/agglomerated dyes and revealed a drying-induced decrease in the first component. The thickness of the vesicle wall was estimated from a relationship between vesicle radius and the drying kinetics obtained from the amplitude ratio of the first and second lifetime components. Microdomains in Block Copolymer Monoliths. Block copolymers consisting of incompatible fragments can offer monolithic materials comprising periodic microdomains that have uniform and predicable sizes (5−100 nm) and morphologies upon microphase separation.64 These monolithic materials have been examined for uses in nanolithography,74 energy-related technologies,75 chemical separations,76−78 and chemical sensing.77,79 SMT has been used to gain information on the properties of individual microdomains under ambient conditions by tracking the motions of individual fluorescent molecules selectively doped into one of the microdomains with higher permeability. Previously, SMT was mainly used to assess the morphologies of individual microdomains in thin block copolymer films.80−82 More recently, SMT has been used for quantitative assessment of the permeability of individual microdomains from the diffusion coefficients of single molecules that selectively partitioned into the microdomains. Tran-Ba and co-workers evaluated the contribution of material heterogeneity to overall microdomain permeability by measuring ensemble and single-molecule diffusion within identical micrometer-scale sample regions (Figure 5).83 They prepared thin films of PS-b-PEO comprising cylindrical PEO microdomains (∼30 nm in diameter) that were elongated along the penetration direction of 1,4-dioxane vapor.81 Optical studies were accomplished by doping the films with sulforhodamine B. Fluorescence recovery after photobleaching (FRAP) was first used to determine the ensemble diffusion coefficient of sulforhodamine B molecules across the entire focal depth, in addition to the average orientation and long-range (>100 μm) connectivity of the microdomains (Figure 5a,c,d). The majority of sulforhodamine B molecules were subsequently photobleached and SMT data were then acquired to obtain the diffusion coefficients of single molecules across the entire film thickness, in addition to the orientations of individual

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SOLUTION−POLYMER INTERFACES An in-depth understanding of molecular interactions at solution−polymer interfaces is crucial to improving the efficiency of chemical separation and sensing techniques such as chromatography,88 microchip electrophoresis,89 and electrochemical sensors based on polymer-modified electrodes.79 For the last 2 decades, single-molecule fluorescence methods have been successfully employed to investigate molecular adsorption/desorption onto surfaces relevant to these applications.88 More recently, SMT has been used to measure the dynamic behavior of single small molecules and macromolecules at solution−solid interfaces, which often involves intermittent fast and slow/negligible diffusion processes attributable to desorption-mediated flights and adsorption/in-plane diffusion.90 For the last 3 years, advanced fluorescence microscopic techniques have been employed to gain more detailed information on dynamic behavior of single molecules/nanoparticles at aqueous interfaces with polymer films, polymer brushes, and filter membranes. Interfacial Dynamics of Polymer Molecules. The interfacial behavior of flexible polymer chains plays an essential I



Review

thin PS-b-PMMA film (Figure 6a). Fluorescently labeled dextran (MW = 10 kg/mol; 2 nm in hydrodynamic radius), which had a much higher affinity to PS than PMMA, was used as a probe molecule. The step-size distribution obtained from SMT data on nanopatterned surfaces reflected the slow inplane diffusion from molecules confined to the PS patterns and desorption-mediated flights from one pattern to another. In contrast, the step-size distribution obtained from a homogeneous PS surface exhibited the presence of an additional diffusion mode associated with slow, continuous in-plane diffusion. This additional mode was not observed on the nanopatterned surface because dextran molecules were eliminated from the PMMA matrix. They also investigated the effects of topographic heterogeneity on the surface diffusion of isolated PEG molecules using SMT.92 A PS-b-PMMA-derived nanoporous template film was used to fabricate a hydrophobic organosilane-coated silica surface patterned with a hexagonal pillar array (24 nm in pillar diameter, 37 nm in lattice spacing, 3∼7 nm in pillar height) (Figure 6b). Fluorescently labeled PEG molecules (MW = 40 kg/mol; 7.5 nm in hydrodynamic radius) on the nanotopographically patterned surface exhibited intermittent hopping-immobilization behavior reflecting desorption-mediated surface diffusion. Interestingly, the higher pillars induced an increase in the fraction of immobile molecules with longer adsorption times between hopping events, and also the reduction of diffusivity of mobile molecules. These observations were explained by obstruction to surface diffusion created by the topographic structures owing to the existence of strong interaction sites and/or the entropic stabilization of the polymer molecules in the vicinity of the pillars. Furthermore, Wang and co-workers systematically investigated the diffusion behavior of five fluorescently labeled PEG molecules of different molecular weights at aqueous interfaces with three types of PDMS oil of different viscosities using SMT (Figure 6c).93 PEG molecules adsorbed/desorbed at a water− oil interface could be selectively observed due to their slow dynamic motions, in contrast to ones in bulk water that were completely blurred due to their fast diffusion. Interfacial PEG molecules exhibited Brownian diffusion for low viscosity oil, and intermittent hopping diffusion for high viscosity oil, as observed with small molecules and biomolecules.94,95 Interestingly, the dependence of the diffusion coefficient (D) on molecular weight (M) for these PDMS oils was D ∝ M−2/3 rather than D ∝ M−1/2 as observed in water, suggesting that the PEG chains had expanded polymer conformations upon adsorption at the water−oil interfaces. Meanwhile, Aloi et al. demonstrated SLM imaging of 100 nm scale particles, emulsions, and air bubbles by taking advantage of the reversible adsorption/desorption of fluorescently labeled PEG molecules onto liquid−solid, liquid−liquid, and gas− liquid interfaces.96 On the other hand, Morrin and Schwartz revealed three regimes of polymer surface dynamics (i.e., site-blocking, crowding, and brush regimes) controlled by PEG adsorption onto a hydrophobic organosilane-coated silica surface.97 SMT measurements were carried out for a trace amount of fluorescein-labeled PEG (1 × 10−6 mg/mL) in phosphate buffered saline solutions containing different concentrations of unlabeled PEG (3 × 10−3 ∼ 100 mg/mL) of the same molecular weight (10 kg/mol). In the site-blocking regime at low PEG concentrations, an increase in effective diffusion coefficient and a decrease in surface adsorption time were

