Polarization-resolved single-molecule tracking reveals strange dynamics of fluorescent tracers through a deep rubbery polymer network.

Tracking the movement of fluorescent single-molecule (SM) tracers has provided several new insights into the local structure and dynamics in complex environments such as soft materials and biological systems. However, SM tracking (SMT) remains unreliable at molecular length scales, as the localization error (LE) of SM trajectories (∼30-50 nm) is considerably larger than the size of molecular tracers (∼1-2 nm). Thus, instances of tracer (im)mobility in heterogeneous media, which provide indicators for underlying anomalous-transport mechanisms, remain obscured within the realms of SMT. Since the translation of passive tracers in an isotropic media is associated with fast dipolar rotation, we propose that authentic pauses within the LE can be revealed by probing the hindrance of SM reorientational dynamics. Here, we demonstrate how polarization-resolved SMT (PR-SMT) can provide emission anisotropy at each super-localized position, thereby revealing the tumbling propensity of SMs during random walks. For rhodamine 6G tracers undergoing heterogeneous transport in a hydrated polyvinylpyrrolidone (PVP) network, analysis of PR-SMT trajectories enabled us to discern instances of genuine immobility and localized motion within the LE. Our investigations on 100 SMs in (plasticized) PVP films reveal a wide distribution of dwell times and pause frequencies, demonstrating that most probes intermittently experience complete translational and rotational immobilization. This indicates that tracers serendipitously encounter compact, rigid polymer cavities during transport, implying the existence of nanoscale glass-like domains sparsely distributed in a predominantly deep-rubbery polymer network far above the glass transition.

Cooperative Supramolecular Block Copolymerization for the Synthesis of Functional Axial Organic Heterostructures.

Supramolecular block copolymerzation with optically or electronically complementary monomers provides an attractive bottom-up approach for the non-covalent synthesis of nascent axial organic heterostructures, which promises to deliver useful applications in energy conversion, optoelectronics, and catalysis. However, the synthesis of supramolecular block copolymers (BCPs) constitutes a significant challenge due to the exchange dynamics of non-covalently bound monomers and hence requires fine microstructure control. Furthermore, temporal stability of the segmented microstructure is a prerequisite to explore the applications of functional supramolecular BCPs. Herein, we report the cooperative supramolecular block copolymerization of fluorescent monomers in solution under thermodynamic control for the synthesis of axial organic heterostructures with light-harvesting properties. The fluorescent nature of the core-substituted naphthalene diimide (cNDI) monomers enables a detailed spectroscopic probing during the supramolecular block copolymerization process to unravel a nucleation-growth mechanism, similar to that of chain copolymerization for covalent block copolymers. Structured illumination microscopy (SIM) imaging of BCP chains characterizes the segmented microstructure and also allows size distribution analysis to reveal the narrow polydispersity (polydispersity index (PDI) ≈ 1.1) for the individual block segments. Spectrally resolved fluorescence microscopy on single block copolymerized organic heterostructures shows energy migration and light-harvesting across the interfaces of linearly connected segments. Molecular dynamics and metadynamics simulations provide useful mechanistic insights into the free energy of interaction between the monomers as well as into monomer exchange mechanisms and dynamics, which have a crucial impact on determining the copolymer microstructure. Our comprehensive spectroscopic, microscopic, and computational analyses provide an unambiguous structural, dynamic, and functional characterization of the supramolecular BCPs. The strategy presented here is expected to pave the way for the synthesis of multi-component organic heterostructures for various functions.

Non-Gaussian subdiffusion of single-molecule tracers in a hydrated polymer network.

Single molecule tracking experiments inside a hydrated polymer network have shown that the tracer motion is subdiffusive due to the viscoelastic environment inside the gel-like network. This property can be related to the negative autocorrelation of the instantaneous displacements at short times. Although the displacements of the individual tracers exhibit Gaussian statistics, the displacement distribution of all the trajectories combined from different spatial locations of the polymer network exhibits a non-Gaussian distribution. Here, we analyze many individual tracer trajectories to show that the central portion of the non-Gaussian distribution can be well approximated by an exponential distribution that spreads sublinearly with time. We explain all these features seen in the experiment by a generalized Langevin model for an overdamped particle with algebraically decaying correlations. We show that the degree of non-Gaussianity can change with the extent of heterogeneity, which is controlled in our model by the experimentally observed distributions of the motion parameters.

