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The high volatility, water‐immiscibility, and light/oxygen‐sensitivity of most aroma compounds represent a challenge to their incorporation in liquid consumer products. Current encapsulation methods entail the use of petroleum‐based materials, initiators, and crosslinkers as well as mixing, heating, and purification steps. Hence, more efficient and eco‐friendly approaches to encapsulation must be sought. Herein, we propose a simple method by making use of a pre‐formed amphiphilic polymer and employing the Hansen Solubility Parameters approach to determine which fragrances could be encapsulated by spontaneous coacervation in water. The coacervates do not precipitate as solids but they remain suspended as colloidally stable liquid microcapsules, as demonstrated by fluorescence correlation spectroscopy. The effective encapsulation of fragrance is proven through confocal Raman spectroscopy, while the structure of the capsules is investigated by means of cryo FIB/SEM, confocal laser scanning microscopy, and small‐angle X‐ray scattering., The use of a PEG‐g‐PVAc amphiphilic graft copolymer together with hydrophobic fragrances in aqueous medium leads to the spontaneous formation of colloidally stable suspensions of liquid coacervate droplets. Arrows indicate the local turbulences sustaining the spontaneous mixing of the components. The bottom panel shows the confocal Raman microspectroscopy mapping of a multicompartment coacervate droplet with carvone as fragrance.

Polymersomes are vesicular structures self-assembled from amphiphilic block copolymers and are considered an alternative to liposomes for applications in drug delivery, immunotherapy, biosensing, and as nanoreactors and artificial organelles. However, the limited availability of systematic stability, protein fouling (protein corona formation), and blood circulation studies hampers their clinical translation. Poly(2-oxazoline)s (POx) are valuable antifouling hydrophilic polymers that can replace the current gold-standard, poly(ethylene glycol) (PEG), yet investigations of POx functionality on nanoparticles are relatively sparse. Herein, a systematic study is reported of the structural, dynamic and antifouling properties of polymersomes made of poly(2-methyl-2-oxazoline)-block-poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline) (PMOXA-b-PDMS-b-PMOXA). The study relates in vitro antifouling performance of the polymersomes to atomistic molecular dynamics simulations of polymersome membrane hydration behavior. These observations support the experimentally demonstrated benefit of maximizing the length of PMOXA (degree of polymerization (DP) > 6) while keeping PDMS at a minimal length that still provides sufficient membrane stability (DP > 19). In vitro macrophage association and in vivo blood circulation evaluation of polymersomes in zebrafish embryos corroborate these findings. They further suggest that single copolymer presentation on polymersomes is outperformed by blends of varied copolymer lengths. This study helps to rationalize design rules for stable and low-fouling polymersomes for future medical applications.

Fluorescence dynamics of gold nanoclusters (Au9 and Au25 ) are studied in the complex and crowded environment of a triblock co-polymer (F127) hydrogel and inside cervical cancer cell, HeLa. In the hydrogel, spherical micelles of F127 remain immobilized with a hydrophobic core (PPO) and a hydrophilic corona (PEO) region. The fluorescence anisotropy decay suggests that the timescale of rotational relaxation in the hydrogel is similar to that in bulk water (viscosity ∼1 cP). From fluorescence correlation spectroscopy (FCS) it is inferred that the local viscosity in the hydrogel is 12 cP for Au9 and 18 cP for Au23 . These results indicate that gold nanoclusters (AuNCs) localize in the corona region of the hydrogel. Evidently, frictions against rotation and translation are different inside the gel. It is suggested that rotation of the AuNCs senses the immediate water-like "void" region while translation motion involves in-and-out movement of the AuNCs at the periphery of the gel. Finally, the gold nanoclusters are used for cell imaging and estimation of intracellular viscosity of HeLa cells.