Figure 6. (a) Schematic illustration of adsorbed dextran chains (red) on a hexagonal PS nanopatterned surface (gray circles) surrounded by a continuous PMMA phase (turquoise matrix), which are shown in the inset SEM image (scale bar: 100 nm). The 2D in-plane motion of dextran molecules is confined within the isolated PS domains. Reproduced from Wang, D.; Chin, H.-Y.; He, C.; Stoykovich, M. P.; Schwartz, D. K. ACS Macro Lett. 2016, 5, 509−514 (ref 91). Copyright 2016 American Chemical Society. (b) Schematic illustration of PEG chains (colorful coils) adsorbed on a silica-based pillar array fabricated from a PS-b-PMMA film, which is shown in the inset SEM image. Reproduced from Wang, D.; He, C.; Stoykovich, M. P.; Schwartz, D. K. ACS Nano 2015, 9, 1656−1664 (ref 92). Copyright 2015 American Chemical Society. (c) Schematic illustration of adsorbed PEG molecules in a swollen (loop-train-tail) interfacial conformation (red) and a PEG molecule in bulk solution (black). The inset illustrates a TIR fluorescence microscopy setup for measurements at the interface between PDMS oil and water, in which a laser beam (blue lines) is totally reflected at the interface to generate an evanescent field for photoexcitation of fluorescently labeled PEG molecules. Reproduced from Wang, D.; Hu, R.; Mabry, J. N.; Miao, B.; Wu, D. T.; Koynov, K.; Schwartz, D. K. J. Am. Chem. Soc. 2015, 137, 12312−12320 (ref 93). Copyright 2015 American Chemical Society.

role in many chemical separations and sensing methods. As described above, small molecules and larger macromolecules showed intermittent hopping and slow/negligible diffusion behavior at solution/solid interfaces that was reflective of a desorption-mediated mechanism for surface diffusion.90 However, it was unclear whether the latter behavior of isolated polymer molecules at uniform surfaces originated from complete immobilization or slow in-plane diffusion. Wang and co-workers compared SMT data recorded on homogeneous and nanopatterned surfaces to clarify the contribution of in-plane surface diffusion of isolated dextran molecules to the slow/negligible diffusion behavior.91 The nanopatterned surface consisted of hexagonally arranged circular PS patterns (30 nm in diameter) that were surrounded by a PMMA matrix in a J



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permeable for molecules. The density of strong surface binding sites involving long-term adsorption was higher below the LCST, suggesting the chemical/physical heterogeneity of the solvent-swollen PNIPAM brush and participation by the underlying silica surface, to which the molecules could penetrate. Furthermore, it is important to understand molecular penetration into the interfacial region of a polymer film under dynamic solution flow conditions, because it could affect band broadening in chromatographic separations. Polymer brushes were used as model polymer films for the investigation of laminar-flow-induced molecular penetration. Wang and coworkers demonstrated the quantification of the penetration depth of a convective flow field within the near-interface region of a polymer brush by coupling FRET and TIR fluorescence microscopy measurements.103 They measured the quenching behavior of surface-immobilized donor probes induced by acceptor probes that penetrated from a flowing solution into a densely grafted PNIPAM brush swollen by a good solvent, methanol, (∼225 nm thick) at different flow rates. The resulting fluorescence decay curves were fitted by a coupled Taylor−Aris−Fickian diffusion model to estimate apparent diffusion coefficients of acceptor molecules within the polymer brush. They revealed that faster flow rates afforded faster acceptor diffusion in the polymer layer and also acceptor penetration to greater depths into the polymer. The penetration depth was up to 60% of the whole brush thickness at higher flow rates. Subsequently, Wang and Pemberton examined the effects of solvent quality on the slip flow penetration into the PNIPAM brush using the same method.104 Good, theta, and poor solvents for the brush were prepared by mixing methanol and water in different ratios on the basis of the conosolvency phenomena for PNIPAM. The depth of slip flow penetration was deeper in the order of good, theta, and poor solvents, reflecting changes to the conformation of polymer brushes in response to solvent quality. Dynamic Molecular Behavior at Synthetic Polymer Surfaces. SMT and STReM were employed to measure the detailed dynamic behavior of protein molecules at aqueous interfaces with thin films of synthetic polymers, PES blends,105 nylon-6,6,36,106,107 and PS.108 These polymers are used to produce protein filtration membranes and cell culture dishes. As model molecules, fluorescently labeled lysozyme106,108 and/ or α-lactalbumin,36,106,107 which have a positive and negative net charge at neutral pH, respectively, were used in addition to bovine serum albumin (BSA) and human monoclonal antibody (IgG).105 Langdon et al. employed single-molecule TIR fluorescence microscopy to study the adsorption behavior of fluorescently labeled BSA and IgG onto thin films of PES blended with PVP, PVAc-co-PVP, and PEGM.105 These films had similar thicknesses (∼30 nm), hydrophobicity (water contact angle of 50∼60°), and surface RMS roughness (≤1 nm2), and their surfaces were negatively charged. These authors showed the presence of anomalous strong surface sites where proteins preferentially adsorbed and were retained for a longer period of time. Interestingly, PVAc-co-PVP/PES exhibited shorter protein residence times, attributable to the higher spatial homogeneity in surface chemical composition. Tauzin et al. investigated the adsorption/desorption behavior of the charged protein molecules on nylon-6,6 surfaces grafted with charged polymers using SMT.106 They