Molecular Intercalation and Electronic Two Dimensionality in Layered Hybrid Perovskites.

In layered hybrid perovskites, such as (BA)2 PbI4 (BA=C4 H9 NH3 ), electrons and holes are considered to be confined in atomically thin two dimensional (2D) Pb-I inorganic layers. These inorganic layers are electronically isolated from each other in the third dimension by the insulating organic layers. Herein we report our experimental findings that suggest the presence of electronic interaction between the inorganic layers in some parts of the single crystals. The extent of this interaction is reversibly tuned by intercalation of organic and inorganic molecules in the layered perovskite single crystals. Consequently, optical absorption and emission properties switch reversibly with intercalation. Furthermore, increasing the distance between inorganic layers by increasing the length of the organic spacer cations systematically decreases these electronic interactions. This finding that the parts of the layered hybrid perovskites are not strictly electronically 2D is critical for understanding the electronic, optical, and optoelectronic properties of these technologically important materials.

Insights on heterogeneity in blinking mechanisms and non-ergodicity using sub-ensemble statistical analysis of single quantum-dots.

Photo-luminescence (P-L) intermittency (or blinking) in semiconductor nanocrystals (NCs), a phenomenon ubiquitous to single-emitters, is generally considered to be temporally random intensity fluctuations between "bright" ("On") and "dark" ("Off") states. However, individual quantum-dots (QDs) rarely exhibit such telegraphic signals, and yet, a vast majority of single-NC blinking data are analyzed using a single fixed threshold which generates binary trajectories. Furthermore, while blinking dynamics can vary dramatically over NCs in the ensemble, the extent of diversity in the exponents (mOn/Off) of single-particle On-/Off-time distributions (P(tOn/Off)), often used to validate mechanistic models of blinking, remains unclear due to a lack of statistically relevant data sets. Here, we subclassify an ensemble of QDs based on the emissivity of each emitter and subsequently compare the (sub)ensembles' behaviors. To achieve this, we analyzed a large number (>1000) of blinking trajectories for a model system, Mn+2 doped ZnCdS QDs, which exhibits diverse blinking dynamics. An intensity histogram dependent thresholding method allowed us to construct distributions of relevant blinking parameters (such as mOn/Off). Interestingly, we find that single QD P(tOn/Off)s follow either truncated power law or power law, and their relative proportion varies over subpopulations. Our results reveal a remarkable variation in mOn/Off amongst as well as within subensembles, which implies multiple blinking mechanisms being operational amongst various QDs. We further show that the mOn/Off obtained via cumulative single-particle P(tOn/Off) is distinct from the weighted mean value of all single-particle mOn/Off, evidence for the lack of ergodicity. Thus, investigation and analyses of a large number of QDs, albeit for a limited time span of a few decades, are crucial to characterize the spatial heterogeneity in possible blinking mechanisms.

Single-Particle Tracking To Probe the Local Environment in Ice-Templated Crosslinked Colloidal Assemblies.