VASCULAR-RELATED NAC-DOMAIN7 (VND7) is the master transcription factor for vessel element differentiation in Arabidopsis thaliana. To identify the cis-acting sequence(s) bound by VND7, we employed fluorescence correlation spectroscopy (FCS) to find VND7-DNA interactions quantitatively. This identified an 18-bp sequence from the promoter of XYLEM CYSTEINE PEPTIDASE1 (XCP1), a direct target of VND7. A quantitative assay for binding affinity between VND7 and the 18-bp sequence revealed the core nucleotides contributing to specific binding between VND7 and the 18-bp sequence. Moreover, by combining the systematic evolution of ligands by exponential enrichment (SELEX) technique with known consensus sequences, we defined a motif termed the Ideal Core Structure for binding by VND7 (ICSV). We also used FCS to search for VND7 binding sequences in the promoter regions of other direct targets. Taking these data together, we proposed that VND7 preferentially binds to the ICSV sequence. Additionally, we found that substitutions among the core nucleotides affected transcriptional regulation by VND7 in vivo, indicating that the core nucleotides contribute to vessel-element-specific gene expression. Furthermore, our results demonstrate that FCS is a powerful tool for unveiling the DNA-binding properties of transcription factors.

Osteoarthritis is a chronic joint disease characterized by articular cartilage degeneration, pain, and disability. As an avascular tissue, the movement of water and solutes through the tissue is critical to cartilage health and function, and early changes in solute diffusivity due to micro-scale changes in the properties of cartilage's extracellular matrix might precede clinical symptoms. A diagnostic technique for quantifying alteration to the diffusive environment of cartilage that precedes macroscopic changes may allow for the earlier identification of osteoarthritic disease, facilitating earlier intervention strategies. Toward this end, we used two confocal microscopy-based correlation spectroscopy techniques, fluorescence correlation spectroscopy and raster image correlation spectroscopy, to quantify the diffusion of two small solutes, fluorescein and 3k dextran, within human osteoarthritic articular cartilage. Our goal was to determine if these relatively simple optical correlation spectroscopy techniques could detect changes in solute diffusivity associated with increasing cartilage damage as assessed by International Cartilage Repair Society scoring guidelines, and if these measures are correlated with mechanical and compositional measures of cartilage health. Our data show a modest, yet significant increase in solute diffusivity and cartilage permeability with increasing osteoarthritis score (grades 0-2), with a strong correlation between diffusion coefficients, permeability, and cartilage composition. The described correlation spectroscopy techniques are quick, simple, and easily adapted to existing laboratory workflow and equipment. Furthermore, the minimal solute concentrations and laser powers required for analysis, combined with recent advances in arthroscopic microscopy, suggest correlation spectroscopy techniques as translational candidates for development into early OA diagnosis tools. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:3256-3267, 2018.

This review focuses on fluorescence spectroscopy techniques for the investigation of electrophoretic separations. Fluorescence has been used as a sensitive detector for capillary, gel, and microchip electrophoresis for decades. However, advanced fluorescence methods can be used to study transport, interfacial phenomena, intermolecular and affinity interactions, and other processes that occur during separation. This so-called spectroscopic toolkit can be implemented to understand fundamental behavior in electrophoresis and electrokinetic chromatography. Techniques such as fluorescence recovery after photobleaching, fluorescence correlation spectroscopy, and fluorescence anisotropy are discussed in relation to electrophoretic separations. Newer methods such as super-resolution microscope are also introduced.

