Organic J-Aggregate Nanodots with Enhanced Light Absorption and Near-Unity Fluorescence Quantum Yield.

Development of biocompatible fluorophores with small size, bright fluorescence, and narrow spectrum translate directly into major advances in fluorescence imaging and related techniques. Here, we discover that a small donor-acceptor-donor-type organic molecule consisting of a carbazole (Cz) donor and benzothiazole (BT) acceptor (CzBTCz) assembles into quasi-crystalline J-aggregates upon a formation of ultrasmall nanoparticles. The 3.5 nm CzBTCz Jdots show a narrow absorption spectrum (fwhm = 27 nm), near-unity fluorescence quantum yield (ϕfl = 0.95), and enhanced peak molar extinction coefficient. The superior spectroscopic characteristics of the CzBTCz Jdots result in two orders of magnitude brighter photoluminescence of the Jdots compared with semiconductor quantum dots, which enables continuous single-Jdots imaging over a 1 h period. Comparison with structurally similar CzBT nanoparticles demonstrates a critical role played by the shape of CzBTCz on the formation of the Jdots. Our findings open an avenue for the development of a new class of fluorescent nanoparticles based on J-aggregates.

Millimeter-Deep Detection of Single Shortwave-Infrared-Emitting Polymer Dots through Turbid Media.

Fluorescence imaging at longer wavelengths, especially in the shortwave-infrared (SWIR: 1000-1700 nm) region, leads to a substantial decrease in light attenuation, scattering, and background autofluorescence, thereby enabling enhanced penetration into biological tissues. The limited selection of fluorescent probes is a major bottleneck in SWIR fluorescence imaging. Here, we develop SWIR-emitting nanoparticles composed of donor-acceptor-type conjugated polymers. The bright SWIR fluorescence of the polymer dots (primarily attributable to their large absorption cross-section and high fluorescence saturation intensity (as high as 113 kW·cm-2)) enables the unprecedented detection of single particles as small as 14 nm through millimeter-thick turbid media. Unlike most SWIR-emitting nanomaterials, which have an excited-state lifetime in the range of microseconds to milliseconds, our polymer dots exhibit a subnanosecond excited-state lifetime. These characteristics enable us to demonstrate new time-gated single-particle imaging with a high signal-to-background ratio. These findings expand the range of potential applications of single-particle deep-tissue imaging.

Allosteric inhibition of individual enzyme molecules trapped in lipid vesicles.

Enzymatic inhibition by product molecules is an important and widespread phenomenon. We describe an approach to study product inhibition at the single-molecule level. Individual HRP molecules are trapped within surface-tethered lipid vesicles, and their reaction with a fluorogenic substrate is probed. While the substrate readily penetrates into the vesicles, the charged product (resorufin) gets trapped and accumulates inside the vesicles. Surprisingly, individual enzyme molecules are found to stall when a few tens of product molecules accumulate. Bulk enzymology experiments verify that the enzyme is noncompetitively inhibited by resorufin. The initial reaction velocity of individual enzyme molecules and the number of product molecules required for their complete inhibition are broadly distributed and dynamically disordered. The two seemingly unrelated parameters, however, are found to be substantially correlated with each other in each enzyme molecule and over long times. These results suggest that, as a way to counter disorder, enzymes have evolved the means to correlate fluctuations at structurally distinct functional sites.

Shielding Effects Provide a Dominant Mechanism in J-Aggregation-Induced Photoluminescence Enhancement of Carbon Nanotubes.

The unique photophysical properties of single-walled carbon nanotubes (SWCNTs) exhibit great potential for bioimaging applications. This led to extensive exploration of photosensitization methods to improve their faint shortwave infrared (SWIR) photoluminescence. Here, we report the mechanisms of SWCNT-assisted J-aggregation of cyanine dyes and the associated photoluminescence enhancement of SWCNTs in the SWIR spectral region. Surprisingly, we found that excitation energy transfer between the cyanine dyes and SWCNTs makes a negligible contribution to the overall photoluminescence enhancement. Instead, the shielding of SWCNTs from the surrounding water molecules through hydrogen bond-assisted macromolecular reorganization of ionic surfactants triggered by counterions and the physisorption of the dye molecules on the side walls of SWCNTs play a primary role in the photoluminescence enhancement of SWCNTs. We observed 2 orders of magnitude photoluminescence enhancement of SWCNTs by optimizing these factors. Our findings suggest that the proper shielding of SWCNTs is the critical factor for their photoluminescence enhancement, which has important implications for their application as imaging agents in biological settings.

Detection and quantification through a lipid membrane using the molecularly controlled semiconductor resistor.