We use single-particle tracking to investigate colloidal dynamics in hybrid assemblies comprising colloids enmeshed in a crosslinked polymer network. These assemblies are prepared using ice templating and are macroporous monolithic structures. We investigate microstructure-property relations in assemblies that appear chemically identical but show qualitatively different mechanical response. Specifically, we contrast elastic assemblies that can recover from large compressive deformations with plastic assemblies that fail on being compressed. Particle tracking provides insights into the microstructural differences that underlie the different mechanical response of elastic and plastic assemblies. Since colloidal motions in these assemblies are sluggish, particle tracking is especially sensitive to imaging artifacts such as stage drift. We demonstrate that the use of wavelet transforms applied to trajectories of probe particles from fluorescence microscopy eliminates stage drift, allowing a spatial resolution of about 2 nm. In elastic and plastic scaffolds, probe particles are surrounded by other particles-thus, their motion is caged. We present mean square displacement and van Hove distributions for particle motions and demonstrate that plastic assemblies are characterized by significantly larger spatial heterogeneity when compared with the elastic sponges. In elastic assemblies, particle diffusivities are peaked around a mean value, whereas in plastic assemblies, there is a wide distribution of diffusivities with no clear peak. Both elastic and plastic assemblies show a frequency independent solid modulus from particle tracking microrheology. Here too, there is a much wider distribution of modulus values for plastic scaffolds as compared to elastic, in contrast to bulk rheological measurements where both assemblies exhibit a similar response. We interpret our results in terms of the spatial distribution of crosslinks in the polymer mesh in the colloidal assemblies.

Fluorescence Blinking Beyond Nanoconfinement: Spatially Synchronous Intermittency of Entire Perovskite Microcrystals.

Abrupt fluorescence intermittency or blinking is long recognized to be characteristic of single nano-emitters. Extended quantum-confined nanostructures also undergo spatially heterogeneous blinking; however, there is no such precedent in dimensionally unconfined (bulk) materials. Herein, we report multi-level blinking of entire individual organo-lead bromide perovskite microcrystals (volume=0.1-3 µm3 ) under ambient conditions. Extremely high spatiotemporal correlation (>0.9) in intracrystal emission intensity fluctuations signifies effective communication amongst photogenerated carriers at distal locations (up to ca. 4 µm) within each crystal. Fused polycrystalline grains also exhibit this intriguing phenomenon, which is rationalized by correlated and efficient migration of carriers to a few transient nonradiative traps, the nature and population of which determine blinking propensity. Observation of spatiotemporally correlated emission intermittency in bulk semiconductor crystals opens the possibility of designing novel devices involving long-range (mesoscopic) electronic communication.

Bioactive polymersomes self-assembled from amphiphilic PPO-glycopolypeptides: synthesis, characterization, and dual-dye encapsulation.

Glycopolypeptide-based polymersomes have promising applications as vehicles for targeted drug delivery because they are capable of encapsulating different pharmaceuticals of diverse polarity as well as interacting with specific cell surfaces due to their hollow structural morphology and bioactive surfaces. We have synthesized glycopolypeptide-b-poly(propylene oxide) by ROP of glyco-N-carboxyanhydride (NCA) using the hydrophobic amine-terminated poly(propylene oxide) (PPO) as the initiator. This block copolymer is composed of an FDA-approved PPO hydrophobic block in conjugation with hydrophilic glycopolypeptides which are expected to be biocompatible. We demonstrate the formation of glycopolypeptide-based polymersomes from the self-assembly of glycopolypeptide-b-poly(propylene oxide) in which the presence of an ordered helical glycopolypeptide segment is required for their self-assembly into spherical nanoscale (∼50 nm) polymersomes. The polymersomes were characterized in detail using a variety of techniques such as TEM, AFM, cryo-SEM, and light-scattering measurements. As a model for drugs, both hydrophobic (RBOE) and hydrophilic (calcein) dyes have been incorporated within the polymersomes from solution. To substantiate the simultaneous entrapment of the two dyes, spectrally resolved fluorescence microscopy was performed on the glycopeptide polymersomes cast on a glass substrate. We show that it is possible to visualize individual nanoscale polymersomes and effectively probe the dyes' colocalization and energy-transfer behaviors therein as well as investigate the variation in dual-dye encapsulation over a large number of single polymersomes. Finally, we show that the galactose moieties present on the surface can specifically recognize lectin RCA120, which reveals that the polymersomes' surface is indeed biologically active.

Colloidal CsPbBr3 Perovskite Nanocrystals: Luminescence beyond Traditional Quantum Dots.

