Fluorescent Dye Molecules Research Articles

Translational Diffusion of a Fluorescent Tracer Molecule in Nanoconfined Water.

Diffusion of tracer dye molecules in water confined to the nanoscale is an important subject with a direct bearing on many technological applications. It is not yet clear, however, if the dynamics of water in hydrophilic as well as hydrophobic nanochannels remains bulk-like. Here, we present diffusion measurement of a fluorescent dye molecule in water confined to the nanoscale between two hydrophilic surfaces whose separation can be controlled with a precision of less than a nm. We observe that the fluorescence intensities correlate over fast (∼30 μs) and slow (∼1000 μs) time components. The slow time scale is due to adsorption of fluorophores to the confining walls, and it disappears in the presence of 1 M salt. The fast component is attributed to diffusion of dye molecules in the gap. It is found to be bulk-like for sub-10 nm separations and indicates that the viscosity of water under confinement remains unaltered up to a confinement gap as small as ∼5 nm. Our findings contradict some of the recent measurements of diffusion under nanoconfinement; however, they are consistent with many estimates of self-diffusion using molecular dynamics simulations and measurements using neutron scattering experiments.

Copper‐Catalyzed Covalent Dimerization of Near‐Infrared Fluorescent Cyanine Dyes: Synergistic Enhancement of Photoacoustic Signals for Molecular Imaging of Tumors

AbstractInvited for this month's cover is the group of Prof. Samuel Achilefu at the Washington University School of Medicine and colleagues at the Pohang University of Science and Technology. The cover picture shows copper (the hammer) catalyzing the assembly of monomeric near‐infrared fluorescent dye molecules, cypate (yellow glow), into covalently‐linked dimeric molecules (merged red balls). More information can be found in the Communication at 10.1002/anse.202100045.

Front Cover: Copper‐Catalyzed Covalent Dimerization of Near‐Infrared Fluorescent Cyanine Dyes: Synergistic Enhancement of Photoacoustic Signals for Molecular Imaging of Tumors (Anal. Sens. 1/2022)

The cover picture shows copper (hammer)-catalyzes the assembly of monomeric near-infrared fluorescent dye molecules, cypate (yellow glow), into covalently-linked dimeric molecules (merged red balls). Dimerization induces fluorescence quenching due to enhanced photothermal dissipation (red hot flashes), which generates acoustic waves. Decorating the dimers with a tumor-targeting agent, LS301, facilitates selective tumor uptake of the probes, which are detectable in vivo by photoacoustic imaging. More information can be found in the Communication by Samuel Achilefu, Chulhong Kim and co-workers.

Investigating nucleation and growth phenomena in microfluidic supercritical antisolvent process by coupling in situ fluorescence spectroscopy and direct numerical simulation

High-pressure microfluidic systems exhibit favorable capacity to enhance mixing quality compared to conventional macroscale liquid systems. These mixing conditions are very interesting for the preparation of fluorescent organic nanoparticles by supercritical antisolvent process. In this work, fundamental mechanisms of microfluidic supercritical antisolvent process (µSAS) including thermodynamics, hydrodynamics, nucleation/growth phenomena, are investigated using a coupled experimental/simulation approach. Specifically, we determined experimentally the particle precipitation field in a CO2 / solvent medium using a fluorescent organic dye molecule which presents an enhancement of fluorescence intensity in its aggregated state (AIE effect). The results of the direct numerical simulation considering all the physical phenomena are compared with the experimental data for validation and deep understanding of the mechanisms. It is shown that despite ultra-short mixing time, the supersaturation field showed some fluctuation leading to variation of the nucleation times.

Anthryl-cinnamonitrile-based supramolecular artificial light-harvesting systems with high efficiency fabricated in aqueous solution

Two highly efficient artificial light-harvesting systems (ALHSs) in water have been successfully fabricated through the supramolecular assembly of an anthryl-cinnamonitrile derivative (ABTA), a water-soluble pillar[5]arene (WP5), and two conventional fluorescent dye molecules (Nile Red (NiR) and sulforhodamine (SR101)). The fabricated ALHSs display efficient energy transfer efficiency (85.7% for WP5⊃ABTA-NiR and 83.2% for WP5⊃ABTA-SR101) and possess ultrahigh donor-acceptor molar ratio ([ABTA]/[NiR] = 250:1 and [ABTA]/[SR101] = 250:1). Significantly, the formed WP5⊃ABTA nanoparticles enhance the aggregation-induced emission ability of ABTA and function as a brilliant donor to transfer the harvested energy to acceptors NiR and SR101 in aqueous solution with high antenna effects (21.8 for WP5⊃ABTA-NiR and 26.1 for WP5⊃ABTA-SR101).