Lipids and proteins in the plasma membrane are laterally heterogeneous and formalised as lipid rafts featuring unique biophysical properties. However, the self-assembly mechanism of lipid raft cannot be revealed even its physical properties and components were determined in specific physiological processes. In this study, two-photon generalised polarisation imaging and fluorescence correlation spectroscopy were used to study the fusion of lipid rafts through the membrane phase and the lateral diffusion of lipids in living breast cancer cells. A self-assembly model of lipid rafts associated with lipid diffusion and membrane phase was proposed to demonstrate the lipid sorting ability of lipid rafts in the plasma membrane. The results showed that the increased proportion of slow subdiffusion of GM1 -binding cholera toxin B-subunit (CT-B) was accompanied with an increased liquid-ordered domain during the β-estradiol-induced fusion of lipid rafts. And slow subdiffusion of CT-B was vanished with the depletion of lipid rafts. Whereas the dialkylindocarbocyanine (DiIC18 ) diffusion was not specifically regulated by lipid rafts. This study will open up a new insight for uncovering the self-assembly of lipid rafts in specific pathophysiological processes.

Semiconductor quantum dots (QDs) are a new nano-material, and their unique optical properties have become a focal point of research in both academia and industry. In this study, we studied photoluminescence (PL) intermittence (or 'blinking') behaviors of individual QDs prepared with different chiral ligands by using single molecule microscopy and single molecule fluorescence correlation spectroscopy (FCS). We found that the chirality of N-isobutyryl-d/l-cysteine (D/L-NIC) as surface stabilizers significantly influences PL blinking behaviors of cadmium telluride (CdTe) QDs synthesized in aqueous solution. The 'on time' distribution and the power-law exponent analyses show that the D-NIC more efficiently suppresses the blinking of QDs over L-NIC. Ensemble spectroscopies verfied that the remarkably-different blinking behaviors of QDs induced by ligand chirality were attributed to the different number of chiral ligands bound in the surface of QDs.

In this study, interactions of synthesized copper nanoclusters (CuNCs) with a model transport protein, human serum albumin (HSA), have been systematically investigated by using various spectroscopic approaches. The interactions give rise to the formation of "protein-corona" like assemblies and the luminescence properties (both steady-state and time-resolved) are enhanced due to gradual adsorption of the protein on the surface of the NCs. The associated thermodynamics and binding parameters have been estimated resorting to luminescent experimental techniques as well as isothermal titration calorimetry (ITC) studies, indicating that every NC is surrounded by (4±1) protein molecules. The adsorption of HSA on the surface of the NCs has been characterized by dynamic light scattering (DLS) and time-resolved anisotropy measurements. Finally, fluorescence correlation spectroscopy (FCS) data substantiate the emergence of new "protein-corona" like assemblies resulting in slower translational diffusion motions and concomitant rise of the hydrodynamic diameters.

Opioid receptors are important pharmacological targets for the management of numerous medical conditions (eg, severe pain), but they are also the gateway to the development of deleterious side effects (eg, opiate addiction). Opioid receptor signaling cascades are well characterized. However, quantitative information regarding their lateral dynamics and nanoscale organization in the plasma membrane remains limited. Since these dynamic properties are important determinants of receptor function, it is crucial to define them. Herein, the nanoscale lateral dynamics and spatial organization of kappa opioid receptor (KOP), wild type mu opioid receptor (MOPwt), and its naturally occurring isoform (MOPN40D) were quantitatively characterized using fluorescence correlation spectroscopy and photoactivated localization microscopy. Obtained results, supported by ensemble-averaged Monte Carlo simulations, indicate that these opioid receptors dynamically partition into different domains. In particular, significant exclusion from GM1 ganglioside-enriched domains and partial association with cholesterol-enriched domains was observed. Nanodomain size, receptor population density and the fraction of receptors residing outside of nanodomains were receptor-specific. KOP-containing domains were the largest and most densely populated, with the smallest fraction of molecules residing outside of nanodomains. The opposite was true for MOPN40D. Moreover, cholesterol depletion dynamically regulated the partitioning of KOP and MOPwt, whereas this effect was not observed for MOPN40D.