observed with increasing PEG concentration due to the passivation of strong adsorption sites by unlabeled PEG. In the crowding regime at intermediate PEG concentrations, a decrease in effective diffusion coefficient and an increase in surface adsorption time were observed with increasing PEG concentration due to enhanced interactions with neighboring PEG chains. In the brush regime at high PEG concentrations, an increase in effective diffusion coefficient and a decrease in surface adsorption time were observed with increasing PEG concentration, reflecting the formation of the highly dense layer of physisorbed PEG molecules with extended conformations on the entire surface. Dynamic Molecular Behavior at Polymer Brushes. Synthetic polymer brushes have been explored to design functional surfaces for various applications including chemical separations and antifouling coatings due to the tunability of their thickness and density.98 Molecular adsorption, desorption, and diffusion at brush−solution interfaces are key processes in these applications and thus have recently been investigated at the single-molecule level. Faulon Marruecos and co-workers showed the influence of PEG brush density on protein adsorption and unfolding using single-molecule FRET methods.99 Recombinant fibronectin was dual-labeled by a FRET donor (Alexa Fluor 555) and an acceptor (Alexa Fluor 647) to assess its adsorption and conformation on PEG brushes (MW = 5 kg/mol) that were prepared via a graft-to approach. Single-molecule FRET data showed that highly dense brushes exhibited a reduction in overall adsorption rate of fibronectin molecules, which was interpreted to reflect the antifouling tendencies of PEG modification. More interestingly, the higher density brushes enhanced the unfolding of adsorbed fibronectin molecules and retained the unfolded proteins for longer residence times. The adsorption−desorption behavior of fibronectin molecules was reversible due to negligible protein−protein interactions at the very low fibronectin surface coverage (∼10−6) used for the single-molecule FRET experiments, in contrast to irreversible protein accumulation observed at much higher coverage with conventional ensemble-averaged methods. Subsequently, they investigated the local hydrophobicity in PEG brushes using SMT with fluorescent solvatochromic nitrobenzoxadiazole, which yielded red-shifted emission in more polar environments.100 SMT data were simultaneously recorded in two spectral bands to estimate the local hydrophobicity of a grafted surface from the emission ratio, with subdiffraction resolution.101 Such two-color SMT measurements clarified that the brush hydrophobicity was spatially and temporally heterogeneous and was higher at higher surface brush density, which could not be recognized from static contact angle measurements. The presence of more nanoscale hydrophobic niches at the higher brush density could be correlated to the enhanced unfolding of fibronectin molecules. In addition, Chin and co-workers measured the effects of temperature on the desorption-mediated surface diffusion of small molecules and polymers at aqueous interfaces with thermoresponsive polymer brushes grown via radical polymerization.102 SMT was employed to measure the surface diffusion of fluorescently labeled fatty acid and dextran (MW = 10 kg/ mol) on a PNIPAM brush below and above the lower critical solution temperature (LCST) (∼32 °C). Regardless of molecular size, molecular surface mobility was distinctly higher above the LCST, reflecting the collapse of the brush to give a more hydrophobic dense polymer surface that was less K



Review

and Schwartz demonstrated SPT measurements of fluorescently labeled 40 nm PS particles diffusing inside a nitrocellulose membrane comprising ∼3 μm pores filled with an index-matching solution based on thiodiethanol (n = 1.52) and Triton X-100 (n = 1.51).111 They observed a significant reduction and wide distribution of particle diffusion coefficients in the nitrocellulose membrane as compared with a glass fiber membrane having similar nominal pore size and porosity. The diffusion behavior reflected the geometric obstruction of particle diffusion by the complex pore morphology as shown by a confocal image, particle adsorption onto the pore walls and hydrodynamic drag between the particles and pore walls. Subsequently, Skaug et al. measured the diffusion of fluorescently labeled PS particles of different diameters (40, 100, and 200 nm) within a polymer matrix comprising disordered 0.1∼10 μm pores that were formed from a UV-cured optical adhesive.112 They observed spatially dependent, heterogeneous particle diffusion behavior as well as a strong particle size dependence of particle accessibility to the void space. These observations were attributable to a kinetic mechanism based on restricted particle motions under the influences of spatially and temporally heterogeneous hydrodynamic effects. Cai and Schwartz measured single particle tracking trajectories moving within a porous PVDF filtration membrane under convective flow conditions to assess the spatiotemporal heterogeneity of particle pathways and retention.113 They measured fluorescently labeled PS particles (40 and 200 nm in diameter) diffusing in a PVDF membrane (n = 1.42) comprising ∼0.65 μm pores that were filled with an indexmatching solution based on 2-propanol and Triton X-100. Higher tortuosity was obtained for the larger particles under slower flow, indicating that the pathway tortuosity depended on flow rate and particle size in addition to structural material properties. SPT data also revealed the presence of highly confined regions that exhibited more meandering trajectories and longer residence times, enhancing the heterogeneity in pathway tortuosity and retention time. Furthermore, they discussed mechanisms of membrane fouling based on the PS tracer particles for PVDF (∼0.65 μm pores, n = 1.42) and PTFE (∼0.45 μm pores, n = 1.36) membranes.114 SPT measurements were carried out in index-matching solutions based on alcohols and Triton X-100 in which “stickiness”, which represented the feasibility of particle aggregation and particle adsorption onto the membranes, was controlled by the surfactant concentration. They could directly visualize different mechanisms behind membrane fouling in more and less sticking conditions. The former condition offered rapid fouling but exhibited constant average particle velocity and tortuosity due to pore blocking by particle aggregates. In contrast, the less sticking condition gave slower fouling but showed the reduction of particle velocity and an increase in pathway tortuosity as fouling progressed, reflecting gradual adsorption of particles onto the membrane surface. These phenomena were observed regardless of the membrane materials and particle sizes. Meanwhile, Parrish et al. employed SPT to investigate detailed nanoparticle dynamics in thermoresponsive hydrogels.115 Diffusional motions of single PEG-grafted quantum dots (10 nm in hydrodynamic diameter) were measured in PNIPAM hydrogels with different cross-linking densities at different temperatures around their volume phase transition temperature (VPTT). They showed the presence of two