Traditional CdSe-based colloidal quantum dots (cQDs) have interesting photoluminescence (PL) properties. Herein we highlight the advantages in both ensemble and single-nanocrystal PL of colloidal CsPbBr3 nanocrystals (NCs) over the traditional cQDs. An ensemble of colloidal CsPbBr3 NCs (11 nm) exhibits ca. 90 % PL quantum yield with narrow (FWHM=86 meV) spectral width. Interestingly, the spectral width of a single-NC and an ensemble are almost identical, ruling out the problem of size-distribution in PL broadening. Eliminating this problem leads to a negligible influence of self-absorption and Förster resonance energy transfer, along with batch-to-batch reproducibility of NCs exhibiting PL peaks within ±1 nm. Also, PL peak positions do not alter with measurement temperature in the range of 25 to 100 °C. Importantly, CsPbBr3 NCs exhibit suppressed PL blinking with ca. 90 % of the individual NCs remain mostly emissive (on-time >85 %), without much influence of excitation power.

Spectrally resolved optical microscopy using a transmission grating spectrograph: importance of spatial selection.

Spectral imaging using transmission grating (TG) based spectrographs has not been effectively utilized for spatially resolved absorption measurements of solid samples, and the role of spatial selection in spectral characteristics remains unexplored. We describe a simple yet efficient method to obtain reliable absorption spectra at sub micrometer spatial resolution for both non-fluorescent and fluorescent samples using a combination of a slit, TG and CCD detector coupled to an optical microscope. In this spectrally resolved optical microscopy (SROM) setup, the adjustable slit located before the TG enabled us to demonstrate that spatial selection greater than ~1 µm is associated with a loss of spectral features, line broadening, and in certain situations, a shift in peak positions. We show that the use of near diffraction-limited slit-width is imperative for determination of reliable absorption profiles when a TG-CCD based spectrograph is used for spatially resolved spectroscopy measurements. The importance of high spatial selection in SROM becomes more apparent for heterogeneous samples with multiple absorbing species present in microscopically phase-separated regions, allowing for the identification of spectral signatures of individual components which cannot be resolved using ensemble measurements. This method can even be used to estimate the relative concentrations of absorbing species within sub-cellular regions, and therefore has the potential to map marker distribution in cellular environments.

Introduction to fundamental processes in optical nanomaterials.

An introduction to the joint Nanoscale and Chemical Communications (ChemComm) themed collection focused on fundamental processes in optical nanomaterials that features a series of articles describing the properties of this versatile class of materials while highlighting some of their potential applications.

Sensitized Emission Imaging Allows Nanoscale Surface Polarity Mapping of α-Synuclein Amyloid Fibrils.

When misfolded, α-Synuclein (α-Syn), a natively disordered protein, aggregates to form amyloid fibrils responsible for the neurodegeneration observed in Parkinson's disease. Structural studies revealed distinct molecular packing of α-Syn in different fibril polymorphs and variations of interprotofilament connections in the fibrillar architecture. Fibril polymorphs have been hypothesized to exhibit diverse surface polarities depending on the folding state of the protein during aggregation; however, the spatial variation of surface polarity in amyloid fibrils remains unexplored. To map the local polarity (or hydrophobicity) along α-Syn fibrils, we visualized the spectral characteristics of two dyes with distinct polarities-hydrophilic Thioflavin T (ThT) and hydrophobic Nile red (NR)─when both are bound to α-Syn fibrils. Dual-channel fluorescence imaging reveals uneven partitioning of ThT and NR along individual fibrils, implying that relatively more polar/hydrophobic patches are spread over a few hundred nanometers. Remarkably, spectrally resolved sensitized emission imaging of α-Syn fibrils provides unambiguous evidence of energy transfer from ThT to NR, implying that dyes of dissimilar polarity are in close proximity. Furthermore, spatially resolved fluorescence spectroscopy of the solvatochromic probe NR allowed us to quantitatively map the range and variation of the polarity parameter ET30 along individual fibrils. Our results suggest the existence of interlaced polar and nonpolar nanoscale domains throughout the fibrils; however, the relative populations of these patches vary considerably over larger length scales likely due to heterogeneous packing of α-Syn during fibrilization and dissimilar exposed polarities of polymorphic segments. The employed method may provide a foundation for imaging modalities of other similar structurally unresolved systems with diverse hydrophobic-hydrophilic topology.