The cytotoxic synapse formed between cytotoxic T lymphocytes or natural killer cells expressing CD95L and target cells with CD95 on their surface is a key pathway for apoptosis induction by the immune system. Despite similarities with the immune synapse in antigen presenting cells, little is known about the role of the spatiotemporal organization of agonistic proteins/receptor interactions for CD95 signaling. Here, we have developed an artificial cytotoxic synapse to examine how mobility and geometry of an anti-CD95 agonistic antibody affect receptor aggregation and mobility, ie the first step of receptor activation. By measuring the distribution, diffusion coefficient, and fraction of immobile CD95 receptor in living cells, we show that at short times, the initial activation of CD95 occurs locally and is limited to the contact region of the cytotoxic synapse. This anisotropic activation of apoptotic signaling supports a role for confined interactions on the efficiency of signal transduction that may have implications for biomedical applications of extrinsic apoptosis induction. Fil: Sánchez, María Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentina Fil: Murad, Fabronia. Universität Tübingen; Alemania Fil: Gülcüler Balta, Gülce S.. German Cancer Research Center; Alemania. Heidelberg University; Alemania Fil: Martin Villalba, Ana. German Cancer Research Center; Alemania Fil: García Sáez, Ana J.. Universität Tübingen; Alemania Fil: Carrer, Dolores Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentina

Because of its importance in viral replication, the M2 proton channel of the influenza A virus has been the focus of many studies. Although we now know a great deal about the structural architecture underlying its proton conduction function, we know little about its conformational dynamics, especially those controlling the rate of this action. Herein, we employ a single-molecule fluorescence method to assess the dynamics of the inter-helical channel motion of both full-length M2 and the transmembrane domain of M2. The rate of this motion depends not only on the identity of the channel and membrane composition but also on the pH in a sigmoidal manner. For the full-length M2 channel, the rate is increased from approximately 190 μs−1 at high pH to approximately 80 μs−1 at low pH, with a transition midpoint at pH 6.1. Because the latter value is within the range reported for the conducting pKa value of the His37 tetrad, we believe that this inter-helical motion accompanies proton conduction.

The structural dynamics of a DNA hairpin (Hp) are studied in the absence and presence of the two natural osmolytes trimethylamine-N-oxide (TMAO) and urea at ambient and extreme environmental conditions, including high pressures and high temperatures, by using single-molecule Förster resonance energy transfer and fluorescence correlation spectroscopy. The effect of pressure on the conformational dynamics of the DNA Hp is investigated on a single-molecule level, providing novel mechanistic insights into its conformational conversions. Different from canonical DNA duplex structures of similar melting points, the DNA Hp is found to be rather pressure sensitive. The combined temperature and pressure dependent data allow dissection of the folding free energy into its enthalpic, entropic, and volumetric contributions. The folded conformation is effectively stabilized by the compatible osmolyte TMAO not only at high temperatures, but also at high pressures and in the presence of the destabilizing co-solute urea.

Fluorescence fluctuation-based methods are non-invasive microscopy tools especially suited for the study of dynamical aspects of biological processes. These methods examine spontaneous intensity fluctuations produced by fluorescent molecules moving through the small, femtoliter-sized observation volume defined in confocal and multiphoton microscopes. The quantitative analysis of the intensity trace provides information on the processes producing the fluctuations that include diffusion, binding interactions, chemical reactions and photophysical phenomena. In this review, we present the basic principles of the most widespread fluctuation-based methods, discuss their implementation in standard confocal microscopes and briefly revise some examples of their applications to address relevant questions in living cells. The ultimate goal of these methods in the Cell Biology field is to observe biomolecules as they move, interact with targets and perform their biological action in the natural context. © 2016 IUBMB Life, 69(1):8-15, 2017.