observed the electrostatic enhancement of the interfacial adsorption of lysozyme and α-lactalbumin on anionic and cationic polymer-grafted surfaces, respectively. Interestingly, lysozyme molecules on the anionic polymer grafted surface exhibited desorption-mediated surface diffusion, in contrast to α-lactalbumin molecules on cationic polymer-grafted surfaces that showed complete immobilization upon adsorption. In the former system, the adsorption frequency and desorptionmediated diffusion were enhanced for longer surface grafting times. Subsequently, Shen et al. investigated the adsorption of α-lactalbumin molecules on flat and porous nylon-6,6 surfaces.107 Porous films comprising micrometer-scale pores (15−20 nm deep) were obtained as a result of phase separation in a polymer solution of a formic acid−ethanol mixture. On both surfaces, adsorption kinetics followed a monolayer adsorption model, suggesting the negligible involvement of protein aggregation and/or multilayered adsorption at the very low protein concentration used for the SMT experiments, in contrast to ensemble data measured at a much higher concentration. The porous films exhibited significantly slower adsorption kinetics and higher adsorption site density than the flat films, which were explained by steric effects and larger surface area. Furthermore, Wang and coworkers employed STReM to gain more detailed information on adsorption/desorption behavior of α-lactalbumin molecules on a flat nylon-6,6 surface.36 STReM provided quantitative information on adsorption events and surface dwell time at higher time resolution (5 ms) as compared with regular SMT (100 ms) (Figure 3b). As a result, STReM data resolved a quicker process involving the formation of tightly folded αlactalbumin molecules upon its adsorption onto the nylon-6,6 surface. In addition, this study showed the accelerated desorption of α-lactalbumin molecules at higher ionic strength, which was explained by the adsorption-induced formation of tightly folded molecules. In addition to nylon-6,6, protein adsorption on PS surfaces were also investigated using SMT. Moringo et al. investigated the effects of PS surface functionalization on the surface dynamics of single lysozyme molecules.108 The surface of PS thin films were converted to be more hydrophilic by UV exposure, oxygen plasma treatment, and the grafting of hydrophilic polymer chains. SMT results showed that an increase in surface hydrophilicity led to the reduction of protein adsorption frequency and the occurrence of desorption-mediated surface diffusion. The authors explained these results by the reduction of hydrophobic adsorption sites as a result of the formation of a water hydration layer at the hydrophilic surface. Dynamic Nanoparticle Behavior in Porous Materials. Porous membranes based on synthetic polymers have been widely employed for various applications including filtration and water treatment.109 Detailed, static nanoscale porous structures derived from synthetic polymers have been routinely measured using SEM. It is however challenging to understand detailed mass transport mechanisms within polymer membranes due to the spatial and temporal heterogeneity in the three-dimensional morphologies and chemical compositions of the pore network. SPT has been employed for in situ mass transport measurements with porous polymer matrixes using an indexmatching technique, in which the pores were filled with a liquid having a refractive index close to that of the solid matrix to reduce light scattering from solid-void boundaries.110 Skaug L



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localization mechanisms for quantum dot confinement into the hydrogel matrix, including attractive chemical interactions with flexible PNIPAM chains at the shorter distance range and steric entrapment by the cross-linked gel matrix.

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CONJUGATED POLYMERS Organic polymers incorporating conjugation that extends across multiple monomer units represent unique materials

Figure 8. Fluorescence images showing the motions of three MEHPPV aggregates in which aggregates 1 and 2 coalesce into a single aggregate, C, toward the end of the series. The plots show the trajectories of aggregates 1 and 2 and their intensities as a function of time. Reproduced from In Situ Optical Imaging of the Growth of Conjugated Polymer Aggregates, Yang, J.; Park, H.; Kaufman, L. J. Angew. Chem. Int. Ed., Vol. 57, Issue 7 (ref 125). Copyright 2018 Wiley.

Figure 7. (a) Top: EL-STED microscope with wavelengthdiscriminated detection. Passing a current across an electroluminescent polymer film provides wide-field excitation and the emission is passed through a pinhole in the image plane and detected using a single-photon diode or spectrometer. A “doughnut” CW depletion beam is then focused to fill the field-of-view defined by the pinhole, quenching the majority of emissive excited states. Excitation sources can be changed from optical to electrical, allowing PL and EL images to be taken sequentially. Bottom: Spectra of MEH-PPV EL and the depletion laser, tuned to the red-side of the emission profile. This prevents absorption of the depletion beam by the sample while still allowing for efficient depletion. (b) EL-STED image of a small defect in an operating organic light-emitting diode. Blue and red are low and high intensity, respectively. (c) Same region in the same device imaged with PL-STED-spectral imaging. Blue and red are believed to be regions of relatively less dense- and more dense-packed chains, probably reflecting varying degrees of annealing during device preparation. Reprinted by permission from Macmillan Publishers Ltd.: NATURE COMMUNICATIONS, King, J. T.; Granick, S. Nat. Commun. 2016, 7, 11691 (ref 121). Copyright 2016.