Storing and Reading Information in Mixtures of Fluorescent Molecules.

The rapidly increasing use of digital technologies requires the rethinking of methods to store data. This work shows that digital data can be stored in mixtures of fluorescent dye molecules, which are deposited on a surface by inkjet printing, where an amide bond tethers the dye molecules to the surface. A microscope equipped with a multichannel fluorescence detector distinguishes individual dyes in the mixture. The presence or absence of these molecules in the mixture encodes binary information (i.e., “0” or “1”). The use of mixtures of molecules, instead of sequence-defined macromolecules, minimizes the time and difficulty of synthesis and eliminates the requirement of sequencing. We have written, stored, and read a total of approximately 400 kilobits (both text and images) with greater than 99% recovery of information, written at an average rate of 128 bits/s (16 bytes/s) and read at a rate of 469 bits/s (58.6 bytes/s).

Design of Over-1000 nm Near-Infrared Fluorescent Polymeric Micellar Nanoparticles by Matching the Solubility Parameter of the Core Polymer and Dye.

Polymeric micellar nanoparticles (PNPs) encapsulating over-thousand-nanometer (OTN) near-infrared (NIR) fluorescent dye molecules in block polymers having hydrophobic and hydrophilic chains are promising agents for the dynamic imaging of deep tissue. To achieve OTN-NIR fluorescent PNPs (OTN-PNPs) having high brightness, it is crucial to increase the affinity between the core polymer and dye molecules by matching their polarities; thus, criteria and methods to evaluate the affinity are required. In this study, we used the Hansen solubility parameter (HSP), including the polarity term, to evaluate the affinity between the two substances. HSP values of the OTN-NIR fluorescent dye IR-1061 and four core polymers, poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), poly(ε-caprolactone) (PCL), and polystyrene (PSt), were calculated using the Hansen solubility sphere method and molecular group contribution method, respectively. The relative energy density between IR-1061 and each core polymer calculated using their HSP values revealed that the affinities of PLGA and PLA for IR-1061 are higher than those of PCL and PSt. Therefore, OTN-PNPs composed of PLGA, PLA, and PCL core polymers were prepared and compared. The OTN-PNPs having PLGA and PLA cores could be loaded with larger amounts of IR-1061, had higher photoluminescence intensities, and showed higher stability in phosphate buffered saline than those having PCL cores. Moreover, the OTN-PNPs having PLGA or PLA cores were used for the dynamic imaging of live mice. Thus, matching the solubility parameters of the core polymer and dye molecule is a useful approach for designing high-performance OTN-NIR fluorescent probes.

Micromotor-based localized electroporation and gene transfection of mammalian cells

Herein, we studied localized electroporation and gene transfection of mammalian cells using a metallodielectric hybrid micromotor that is magnetically and electrically powered. Much like nanochannel-based, local electroporation of single cells, the presented micromotor was expected to increase reversible electroporation yield, relative to standard electroporation, as only a small portion of the cell's membrane (in contact with the micromotor) is affected. In contrast to methods in which the entire membrane of all cells within the sample are electroporated, the presented micromotor can perform, via magnetic steering, localized, spatially precise electroporation of the target cells that it traps and transports. In order to minimize nonselective electrical lysis of all cells within the chamber, resulting from extended exposure to an electrical field, magnetic propulsion was used to approach the immediate vicinity of the targeted cell, after which short-duration, electric-driven propulsion was activated to enable contact with the cell, followed by electroporation. In addition to local injection of fluorescent dye molecules, we demonstrated that the micromotor can enhance the introduction of plasmids into the suspension cells because of the dielectrophoretic accumulation of the plasmids in between the Janus particle and the attached cell prior to the electroporation step. Here, we chose a different strategy involving the simultaneous operation of many micromotors that are self-propelling, without external steering, and pair with cells in an autonomic manner. The locally electroporated suspension cells that are considered to be very difficult to transfect were shown to express the transfected gene, which is of significant importance for molecular biology research.