Biological responses of cells and organisms to nanoparticle exposure crucially depend on the properties of the protein adsorption layer ("protein corona") forming on nanoparticle surfaces and their characterization is a crucial step toward a deep, mechanistic understanding of their build-up. Previously, adsorption of one type of model protein on nanoparticles was systematically studied in situ by using fluorescence correlation spectroscopy. Here, the first such study of interactions is presented between water-solubilized CdSe/ZnS quantum dots (QDs) and a complex biofluid, human blood serum. Despite the large number of proteins in serum, a protein layer of well-defined (average) thickness forming on QD surfaces is observed. Both the thickness and the apparent binding affinity depend on the type of QD surface ligand. Kinetic experiments reveal that the protein corona formed from serum is irreversibly bound, whereas the one formed from human serum albumin was earlier observed to be reversible. By using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry, the most abundant serum proteins contributing to the formation of a hard corona on the QDs are identified.

We explored the feasibility of using confocal fluorescence correlation spectroscopy to study small nanoparticle diffusion in hundred-nanometer-sized cylindrical pores. By modeling single particle diffusion in tube-like confined three-dimensional space aligned parallel to the confocal optical axis, we showed that two diffusion dynamics can be observed in both original intensity traces and the autocorrelation functions (ACFs): the confined two-dimensional lateral diffusion and the unconfined one-dimensional (1D) axial diffusion. The separation of the axial and confined lateral diffusion dynamics provides an opportunity to study diffusions in different dimensions separately. We further experimentally studied 45 nm carboxylated polystyrene particles diffusing in 300 nm alumina pores. The experimental data showed consistency with the simulation. To extract the accurate axial diffusion coefficient, we found that a 1D diffusion model with a Lorentzian axial collection profile needs to be used to analyze the experimental ACFs. The diffusion of the 45 nm nanoparticles in polyethyleneglycol-passivated 300 nm pores slowed down by a factor of ∼2, which can be satisfactorily explained by hydrodynamic frictions.

The complexity of biological processes requires experimental techniques which are able to resolve events on appropriate temporal and spatial scales. As all biological processes are ultimately driven by the dynamics and interactions of individual molecules, studies on the single-molecule level provide important insights about a large variety of parameters at thermodynamic equilibrium and without ensemble averaging. In the life sciences, single-molecule experiments are preferentially performed using fluorescence light microscopy owing to its high sensitivity, its temporal resolution and its ability to address live and thus dynamic specimen. By today, a range of single-molecule techniques such as single-pair Forster resonance energy transfer (FRET), single-molecule tracking and different counting techniques are readily available to characterise molecular interactions, conformational dynamics, complex stoichiometries and translational mobilities in biological systems both in vitro and in situ. Key Concepts Single-molecule analysis of biological processes allows to observe the behaviour of individual molecules rather than ensemble averages. Owing to its sensitivity, fluorescence microscopy is the dominating single-molecule technique used in the life sciences. Detection of individual fluorescently labelled biomolecules requires dilution of the sample and spatial restriction of the observation volume. A variety of single-molecule fluorescence microscopy techniques is now being routinely applied in many laboratories. The recent introduction of single-molecule DNA sequencers illustrates the commercial impact of single-molecule techniques in the life sciences. The absolute stoichiometry of individual macromolecular complexes can be determined by different counting methods such as photobleaching step analysis. Single-pair Forster resonance energy transfer (spFRET) can be used to measure molecular interactions and intramolecular distances at the nanometre level. Tracking of individual molecules in their native context helps understanding how their behaviour is influenced by interactions with other molecules and by cellular processes. Among other things, fluorescence correlation spectroscopy (FCS) can be used to investigate the diffusion of individual molecular species and the interaction of multiple species with each other. Keywords: single-molecule fluorescence spectroscopy; protein conformation; protein–protein interactions; protein dynamics; live-cell experiments; molecular heterogeneities; single-pair Forster resonance energy transfer; fluorescence correlation spectroscopy; single-molecule/particle tracking

Newly synthesized proteins constitute an important subset of the proteome involved in every cellular process, yet existing chemical tools used to study them have major shortcomings. Herein we report a suite of cell-permeable puromycin analogues capable of being metabolically incorporated into newly synthesized proteins in different mammalian cells, including neuronal cells. Subsequent labeling with suitable bioorthogonal reporters, in both fixed and live cells, enabled direct imaging and enrichment of these proteins. By taking advantage of the mutually orthogonal reactivity of these analogues, we showed multiplexed labeling of different protein populations, as well as quantitative measurements of protein dynamics by fluorescence correlation spectroscopy, could be achieved in live-cell environments.