Figure 9. (a) Fluorescence image of PFBT conjugated polymer nanoparticles. (b) Trajectory of the apparent particle position in time. (c) Model for quenching of a nanoparticle by a mobile hole polaron. Reproduced from Jiang, Y.; Nongnual, T.; Groff, L.; McNeil, J. J. Phys. Chem. C 2018, 122, 1376−1383 (ref 146). Copyright 2018 American Chemical Society.

Conjugated polymers comprise dense multichromophoric systems in which the chromophores often exhibit strong electronic interactions with their neighbors. Much remains to be learned about the structural and organizational origins of these interactions. Their electronic consequences are of paramount importance to applications of these materials, as these help determine the wavelengths of light absorbed and emitted as well as the lifetimes of photogenerated excited states and their quenching by defects and charge carriers. Methods for controlling the assembly of conjugated polymer chains and aggregates must be developed to achieve the full potential of these materials in many of their fundamental and technological applications. All of these issues continue to be widely explored in the literature. As with all organic polymers, the quality of the solvent in which the polymer is dissolved plays a critical role in determining the conformation of the polymer chains and, hence, the degree to which the individual chromophores interact in solution, and in the solid state after they have been

that can be made to conduct electricity and to absorb and emit visible light.116 Figure 1c shows representative conjugated polymers discussed in this review. Thin films of conjugated polymers are now being widely explored for use in organic electronics, as photovoltaics, as light emitting diodes, and as optical and chemical sensors.116 They have unique advantages over traditional inorganic conductors and semiconductors in that they are solution processable and their films can be flexible and self-repairing. CPNs comprising from one to several individual polymer molecules can also be prepared. CPNs are now being explored as luminescent probes in disease diagnosis and biological imaging,117 as light harvesting systems,118 and as probes for super-resolution imaging applications.28 Noteworthy advantages over fluorescent dyes include their higher brightness and greater photostability.28 M



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cast as thin films or precipitated as CPNs or as individual molecules. The aggregates that form as a result of chromophore interactions exhibit absorption and emission spectra that differ markedly from those of the unaggregated species. Their spectral characteristics depend on whether the transition dipoles of the individual chromophores are aligned end-to-end (forming J-aggregates), side-by-side (H-aggregates), or in some intermediate geometry. Aggregates can form from chromophore interactions along a single polymer chain (intrachain aggregates, often J-aggregates) or between different polymer chains (interchain aggregates, often Haggregates). A wide variety of optical microscopic and spectroscopic methods have been employed to better understand the structures formed by conjugated polymers and their resulting electronic and photophysical properties. Representative recent studies are reviewed below. For more comprehensive, historical, or applications-specific information, the reader is referred to the many review articles that have been published.13,118−120 Nanoscale Morphologies of Conjugated Polymer Films. Super-resolution optical microscopy has revealed the submicrometer domain morphologies in conjugated polymer thin films. King and Granick demonstrated the spectral superresolution imaging of an operating organic light-emitting diode based on a 50 nm thick MEH-PPV film using STED microscopy.121 A STED-type microscope (Figure 7a) was built to map the local intensity of electroluminescence (EL) stimulated by electrical current as well as to obtain the local photoluminescence (PL) spectra via two-photon excitation in the absence of an applied current. EL-STED images (Figure 7b) showed heterogeneous EL intensity reflecting the presence of defects. A PL-STED spectral image from the same area (Figure 7c) revealed heterogeneity in chain packing density, as indicated by the variation of the ratio of emission intensities at two wavelengths that was higher for the higher packing density due to a bathochromic shift in the emission. Interestingly, these two images revealed that the brighter EL emission came from regions with more densely packed chains. Polymer Chain Conformation and Aggregation. Aggregation of the chromophores in conjugated polymers plays an important role in governing charge transport in their films,120,122 blinking and bleaching dynamics in CPNs and single molecules, and their absorption and emission characteristics in any of these forms.120 The formation of aggregates along and between conjugated polymer chains is related to polymer conformation and can be induced by solvent evaporation or by exposure to poor solvents. In recent studies, Yang et al. employed measurements of luminescence intensity and optical anisotropy to follow the formation of MEH-PPV aggregates in PMMA films under solvent vapor annealing conditions.123 In this study, the polymer was spin-cast from PMMA solutions in toluene, having different MEH-PPV concentrations. As cast films showed a nearly linear increase in intensity with MEH-PPV concentration, suggesting they were initially composed of isolated, unaggregated polymer chains, in contrast to what is expected when aggregates form. The authors subsequently employed a specially designed apparatus that allowed them to precisely control vapor delivery during solvent vapor annealing.55 Specifically, they monitored polymer swelling during their optical measurements by use of a quartz crystal microbalance. Upon exposure to acetone−chloroform vapor,

clear evidence of aggregate formation was observed. Polarization anisotropy measurements of the aggregates were then made by inserting a rotating polarizer in the excitation beam path and monitoring the fluorescence intensity synchronously with the rotating polarization. The polarization modulation, M, was obtained by fitting the modulated intensity data to eq 2. I(ϕ) = I0[1 = M cos(2(ϕ − ϕ0))]