A metasurface performance enhancer

Metasurfaces The detection and emission of light plays a key role in applications such as free-space communications, biosensing, and optical imaging. Fluorescent dye molecules or nanoparticles in a cavity are typically used for photodetection but can be limited in sensitivity and efficiency. Traverso et al. fabricated a low-loss plasmonic metasurface made from silver nanocubes scattered across the surface of the cavity. Modification of the local optical density of states results in a device that can collect fast-modulated light (>14 gigahertz) with a high acceptance angle (120°) and convert it to a directional source with enhanced overall efficiency (>30%). The approach could be incorporated into the design of other ultrafast optoelectronic components to enhance performance. Optica 8 , 202 (2021).

Aggregation-related quenching of LHCII fluorescence in liposomes revealed by single-molecule spectroscopy

Incorporation of membrane proteins into reconstituted lipid membranes is a common approach for studying their structure and function relationship in a native-like environment. In this work, we investigated fluorescence properties of liposome-reconstituted major light-harvesting complexes of plants (LHCII). By utilizing liposome labelling with the fluorescent dye molecules and single-molecule microscopy techniques, we were able to study truly liposome-reconstituted LHCII and compare them with bulk measurements and liposome-free LHCII aggregates bound to the surface. Our results showed that fluorescence lifetime obtained in bulk and in single liposome measurements were correlated. The fluorescence lifetimes of LHCII were shorter for liposome-free LHCII than for reconstituted LHCII. In the case of liposome-reconstituted LHCII, fluorescence lifetime showed dependence on the protein density reminiscent to concentration quenching. The dependence of fluorescence lifetime of LHCII on the liposome size was not significant. Our results demonstrated that fluorescence quenching can be induced by LHCII – LHCII interactions in reconstituted membranes, most likely occurring via the same mechanism as photoprotective non-photochemical quenching in vivo.

Spectral analysis of organic LED emitters' orientation in thin layers by resonant emission on dielectric stacks.

Purposely tailored thin film stacks sustaining surface waves have been utilized to create a unique link between emission angle and wavelength of fluorescent dye molecules. The knowledge of the thin film stack's properties allows us to derive the intrinsically emitted luminescence spectrum as well as to gain information about the orientation of fluorophores from angularly resolved experiments. This corresponds to replacing all the equipment necessary for polarized spectroscopy with a single smart thin film stack, potentially enabling single shot analyses in the future. The experimental results agree well with those from other established techniques, when analyzing the Rubrene derivative in a 2,4,6-tris(biphenyl-3-yl)-1,3,5-triazine (T2T) host used for the fabrication of optimized organic light-emitting diodes. The findings illustrate how resonant layered stacks can be applied to integrated spectroscopic analyses.

Controllable molecular doping in organic single crystals toward high-efficiency light-emitting devices

Organic single-crystalline semiconductors have drawn significant attention in the area of organic electronic and optoelectronic devices due to their superiorities of highly ordered structure, high carrier mobility and low impurity content. Molecular doping technique has made great progress in improving device performance via optimizing the optical and electrical properties of organic semiconductors. In particular, this technique has been attempted by taking fluorescent dye-molecules as the emissive dopants to tune emission color and improve device performance of organic single crystals. Up to now, there are few reports about the use of molecular doping in organic single crystals to optimize their intrinsic electrical properties. Here, we have introduced the controllable molecular doping as a feasible approach toward manipulating charge carrier transport properties of organic single crystals. Upon optimization of doping concentration, balanced carrier transport can be realized in 5,5′-bis(4-trifluoromethyl phenyl) [2,2’] bithiophene (P2TCF3)-doped 1,4-bis(4-methylstyryl) benzene (BSB–Me) crystals. Organic light-emitting devices (OLEDs) based on these doped crystals achieve a maximum luminance of 423 cd/m2 and current efficiency of 0.48 cd/A. It demonstrates that high-efficiency crystal-based OLEDs are of great significance for the development of organic electronics, especially for display and lighting applications.

Protein-, polymer-, and silica-based luminescent nanomaterial probes for super resolution microscopy: a review.

Super resolution microscopy was developed to overcome the Abbe diffraction limit, which effects conventional optical microscopy, in order to study the smaller components of biological systems. In recent years nanomaterials have been explored as luminescent probes for super resolution microscopy, as many have advantages over traditional fluorescent dye molecules. This review will summarize several different types of nanomaterial probes, covering quantum dots, carbon dots, and dye doped nanoparticles. For the purposes of this review the term “nanoparticle” will be limited to polymer-based, protein-based, and silica-based nanoparticles, including core–shell structured nanoparticles. Luminescent nanomaterials have shown promise as super-resolution probes, and continued research in this area will yield new advances in both materials science and biochemical microscopy at the nanometer scale.