Fluorescence correlation spectroscopy (FCS) is regularly used to study diffusion in non-dilute "crowded" biopolymer solutions, including the interior of living cells. For fluorophores in dilute solution, the relationship between the FCS spectrum G(t) and the diffusion coefficient D is well-established. However, the dilute-solution relationship between G(t) and D has sometimes been used to interpret FCS spectra of fluorophores in non-dilute solutions. Unfortunately, the relationship used to interpret FCS spectra in dilute solutions relies on an assumption that is not always correct in non-dilute solutions. This paper obtains the correct form for interpreting FCS spectra of non-dilute solutions, writing G(t) in terms of the statistical properties of the fluorophore motions. Approaches for applying this form are discussed.

Extracellular and intracellular degradation of nucleic acids remains an issue in non-viral gene therapy. Understanding biodegradation is critical for the rational design of gene therapeutics in order to maintain stability and functionality at the target site. However, there are only limited methods available that allow determining the stability of genetic materials in biological environments. In this context, the decay kinetics of fluorescently labeled plasmid DNA (pDNA) and messenger RNA (mRNA) in undiluted biological samples (i.e., human serum, human ascites, bovine vitreous) and cell extracts is studied using fluorescence correlation spectroscopy (FCS) and single particle tracking (SPT). It is demonstrated that FCS is suitable to follow mRNA degradation, while SPT is better suited to investigate pDNA integrity. The half-life of mRNA and pDNA is approximate to 1-2 min and 1-4 h in biological samples, respectively. The resistance against biodegradation drastically improves by complexation with lipid-based carriers. Taken together, FCS and SPT are able to quantify the integrity of mRNA and pDNA, respectively, as a function of time, both in the extracellular biological fluids and cell extracts. This in turn allows to focus on the important but less understood issue of nucleic acids degradation in more detail and to rationally optimize gene delivery system as therapeutics.

International audience; Two‐photon photoluminescence (TPPL) emission spectra of DNA–gold nanoparticle (AuNP) monoconjugates and the corresponding DNA‐linked AuNP dimers are obtained by photon time‐of‐flight spectroscopy. This technique is combined with two‐photon photoluminescence fluctuation correlation spectroscopy (TPPL–FCS) to simultaneously monitor the optical and hydrodynamic behaviour of these nano‐assemblies in solution, with single‐particle sensitivity and microsecond temporal resolution. In this study, the AuNPs have an average core diameter of 12 nm, which renders their dark‐field plasmonic light scattering too weak for single‐particle imaging. Moreover, as a result of the lack of plasmonic coupling in the dimers, the optical extinction, scattering and photoluminescence spectra of the DNA–AuNP complexes are not sufficiently different to distinguish between monomers and dimers. The use of TPPL–FCS successfully addresses these bottlenecks and enables the distinction between AuNP monomers and AuNP dimers in solution by measurement of their hydrodynamic rotational and translational diffusion.