(2)

where I(ϕ) is the fluorescence intensity as a function of the incident polarization angle, ϕ, and ϕ0 is the polarization angle at which maximum fluorescence is observed. Simultaneously with the aforementioned changes in fluorescence intensity, the polarization modulation depth increased for the isolated chains and remained high for the aggregates, indicating that the polymer chains were present in highly ordered, collapsed conformations. Vapor annealing studies have also been employed to help identify the mechanisms by which conjugated polymer aggregates grow. Early investigations pointed to the importance of Ostwald ripening in the aggregation of MEHPPV dispersed in PMMA.124 More recently, Yang et al. followed the growth of MEH-PPV aggregates in situ by tracking the aggregates as they diffused through PMMA films that were again swollen by exposure to acetone−chloroform vapor.125 Their studies confirmed the importance of Ostwald ripening in aggregate growth but also revealed that some aggregates grew by coalescence of smaller aggregates. Figure 8 shows particle tracking results that depict aggregate growth by coalescence. Polarization-dependent imaging revealed that aggregate growth by Ostwald ripening preserved the order of the polymer chains while particle coalescence led to increased chromophore disorder. Tauber et al. employed 2D polarization imaging to investigate the formation of micrometer-scale domains in binary blend films of conjugated polymers that were examined as bulk heterojunction organic photovoltaics.126 These authors modulated both the excitation and emission polarizations to explore polymer chain alignment and morphology. The blend films consisted of electron donor and acceptor polymers, the latter of which formed fiber-like aggregates comprising highly oriented chains. Solvent annealing induced micrometer-scale aggregate formation, which could lead to the improvement of power conversion efficiency of the photovoltaics. The same method was also used to observe millimeter-scale domains in a liquid-crystalline conjugated polymer film formed on a liquid surface.127 Spectroscopic evidence is required to detect chromophore aggregation and such evidence usually comes from timeresolved measurements of excited state lifetimes or from wavelength-resolved emission spectra. When employed in an imaging modality, such measurements can provide important knowledge on materials heterogeneity. So and co-workers employed FLIM to understand the effects of solvent on the appearance of monomer- and aggregate-like emission in CPNs based on PPV oligomers.128 The fluorescence lifetime was longer at the periphery and shorter in central regions of PPV particles formed from a 90% methanol in methyl tetrahydrofuran solution, attributable to the formation of a “core− shell” structure, with relatively greater aggregation of the PPV chains in central regions. In contrast, PPV particles prepared from an aqueous solution exhibited shorter lifetimes, suggesting a larger fraction of the PPV oligomers formed aggregates. Yu et al. used FLIM and FAIM to reveal the N



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formation of micrometer-scale β-conformational aggregates surrounded by glassy conformational matrix in thin films of polyfluorene derivatives.129 These aggregates exhibited a shorter fluorescence lifetime due to higher energy transfer efficiency and also higher emission anisotropy, reflecting the presence of oriented rigid chain structures, as compared with the amorphous matrix. Baghgar and Barnes employed photoluminescence imaging in the spectral, temporal, and polarization domains to explore the relationships between polymer chain aggregation in crystalline P3HT nanowires and their electronic properties.130 The crystalline nanowires were prepared from dichloromethane/chloroform mixtures and were subsequently diluted and drop cast on glass substrates to obtain isolated nanofibers. Nanofibers prepared from polymers having a range of different molecular weights were studied. Their spectroscopic data showed that intrachain J-aggregates became more prevalent with increasing molecular weight, while interchain Haggregates commonly formed at low molecular weight. In a subsequent study that coupled optical imaging with Kelvin probe force microscopy, these authors further explored P3HT nanofiber properties as a function of polymer chain regioregularity and molecular weight.131 They found that enhanced interchain aggregation led to a decrease in the highest occupied molecular orbital (HOMO) energy of the nanofibers, as observed via surface potential contrast measurements. They also found that increased regioregularity at high molecular weights led to increased chain entanglement and also lowered the HOMO energy. In related studies, other groups sought to better control polymer chain aggregation by adding and manipulating polymer side-chain structures. For example, Eder et al. prepared PPE derivatives having chemically different hexyloxy side chains that behave differently under solvent vapor annealing.132 They showed that the different side chains allowed them to control the prevalence of H- and J-aggregates in single conjugated polymer aggregates, as revealed by wavelength-resolved and polarization-dependent imaging methods. Shao et al. in turn sought to use hydrogen bonding interactions as a means to control polymer chain folding in short BEH-PPV oligomers.133 Their polymers incorporated side chains having carboxylic or urea units. Polarization dependent imaging showed that the polymers with the ureabased side chains exhibited the greatest order. Nakamura et al. investigated the origins of the green emission sometimes observed from PFO instead of its usual blue emission.134 Although studied for many years, the origins of the green emission developed upon UV irradiation in air remained uncertain. Some groups attributed its appearance to oxidation of the polymer chains,135 while others suggested polymer chain cross-linking plays a role.136 An important alternative hypothesis was the formation of aggregates and/or excimers, as described in earlier works.135,137 Nakamura and co-workers used ensemble and single molecule wavelengthand time-resolved spectroscopies to probe PFO emission, with the conjugated polymer molecules dispersed in polystyrene and PMMA matrixes. They obtained evidence for emission from H-type aggregates and the participation of charge-transfer or excimer states in the appearance of the green emission. Both of these factors were concluded to arise from short-range intrachain interactions in the compact chain conformations assumed by the polymers.