Ultranarrow and widely tunable Mn2+-Induced photoluminescence from single Mn-doped nanocrystals of ZnS-CdS alloys.

Extensively studied Mn-doped semiconductor nanocrystals have invariably exhibited photoluminescence over a narrow energy window of width ≤150 meV in the orange-red region and a surprisingly large spectral width (≥180 meV), contrary to its presumed atomic-like origin. Carrying out emission measurements on individual single nanocrystals and supported by ab initio calculations, we show that Mn PL emission, in fact, can (i) vary over a much wider range (∼370 meV) covering the deep green--deep red region and (ii) exhibit widths substantially lower (∼60-75 meV) than reported so far, opening newer application possibilities and requiring a fundamental shift in our perception of the emission from Mn-doped semiconductor nanocrystals.

Sensing Hg(II) in vitro and in vivo using a benzimidazole substituted BODIPY.

A multisignaling Hg(II) sensor based on a benzimidazole substituted BODIPY framework was designed, which displays excellent selectively toward Hg(II) in vitro and in vivo. Optical and fluorogenic measurements in solution reveal that the sensor can detect mercury ions at submicromolar concentrations, with high specificity. The detection of Hg(II) is associated with a blue-shift in optical spectra and a simultaneous increase in the fluorescence quantum yield of the sensor, which is attributed to a decrease in charge delocalization and inhibition of photoinduced electron transfer upon binding to Hg(II). Using several spectroscopic measurements, it is shown that the binding mechanism involves two sensor molecules, where lone pairs of the benzimidazole nitrogen coordinate to a single mercury ion. The utility of this BODIPY sensor to detect Hg(II) in vivo was demonstrated by fluorescence imaging and spectroscopy of labeled human breast adenocarcinoma cells. While average emission intensity of the sensor over a large number of cells increases with incubated mercury concentrations, spatially resolved fluorescence spectroscopy performed on individual cells reveals clear spectral blue-shifts from a subensemble of sensors, corroborating the detection of Hg(II). Interestingly, the emission spectra at various submicrometer locations within cells exhibited considerable inhomogeneity in the extent of blue-shift, which demonstrates the potential of this sensor to monitor the local (effective) concentration of mercury ions within various subcellular environments.

Spectrally Resolved FRET Microscopy of α-Synuclein Phase-Separated Liquid Droplets.

Liquid-liquid phase separation (LLPS) has emerged as an important phenomenon associated with formation of membraneless organelles. Recently, LLPS has been shown to act as nucleation centers for disease-associated protein aggregation and amyloid fibril formation. Phase-separated α-synuclein droplets gradually rigidify during the course of protein aggregation, and it is very challenging to understand the biomolecular interactions that lead to liquid-like to solid-like transition using conventional ensemble measurements. Here, we describe a spectrally-resolved fluorescence microscopy based Förster resonance energy transfer (FRET) imaging to probe interactions of α-synuclein in individual droplets during LLPS-mediated aggregation. By acquiring entire emission spectral profiles of individual droplets upon sequential excitation of acceptors and donors therein, this technique allows for the quantification of sensitized emission proportional to the extent of FRET, which enables interrogation of the evolution of local interactions of donor-/acceptor-labeled α-synuclein molecules within each droplet. The present study on single droplets is not only an important development for studying LLPS but can also be used to investigate self-assembly or aggregation in biomolecular systems and soft materials.

Deciphering modes of long-range energy transfer in perovskite crystals using confocal excitation and wide-field fluorescence spectral imaging.