Triplex DNA has become one of the most useful recognition motifs in the design of new molecular biology tools, therapeutic agents and sophisticated DNA-based nanomaterials because of its direct recognition of natural double-stranded DNA. In this paper, we developed a sensitive and microscale method to study the formation and stability characterization of triplex DNA using fluorescence correlation spectroscopy (FCS). The principle of this method is mainly based on the excellent capacity of FCS for sensitively distinguishing between free single-strand DNA (ssDNA) fluorescent probes and fluorescent probe-double-strand DNA (dsDNA) hybridized complexes. First, we systematically investigated the experimental conditions of triplex DNA formation. Then, we evaluated the equilibrium association constants (K(a)) under different ssDNA probe lengths, composition and pH. Finally, we used FCS to measure the hybridization fraction of a 20-mer perfectly matched ssDNA probe and three single-base mismatched ssDNA probes with 146-mer dsDNA. Our data illustrated that FCS is a useful tool for the direct determination of the thermodynamic parameters of triplex DNA formation and discrimination of a single-base mismatch of triplex DNA without denaturation. Compared with current methods, our method is characterized by high sensitivity, good universality and small sample and reagent requirements. More importantly, our method has the potential to become a platform for triplex DNA research in vitro.

Biocompatible polymer nanoparticles are of high interest in chemical, biological, and medical research as potential drug delivery systems. These nanoparticles must be nontoxic and metabolizable. Poly(lactic acid) (PLA) meets these requirements and therefore is widely used for biomedical applications. The miniemulsion/solvent evaporation procedure is a very efficient approach to produce PLA nanoparticles with controlled size and morphology using preformed polymer. In this paper, the synthesis of PLA-based nanoparticles functionalized with either carboxyl groups or fluorescent molecules is presented. Both functionalities are covalently attached to the particle, thus any physical desorption and leakage of the dye upon storage or experiments performed under physiological conditions, for example during uptake by living cells, can be avoided. The fluorescence properties and stability of the obtained nanoparticles in aqueous solutions with different ionic strength are studied by fluorescence correlation spectroscopy and dynamic light scattering.

Glucocorticoid receptor α (GR) binds to the promoter regions of target genes as a homodimer and activates its transcriptional process. Though the homodimerization is thought to be the initial and essential process, the dissociation constant for homodimerization of GR remains controversial. To quantify homodimerization of (enhanced green fluorescence protein) EGFP–(glucocorticoid receptor) GR, the particle brightness in lysates from single cell was estimated for the fraction of homodimeric EGFP–GR using fluorescence correlation spectroscopy and microwells. Fitting the data with a bimolecular reaction model, the dissociation constant was determined. Moreover slow-diffusion complex was observed. These results suggest that EGFP–GR forms not only a monomer–dimer equivalent state but also a large-molecular-weight complex.

The temperature and pH-dependent diffusion of poly(glycerol monomethacrylate)-block-poly(2-hydroxypropyl methacrylate) nanoparticles prepared via polymerization-induced self-assembly in water is characterized using fluorescence correlation spectroscopy (FCS). Lowering the solution temperature or raising the solution pH induces a worm-to-sphere transition and hence an increase in diffusion coefficient by a factor of between four and eight. FCS enables morphological transitions to be monitored at relatively high copolymer concentrations (10% w/w) compared to those required for dynamic light scattering (0.1% w/w). This is important because such transitions are reversible at the former concentration, whereas they are irreversible at the latter. Furthermore, the FCS data suggest that the thermal transition takes place over a very narrow temperature range (less than 2 °C). These results demonstrate the application of FCS to characterize order-order transitions, as opposed to order-disorder transitions. The temperature and pH-dependent diffusion of poly(glycerol monomethacrylate)-block-poly(2-hydroxypropylmethacrylate)- nanoparticles in water is characterized using fluorescence correlation spectroscopy. Lowering either the solution temperature or pH induces a worm-to-sphere transition and hence an increase in diffusion coefficient by a factor of between four and eight.