In a pair of publications, Park et al., studied the photobleaching dependence of MEH-PPV molecules immobilized in polystyrene films on polymer chain conformation.27,138 In one report,27 they used superlocalization methods to estimate the radius of gyration for each molecule from the positions of the individual emitters. They found that molecules exhibiting stepwise photobleaching dynamics were more compact, incorporating strongly interacting chromophores, while those exhibiting continuous bleaching behavior were more elongated and comprised many independent and simultaneously emitting chromophores. In the second report,138 they used polarization dependent fluorescence excitation to explore how photobleaching impacted polymer chain order and hence how these data could be used to infer chromophore stability. Their polarization modulation results revealed a decrease in apparent chromophore order when collapsed, highly ordered molecules were partially bleached, while an increase in order was observed when molecules of random coil conformation were partly bleached. They also concluded that ordered segments of these molecules were relatively less photostable than less ordered regions. Excited State and Charge Carrier Diffusion. The excited states accessed by photoexcitation of conjugated polymers are formally known as Frenkel excitons, comprising collective excitation of many neighboring chromophores through the coupling of their transition dipoles. The specific geometries of the chromophore assemblies and their electronic interactions lead to the observation of characteristic spectra commonly attributed to the presence of H- or J-aggregates. These classifications have been widely employed in assessing chromophore organization in the work reviewed in the previous section and elsewhere in the literature. Importantly, the optical excitons formed in conjugated polymers are mobile and can migrate along individual polymer molecules or cross between neighboring polymer chains. When a conjugated polymer is oxidized or reduced by chemical, electrochemical, or optical means, charge carriers are incorporated into the polymer. Both hole and electron polarons, which are formally radical cations and radical anions, respectively, are formed. Polarons are mobile and increase the conductivity of the conjugated polymers. They also interact with optical excitons, playing an important role in their quenching, and hence, the intensity of emission observed following excitation by either optical or electronic means. A better understanding of exciton and polaron dynamics has been the focus of many optical microscopic studies of conjugated polymer systems. Exciton Dynamics. Unique optical imaging methods were recently employed by Hou and co-workers to address exciton dynamics and quenching.139 In this study, the authors used photothermal imaging methods140 to acquire optical absorption images while also collecting emission images of single MEH-PPV molecules dispersed in PMMA. The sensitivity of photothermal imaging was enhanced by the use of near-critical xenon (6.26 MPa, 288 K) so that absorption by single molecules could be detected. Their results allowed for quantitative measurements of single molecule absorption cross sections and emission quantum yields. The authors were also able to determine the number of monomers in each molecule. Their observations showed that irradiation of the molecules resulted in a more rapid decline in the emission, compared to parallel changes observed in polymer absorption. They also found that larger (higher MW) molecules had O



Review

Figure 10. (a) Experimental design for in situ fluorescence observation of ring-opening metathesis polymerization (ROMP) for polynorbornene at second-generation Grubbs’ catalysts (1) through the insertion of fluorescently labeled ROMP probes (3). Reproduced from Kinetics of the Same Reaction Monitored over Nine Orders of Magnitude in Concentration: When Are Unique Subensemble and Single-Turnover Reactivity Displayed?, Easter, Q. T.; Blum, S. A. Angew. Chem. Int. Ed., Vol. 57, Issue 37 (ref 149). Copyright 2018 Wiley. (b) TIR fluorescence images of polynorbornene precipitate(s) (top) show bright-green quantized flashes at specific times, corresponding to single monomer reactions of 3. A fluorescence intensity versus time trace (bottom) of the region within the yellow box shows signal spikes attributable to the insertion of individual probes 3 into polymer chains, followed by photobleaching. Reproduced from Single Turnover at Molecular Polymerization Catalysts Reveals Spatiotemporally Resolved Reactions, Easter, Q. T.; Blum, S. A. Angew. Chem. Int. Ed., Vol. 56, Issue 44 (ref 147). Copyright 2017 Wiley. (c) Intensity versus time traces for regions within single aggregates of polynorbornene (green particles), as indicated by the red boxes in the image. Initial vertical increases in fluorescence intensity represent polymer precipitation events. Reproduced from Evidence for Dynamic Chemical Kinetics at Individual Molecular Ruthenium Catalysts, Easter, Q. T.; Blum, S. A. Angew. Chem. Int. Ed., Vol. 57, Issue 6 (ref 148). Copyright 2018 Wiley.

smaller emission yields. They interpreted their observations to reflect the creation of photogenerated sites that quenched the optical excitons. They concluded that more such sites form in the longer molecules, but enhanced participation by exciton− exciton annihilation could also explain their findings. Nabha-Barnea et al. employed NSOM to investigate the degradation of photoluminescence from conjugated organic polymer films on submicrometer length scales. Their films comprised phase-separated blends of FRET donor and acceptor polyfluorene derivatives.141 These studies made use of both the super-resolved imaging capabilities of NSOM and the fact that it simultaneously provided correlated topographic and photoluminescence images, allowing for the polymer domain in which photodegradation was most severe to be determined. The results showed that faster degradation occurred in donor-rich domains during long-term irradiation of the films. Degradation of the photoluminescence likely involved formation of defect sites in the donor-rich domains that trapped the optically prepared excitons. The results also

suggest that energy transfer to the acceptor domains did not enhance the photostability of the donor domains. Hu and co-workers employed single molecule spectroscopic investigations of individual P3HT molecules and aggregates to explore the role of interchain assemblies in governing energy migration within the polymers.142 Observed photoblinking of the single aggregates was used to detect variations in energy migration. They used solvent vapor annealing for conjugated polymers with different side chains and showed that wellordered regioregular polymers having a high degree of interchain packing exhibited long-range interchain energy migration. In contrast, disordered aggregates formed from regiorandom polymers and those with bulky side chains strongly inhibited energy migration. Charge Carrier Dynamics. Fluorescence fluctuations in luminescent CPNs have long been attributed to intermittent fluorescence quenching by photogenerated hole polarons (radical cations)143 and other trapping sites,144 such as chemical or physical defects in the polymer chains. Yu and P