Single-molecule tracking measurement of PDMS layer during curing process

The curing process of poly(dimethylsiloxane) (PDMS) was microscopically investigated by the single-molecule tracking method based on the diffusion motion of fluorescent dye molecules adding to the PDMS layer stored at a temperature of 308K. The PDMS layer was completely cured at 900 min after adding a curing agent. We compared the time- and ensemble-averaged mean square displacements (MSDs) of the tracked molecules at 90, 510, and 900 min after adding the curing agent into the PDMS layer. The discrepancies were observed between the time- and ensemble-averaged MSDs, indicating weak ergodicity breaking. The spatially averaged diffusion coefficient exhibited a two-step decrease: first step was rapid decrease suggesting the extent of crosslinking, and second step was slow one suggesting the increase of crosslink density. The single-molecule trajectory scale analysis revealed the heterogeneous distribution of the diffusion coefficient. By calculating the heat map from the slope of moment scaling spectrum (MSS) of each single-molecule trajectory, cluster structures were recognized. The spatial correlation of the slope of MSS decreased with the time elapsed. These results suggested the existence of the heterogeneous structure in the PDMS layer during the curing process.

Fluorescent Dye Labeling Changes the Biodistribution of Tumor-Targeted Nanoparticles.

Fluorescent dye labeling is a common strategy to analyze the fate of administered nanoparticles in living organisms. However, to which extent the labeling processes can alter the original nanoparticle biodistribution has been so far neglected. In this work, two widely used fluorescent dye molecules, namely, ATTO488 (ATTO) and Sulfo-Cy5 (S-Cy5), have been covalently attached to a well-characterized CXCR4-targeted self-assembling protein nanoparticle (known as T22-GFP-H6). The biodistribution of labeled T22-GFP-H6-ATTO and T22-GFP-H6-S-Cy5 nanoparticles has been then compared to that of the non-labeled nanoparticle in different CXCR4+ tumor mouse models. We observed that while parental T22-GFP-H6 nanoparticles accumulated mostly and specifically in CXCR4+ tumor cells, labeled T22-GFP-H6-ATTO and T22-GFP-H6-S-Cy5 nanoparticles showed a dramatic change in the biodistribution pattern, accumulating in non-target organs such as liver or kidney while reducing tumor targeting capacity. Therefore, the use of such labeling molecules should be avoided in target and non-target tissue uptake studies during the design and development of targeted nanoscale drug delivery systems, since their effect over the fate of the nanomaterial can lead to considerable miss-interpretations of the actual nanoparticle biodistribution.

Enhancement of Luminescence Dissymmetry Factor in Nano-Segregated Phase Generated by Phase Separation between Helical Nanofilaments and Liquid-Crystalline Smectic A Phase

Although several methods exist for the synthesis of circularly polarized luminescent (CPL) materials, the methods are extremely complex and tedious. In recent years, the chiral host-achiral luminescent guest method and the achiral host-achiral luminescent guest method have been employed to fabricate CPL materials; however, the main disadvantage of the latter is the small luminescence dissymmetry factor (glum) that limits the practical applications of the method. Therefore, this study reports on the enhancement of glum in a nano-segregated phase system, generated by the phase separation between helical nanofilaments (HNFs; originating from an achiral bent-core molecule) and a liquid-crystalline (LC) smectic A (SmA) phase (originating from an achiral rod-like mesogen). The observed glum value in the nano-segregated phase between the HNFs and LC SmA phase was larger than that in the nano-segregated phase between the HNFs and LC nematic (N) phase. The enhancement of the glum value was attributed to the order parameter (S) of the dye molecules in the SmA phase being larger than that in the N phase. Therefore, we concluded that the S value of the fluorescent dye molecules, doped into the embedded LC phase between the HNFs, strongly influenced the glum value.

Plasmon-modulated photoluminescence enhancement in hybrid plasmonic nano-antennas

In this work, we performed a systematic study on a hybrid plasmonic system to elucidate a new insight into the mechanisms governing the fluorescent enhancement process. Our lithographically defined plasmonic nanodisks with various diameters act as receiver and transmitter nano-antennas to outcouple efficiently the photoluminescence of the coupled dye molecules. We show that the enhancement of the spontaneous emission rate arises from the superposition of three principal phenomena: (i) metal enhanced fluorescence, (ii) metal enhanced excitation and (iii) plasmon-modulated photoluminescence of the photoexcited nanostructures. Overall, the observed enhanced emission is attributed to the bi-directional near-field coupling of the fluorescent dye molecules to the localized plasmonic field of nano-antennas. We identify the role of exciton–plasmon coupling in the recombination rate of the sp-band electrons with d-band holes, resulting in the generation of particle plasmons. According to our comprehensive experimental analyses, the mismatch between the enhanced emission and the emission spectrum of the uncoupled dye molecules is attributed to the plasmon-modulated photoluminescence of the photoexcited hybrid plasmonic system.