The detection of covalent and noncovalent binding events between molecules and biomembranes is a fundamental goal of contemporary biochemistry and analytical chemistry. Currently, such studies are performed routinely using fluorescence methods, surface-plasmon resonance spectroscopy, and electrochemical methods. However, there is still a need for novel sensitive miniaturizable detection methods where the sample does not have to be transferred to the sensor, but the sensor can be brought into contact with the sample studied. We present a novel approach for detection and quantification of processes occurring on the surface of a lipid bilayer membrane, by monitoring the current change through the n-type GaAs-based molecularly controlled semiconductor resistor (MOCSER), on which the membrane is adsorbed. Since GaAs is susceptible to etching in an aqueous environment, a protective thin film of methoxysilane was deposited on the device. The system was found to be sensitive enough to allow monitoring changes in pH and in the concentration of amino acids in aqueous solution on top of the membrane. When biotinylated lipids were incorporated into the membrane, it was possible to monitor the binding of streptavidin or avidin. The device modified with biotin-streptavidin complex was capable of detecting the binding of streptavidin antibodies to immobilized streptavidin with high sensitivity and selectivity. The response depends on the charge on the analyte. These results open the way to facile electrical detection of protein-membrane interactions.

The Pursuit of Shortwave Infrared-Emitting Nanoparticles with Bright Fluorescence through Molecular Design and Excited-State Engineering of Molecular Aggregates.

Shortwave infrared (SWIR) fluorescence detection gradually becomes a pivotal real-time imaging modality, allowing one to elucidate biological complexity in deep tissues with subcellular resolution. The key challenge for the further growth of this imaging modality is the design of new brighter biocompatible fluorescent probes. This review summarizes the recent progress in the development of organic-based nanomaterials with an emphasis on new strategies that extend the fluorescence wavelength from the near-infrared to the SWIR spectral range and amplify the fluorescence brightness. We first introduce the most representative molecular design strategies to obtain near-infrared-SWIR wavelength fluorescence emission from small organic molecules. We then discuss how the formation of nanoparticles based on small organic molecules contributes to the improvement of fluorescence brightness and the shift of fluorescence to SWIR, with a special emphasis on the excited-state engineering of molecular probes in an aggregate state and spatial packing of the molecules in nanoparticles. We build our discussion based on a historical perspective on the photophysics of molecular aggregates. We extend this discussion to nanoparticles made of conjugated polymers and discuss how fluorescence characteristics could be improved by molecular design and chain conformation of the polymer molecules in nanoparticles. We conclude the article with future directions necessary to expand this imaging modality to wider bioimaging applications including single-particle deep tissue imaging. Issues related to the characterization of SWIR fluorophores, including fluorescence quantum yield unification, are also mentioned.

Photostable polymorphic organic cages for targeted live cell imaging.

Fluorescent microscopy is a powerful tool for studying the cellular dynamics of biological systems. Small-molecule organic fluorophores are the most commonly used for live cell imaging; however, they often suffer from low solubility, limited photostability and variable targetability. Herein, we demonstrate that a tautomeric organic cage, OC1, has high cell permeability, photostability and selectivity towards the mitochondria. We further performed a structure-activity study to investigate the role of the keto-enol tautomerization, which affords strong and consistent fluorescence in dilute solutions through supramolecular self-assembly. Significantly, OC1 can passively diffuse through the cell membrane directly targeting the mitochondria without going through the endosomes or the lysosomes. We envisage that designing highly stable and biocompatible self-assembled fluorophores that can passively diffuse through the cell membrane while selectively targeting specific organelles will push the boundaries of fluorescent microscopy to visualize intricate cellular processes at the single molecule level in live samples.

Geometry-Based Self-Assembly of Histone-DNA Nanostructures at Single-Nucleotide Resolution.

Histones are basic protein monomers capable of interacting with DNA, providing the mechanism of DNA compaction inside the cell nucleus. The well-ordered assembly process of histone and DNA is a potential candidate as the approach for building DNA-protein nanostructures. Here, utilizing the sequence-independent histone-DNA interaction, we present an approach to self-assemble histones and single-stranded DNA (ssDNA) to form well-defined histone-DNA (sHD) nanoparticles and their multidimensional cross-linked complexes (cHD). By using various molecular biology and microscopy techniques, we elucidate the structure of these complexes, and we show that they are formed at carefully controlled conditions of temperature, ionic strength, concentration, and incubation time. We also demonstrate using a set of ssDNA molecular rulers and a geometric accommodation model that the assembly of sHD and cHD particles proceeds with precise geometry so that the number of ssDNA in these particles can be programmed by the length of ssDNA. We further show that the formation of cHD amplifies the effect of the length of ssDNA on the self-assembly, allowing for distinguishing ssDNA of different lengths at single nucleotide resolution. We envision that our geometry-directed approach of self-assembling histone-DNA nanostructures and the fundamental insights can serve as a structural platform to advance building precisely ordered DNA-protein nanostructures.

Controlling photophysical properties of ultrasmall conjugated polymer nanoparticles through polymer chain packing.

Applications of conjugated polymer nanoparticles (Pdots) for imaging and sensing depend on their size, fluorescence brightness and intraparticle energy transfer. The molecular design of conjugated polymers (CPs) has been the main focus of the development of Pdots. Here we demonstrate that proper control of the physical interactions between the chains is as critical as the molecular design. The unique design of twisted CPs and fine-tuning of the reprecipitation conditions allow us to fabricate ultrasmall (3.0-4.5 nm) Pdots with excellent photostability. Extensive photophysical and structural characterization reveals the essential role played by the packing of the polymer chains in the particles in the intraparticle spatial alignment of the emitting sites, which regulate the fluorescence brightness and the intraparticle energy migration efficiency. Our findings enhance understanding of the relationship between chain interactions and the photophysical properties of CP nanomaterials, providing a framework for designing and fabricating functional Pdots for imaging applications.