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molecules without ensemble averaging. They have also revealed the spatiotemporal heterogeneity of polymer properties and the interfacial dynamics of single polymer molecules. We can foresee a number of future research directions in this field, including 3D SMT measurements in/on polymer materials,24 the development of new solvatochromic probes for visualizing and characterizing polymer domains and their properties,42 and the direct visualization of polymer chain growth at the terminal.150 Furthermore, novel microscopic methods developed for biological imaging17 will afford the possibility to reveal various unexplored properties of synthetic polymeric materials.

co-workers originally used super localization imaging of individual PFBT nanoparticles to study this phenomenon.145 They observed apparent motions of the particle centroid and attributed this motion to the hopping of hole polarons between trap sites within the nanoparticles. In this case, polaron motion was manifested as a dark region that moved through the fluorescent spot produced by the CPN. Because of the small size of the CPNs (∼15 nm) and the tightly confined regions over which the polarons could move, these studies required the ability to localize the particle centroid to within ∼1 nm in the sample plane. Their initial study employed a CCD camera for detection of the fluorescence and this limited their time resolution to ∼20 ms. In more recent studies by Jiang and coworkers,146 a sCMOS camera afforded 1 ms time resolution and allowed them to capture most, if not all, of the individual polaron hopping events, as shown in Figure 9. Their results revealed the presence of deep traps at 2−5 nm separations, consistent with the presence of chemical or structural defects in the polymers.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

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Daniel A. Higgins: 0000-0002-8011-2648 Takashi Ito: 0000-0001-7443-3157

IN SITU MONITORING OF POLYMERIZATION REACTIONS Very recently, SLM has been explored for in situ monitoring of polymer growth at individual molecular catalysts. Easter and Blum successfully demonstrated the observation of single insertion events of fluorescently labeled monomers at individual ruthenium catalysts during ring-opening metathesis polymerization (ROMP) for polynorbornene under synthetically relevant conditions (Figure 10a).147 TIR fluorescence microscopy was used to monitor the fluorescence intensity of individual polymer precipitates incorporating multiple catalysts. Here, the experiments were carried out in a heptane solution containing a low concentration of labeled monomer (≤1 pM) with excess unlabeled monomer (≥10 mM). The insertion of a labeled monomer into a polymer chain could be recognized as the appearance of a fluorescent spot, followed by its disappearance due to photobleaching (Figure 10b). Subsequently, the same authors assessed the insertion rate of labeled monomers at individual catalyst-incorporating precipitates at a higher labeled monomer concentration (2 nM).148 A gradual increase in the fluorescence intensity of a polymer precipitate resulted from the insertion of labeled monomers at multiple catalyst sites (Figure 10c). The rate of increase in fluorescence intensity, corresponding to the insertion rate of labeled monomers, was different for individual precipitates possibly due to the variation of the number of active catalysts. More interestingly, many precipitates exhibited abrupt changes in insertion rate, attributable to the dynamic fluctuation of monomer accessibility to catalytic sites. Furthermore, they compared polymer growth rate at different labeled monomer concentrations (10−9 to ∼10−6 M),149 showing that the insertion rate of labeled monomers was higher at the higher labeled monomer concentration.

Notes

The authors declare no competing financial interest. Biographies Herman Coceancigh obtained a B.S. degree in Chemistry from University of Buenos Aires, Argentina, in 2012. He is currently a Ph.D. student in Prof. Takashi Ito’s group at Kansas State University. He was a Fulbright scholar in 2014−2016. His research interests include electrochemically controlled functionalization of porous polymer membranes and single-molecule fluorescence studies of polymer dynamics. Daniel A. Higgins serves as a Professor and Head of the Chemistry Department at Kansas State University (KSU). He received a B.A. in Chemistry from St. Olaf College in 1988 and a Ph.D. in Chemistry from the University of Wisconsin, Madison in 1993. Afterwards, he did postdoctoral research at the University of Minnesota. He has served on the faculty at KSU since 1996. His group employs optical microscopy and single molecule fluorescence detection and tracking methods to probe the properties of chemical gradients and mesoporous silica materials on nanometer length scales. Takashi Ito is a Professor of Chemistry at Kansas State University. He received his B.S. (1993), M.S. (1995), and Ph.D. (1998) degrees in Chemistry from the University of Tokyo. He worked as a Research Associate at Tokyo University of Science and then as a Postdoctoral Research Associate at Texas A&M University before starting his independent career as an Assistant Professor at Kansas State University in 2004. His current research projects involve investigation of mass and charge transport within self-organized nanostructures using electrochemical and microscopic techniques for future development of efficient separation and sensing materials. He currently serves as an editorial board member for The Chemical Record (Wiley).

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ACKNOWLEDGMENTS The authors acknowledge the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the U.S. Department of Energy (Grant DE-SC0002362) for financial support of this work. H.C. thanks the Fulbright Program for financial support.

SUMMARY AND OUTLOOK Recent advances in optical microscopic techniques permit us to measure the detailed morphological, dynamic, and physicochemical properties of synthetic polymers of scientific and technological interest under ambient conditions with spatial resolution beyond the diffraction limit. These methods have not only visualized polymer nanostructures but also revealed the dynamic behavior of polymer chains and probe

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