Excitation energy migration beyond mesoscale is of contemporary interest for both solar photovoltaic and light-emissive devices, especially in context of organometal halide perovskites (OMHPs) which have been shown to have very long (charge carrier) diffusion lengths. While understanding the energy propagation pathways in OMHPs is crucial for further advancement of material design and improvement of opto-electronic features, the simultaneous existence of multiple processes like carrier diffusion, photon recycling, and photon transport makes it often complex to differentiate them. In this study, we unravel the diverse yet dominant excitation energy transfer mode(s) in crystalline MAPbBr3micron-sized 1D rods and plates by localized (confocal) laser excitation coupled with spectrally-resolved wide-field fluorescence imaging. While rarely used, this technique can efficiently probe excitation migration beyond the diffraction limit and can be realized by simple modification of existing epifluorescence microscopy setups. We find that in rods of length below ∼2 microns, carrier diffusion dominates amongst various energy transfer processes. However, the transient non-radiative defects severely inhibit the extent of carrier migration and also temporarily affect the radiative recombination dynamics of the photo-carriers. For MAPbBr3plates of several tens of micrometers, we find that the photoluminescence (PL) spectral characteristics remain unaltered at short distances (< ∼3µm) while at a larger distance, the spectral profile is gradually red-shifted. This implies that carrier diffusion dominates over small distances, while photon recycling,i.e., repeated re-absorption and re-emission of photons, propagates excitation energy transfer over extended length scales with assistance from wave-guided photon transport. Our findings can potentially be used for future studies on the characterization of energy transport mechanisms in semiconductor solids as well as for organic (molecular) self-assembled microstructures.

Tunable Multiplexed Whole-Cell Biosensors as Environmental Diagnostics for ppb-Level Detection of Aromatic Pollutants.

Aromatics such as phenols, benzene, and toluene are carcinogenic xenobiotics which are known to pollute water resources. By employing synthetic biology approaches combined with a structure-guided design, we created a tunable array of whole-cell biosensors (WCBs). The MopR genetic system that has the natural ability to sense and degrade phenol was adapted to detect phenol down to ∼1 ppb, making this sensor capable of directly detecting phenol in permissible limits in drinking water. Importantly, by using a single WCB design, we engineered mutations into the MopR gene that enabled generation of a battery of sensors for a wide array of pollutants. The engineered WCBs were able to sense inert compounds like benzene and xylene which lack active functional groups, without any loss in sensitivity. Overall, this universal programmable biosensor platform can be used to create WCBs that can be deployed on field for rapid testing and screening of suitable drinking water sources.

Investigation of Structural Heterogeneity in Individual Amyloid Fibrils Using Polarization-Resolved Microscopy.

Amyloid fibrils are structurally heterogeneous protein aggregates that are implicated in a wide range of neurodegenerative and other proteopathic diseases. These fibrils exist in a variety of different tertiary and higher-level structures, and this exhibited polymorphism greatly complicates any structural study of amyloid fibrils. In this work, we demonstrate a method of using polarization-resolved microscopy to directly observe the structural heterogeneity of individual amyloid fibrils using amyloid-bound fluorophores. We formulate a mathematical quantity, helical anisotropy, which utilizes the polarized emission of amyloid-bound fluorophores to report on the local structure of individual fibrils. Using this method, we show how model amyloid fibrils generated from short peptides exhibit diverse structural properties both between different fibrils and within a single fibril, in a manner that is replicated for fibrils assembled from longer proteins. Our method represents an accessible and easily adaptable technique by which polymorphism in the structure of amyloid fibrils can be probed. Additionally, the methodology we describe here can be easily extended to the study of other fibrillar and otherwise ordered supramolecular structures.

Self-Assembly of Nicotinic Acid-Conjugated Selenopeptides into Mesotubes.

The study of controlling the morphology for designing advanced supramolecular architectures by tuning the molecular motif at the elemental level has been rarely carried out. Here, we report the synthesis of a nicotinic acid-conjugated selenopeptide, which induced the formation of an unbranched mesoscale elongated tubular morphology. We rationally designed two additional peptides to find out the decisive role played by the nitrogen atom (in nicotinic acid) and selenium (in the peptide backbone) toward the formation of the mesotube. We found that the peptide, devoid of nitrogen, forms a fibrillar structure, whereas the peptide without selenium self-assembled into a cylindrical filled rodlike morphology. Here, we report an entirely different class of peptide inspired from the selenopeptide chemistry that forms a tubular structure and unambiguously establish that both nicotinic acid and selenium are essential toward the formation of such mesotubes.