Non-typical fluorescence studies of excited and ground state proton and hydrogen transfer.

Fluorescence studies of tautomerization have been carried out for various systems that exhibit single and double proton or hydrogen translocation in various environments, such as liquid and solid condensed phases, ultracold supersonic jets, and finally, polymer matrices with single emitters. We focus on less explored areas of application of fluorescence for tautomerization studies, using porphycene, a porphyrin isomer, as an example. Fluorescence anisotropy techniques allow investigations of self-exchange reactions, where the reactant and product are formally identical. Excitation with polarized light makes it possible to monitor tautomerization in single molecules and to detect their three-dimensional orientation. Analysis of fluorescence from single vibronic levels of jet-isolated porphycene not only demonstrates coherent tunneling of two internal protons, but also indicates that the process is vibrational mode-specific. Next, we present bifunctional proton donor-acceptor systems, molecules that are able, depending on the environment, to undergo excited state single intramolecular or double intermolecular proton transfer. For molecules that have donor and acceptor groups located in separate moieties linked by a single bond, excited state tautomerization can be coupled to mutual twisting of the two subunits.

Single-molecule FRET unveils induced-fit mechanism for substrate selectivity in flap endonuclease 1.

Human flap endonuclease 1 (FEN1) and related structure-specific 5'nucleases precisely identify and incise aberrant DNA structures during replication, repair and recombination to avoid genomic instability. Yet, it is unclear how the 5'nuclease mechanisms of DNA distortion and protein ordering robustly mediate efficient and accurate substrate recognition and catalytic selectivity. Here, single-molecule sub-millisecond and millisecond analyses of FEN1 reveal a protein-DNA induced-fit mechanism that efficiently verifies substrate and suppresses off-target cleavage. FEN1 sculpts DNA with diffusion-limited kinetics to test DNA substrate. This DNA distortion mutually 'locks' protein and DNA conformation and enables substrate verification with extreme precision. Strikingly, FEN1 never misses cleavage of its cognate substrate while blocking probable formation of catalytically competent interactions with noncognate substrates and fostering their pre-incision dissociation. These findings establish FEN1 has practically perfect precision and that separate control of induced-fit substrate recognition sets up the catalytic selectivity of the nuclease active site for genome stability.

In Search for the Best Environment for Single Molecule Studies: Photostability of Single Terrylenediimide Molecules in Various Polymer Matrices.

Photobleaching is the main limiting factor in single molecule studies by optical techniques. We investigated the dependence of photostability of terrylene diimide (TDI) derivative on its environment using confocal fluorescence microscopy. Seven different polymers were tested. Depending on the matrix, photobleaching quantum yields vary by 2 orders of magnitude. Their values correlate with parameters characterizing oxygen mobility in polymers: diffusion coefficient and permeability. Poly(vinyl chloride) (PVC) and poly(methyl methacrylate) (PMMA) exhibit the lowest photodestruction quantum yields. Additional enhancement of photostability can be achieved by aging of PVC or by flushing the sample with nitrogen, which confirms the involvement of oxygen in photodestruction. Different character of the time traces of the intensity of emission from single TDI molecules is observed for different polymer matrices, ranging from intense blinking in the least stable polycarbonate, to practically no blinking in the most stable PVC. These results suggest a photodegradation mechanism involving self-sensitized photooxidation in oxygen complexes of TDI.

Enhancing fluorescence by using pluronic block copolymers as carriers of monomeric porphycenes.

Porphycenes, structural isomers of porphyrins, usually exhibit strong fluorescence in organic solvents. However, in water they are practically insoluble or form only weakly emitting aggregates. We show that embedding porphycenes inside pluronic micelles in water solutions leads to the recovery of strong monomeric fluorescence, of which the decay times and quantum yields are similar to those observed for homogeneous solvents. One of the investigated porphycenes serves as a fluorescence sensor of the microenvironment viscosity, revealing that the viscosity inside pluronic micelles is quite high. Using confocal fluorescence microscopy, we obtained images of single pluronic micelles containing monomeric porphycene chromophores.

Imaging of tautomerism in a single molecule.

Fluorescence imaging is used to visualize directly the transfer of two inner hydrogen atoms in single porphycene molecules. This reaction leads to a chemically equivalent but differently oriented structure and hence results in a rotation of the transition dipole moments. By probing single immobilized molecules with an azimuthally polarized laser beam in the focal spot of a confocal microscope we observe ring-like emission patterns, possible only for a chromophore with two nearly orthogonal transition dipole moments. Numerical simulations of the observed emission patterns yield a value of 72 degrees for the angle between the S0-S1 transition moments in the two tautomeric forms.