A dual-emission Acf@bioMOF-1 platform as fluorescence sensor for highly efficient detection of inorganic ions

An anionic bio-MOF-1 with 1D square channels is applied as container for the adsorption of organic dyes owing to the porous structure and charge properties. Through ion exchange, cationic acriflavine (Acf) dye with the minimum size is selected as a fluorescent dye molecule to introduce into the pores of anionic bio-MOF-1 to obtain a self-calibrating dual-emitting fluorescent composite of Acf@bio-MOF-1, which exhibits two characteristic emission peaks at 385 and 582 ​nm. The composite acts as fluorescence sensing probe for the selective and sensitive detection of Fe3+, Al3+ and Cr2O72− among metal cations and anions and has a “turn-off” response to Fe3+ and Cr2O72− as well as a “turn-on” response to Al3+ at the same time. Particularly, Acf@bio-MOF-1 shows higher fluorescence quenching effects of 95% for Cr2O72− and 90% for Fe3+ and low detection limits of 0.25 ​μM for Cr2O72− and 1.33 ​μM for Fe3+, respectively. Such a special encapsulation strategy to form a dual-emission platform can be adopted to the detection of inorganic ions sensitively.

Observation of Fano line shape in directional fluorescence emission mediated by coupled planar waveguide modes and interpretation based on Lorentz reciprocity

Using a reverse attenuated-total-reflection geometry, we measured angle-scan fluorescence emission spectra of all-dielectric multilayer samples containing a waveguide layer doped with fluorescent dye molecules (fluorescent waveguide layer). A sample containing only one fluorescent waveguide layer showed a highly directional emission spectrum with a Lorentzian line shape caused by the radiative decay of an excited planar waveguide mode into a traveling wave in a decoupling prism. Addition of another waveguide layer containing absorptive dye molecules was found to greatly modify the spectrum and generate a Fano line shape in the emission spectrum. The observed Lorentzian and Fano emission spectra could be well reproduced by electromagnetic calculations based on the Lorentz reciprocity theorem. Calculated results of electric field distributions indicate that the Fano line shape is generated by the suppression of local electric fields inside the fluorescent waveguide layer resulting from coupling between two waveguide modes.

Amyloid-β oligomerization monitored by single-molecule stepwise photobleaching.

A major hallmark of Alzheimer's disease is the misfolding and aggregation of the amyloid- β peptide (Aβ). While early research pointed towards large fibrillar- and plaque-like aggregates as being the most toxic species, recent evidence now implicates small soluble Aβ oligomers as being orders of magnitude more harmful. Techniques capable of characterizing oligomer stoichiometry and assembly are thus critical for a deeper understanding of the earliest stages of neurodegeneration and for rationally testing next-generation oligomer inhibitors. While the fluorescence response of extrinsic fluorescent probes such as Thioflavin-T have become workhorse tools for characterizing large Aβ aggregates in solution, it is widely accepted that these methods suffer from many important drawbacks, including an insensitivity to oligomeric species. Here, we integrate several biophysics techniques to gain new insight into oligomer formation at the single-molecule level. We showcase single-molecule stepwise photobleaching of fluorescent dye molecules as a powerful method to bypass many of the traditional limitations, and provide a step-by-step guide to implementing the technique in vitro. By collecting fluorescence emission from single Aβ(1-42) peptides labelled at the N-terminal position with HiLyte Fluor 555 via wide-field total internal reflection fluorescence (TIRF) imaging, we demonstrate how to characterize the number of peptides per single immobile oligomer and reveal heterogeneity within sample populations. Importantly, fluorescence emerging from Aβ oligomers cannot be easily investigated using diffraction-limited optical microscopy tools. To assay oligomer activity, we also demonstrate the implementation of another biophysical method involving the ratiometric imaging of Fura-2-AM loaded cells which quantifies the rate of oligomer-induced dysregulation of intracellular Ca2+ homeostasis. We anticipate that the integrated single-molecule biophysics approaches highlighted here will develop further and in principle may be extended to the investigation of other protein aggregation systems under controlled experimental conditions.