A study was made of the anti-polyelectrolyte effect of a model polyzwitterion at the single-chain level and from the point of view of comparison with inorganic salts and organic inner salts. A well-defined polymer of sulfobetaine was synthesized and fluorescently labeled. Fluorescence correlation spectroscopy was adopted to probe diffusion and chain size of polyzwitterion single chains in extremely dilute aqueous solutions over a broad concentration range of added salts. By the introduction of inorganic salts and organic inner salts, the single chains of polyzwitterions expanded with an increase of salt concentration. This observation indicates that the break-up of the dipole-dipole attraction between the zwitterion units is the mechanism of the anti-polyelectrolyte effect of polyzwitterions. The difference in the strength of the anti-polyelectrolyte effect induced by inorganic salts and organic inner salts also indicates the partial contribution from the asymmetric adsorption of ions onto polyzwitterion chains. (c) 2015 Society of Chemical Industry

Protein aggregation generally results from association between hydrophobic regions of individual monomers. However, additional mechanisms arising from specific interactions, such as intermolecular disulfide bond formation, may also contribute to the process. The latter is proposed to be the initiating pathway for aggregation of immunoglobulin (IgG), which is essential for triggering its immune response. To test the veracity of this hypothesis, we have employed fluorescence correlation spectroscopy to measure the kinetics of aggregation of IgG in separate experiments either allowing or inhibiting disulfide formation. Fluorescence correlation spectroscopy measurements yielded a diffusion time (τ(D)) of ∼200 µsec for Rhodamine-labeled IgG, corresponding to a hydrodynamic radius (R(H)) of 56 A for the IgG monomer. The aggregation kinetics of the protein was followed by monitoring the time evolution of τ(D) under conditions in which its cysteine residues were either free or blocked. In both cases, the progress curves confirmed that aggregation proceeded via the nucleation-dependent polymerization pathway. However, for aggregation in the presence of free cysteines, the lag times were shorter, and the aggregate sizes bigger, than their respective counterparts for aggregation in the presence of blocked cysteines. This result clearly demonstrates that formation of intermolecular disulfide bonds represents a preferred pathway in the aggregation process of IgG. Fluorescence spectroscopy showed that aggregates formed in experiments where disulfide formation was prevented denatured at lower concentration of guanidine hydrochloride than those obtained in experiments where the disulfides were free to form, indicating that intermolecular disulfide bridging is a valid pathway for IgG aggregation.

Most surfaces of engineered nanoparticles (NPs) are reactive toward biomolecules. Therefore, whenever NPs become immersed in biological fluids, proteins and other biomolecules bind to the NP surface, forming an adsorption layer (biomolecular corona) that modifies the NPs' physicochemical properties and subsequent interactions with living systems. Its exploration is a formidable endeavor owing to the enormous diversity of engineered NPs in terms of their physicochemical properties and the vast number of biomolecules available in biofluids that may bind to NPs with widely different strengths. Significant progress has been made in our understanding of the biomolecular corona, but even very basic issues are still controversially debated. In fact, there are divergent views of its microscopic structure and dynamics, even on physical properties, such as its thickness. As an example, there is no agreement on whether proteins form monolayers or multilayers upon adsorption. In our quantitative studies of NP-protein interactions by in situ fluorescence correlation spectroscopy (FCS) with highly defined model NPs and important serum proteins, we have universally observed protein monolayer formation around NPs under saturation or even oversaturation conditions. Here, we critically discuss biomolecular corona characterization using FCS and dynamic light scattering and identify challenges and future opportunities. Further careful, quantitative experiments are needed to elucidate the mechanisms of biomolecular corona formation and to characterize its structure. This article is categorized under: Nanotechnology Approaches to Biology > Nanoscale Systems in Biology.

The effect of solute affinity on solute diffusion in binary liquids well below the lower critical solution temperature (LCST) was studied by using fluorescence correlation spectroscopy. We measured the hydrodynamic radii of a hydrophobic and an amphiphilic fluorescent dye under systematic variation of the relative molar fractions of water/2-butoxyethanol and, for comparison, of water/methanol mixtures, which do not show phase separation. We found that the apparent hydrodynamic radius of the hydrophobic dye almost doubled in water/2-butoxyethanol, whereas it remained largely unchanged for the amphiphilic dye and in water/methanol mixtures. Our results indicate that the translational diffusion of solutes is influenced by transient local solution structures, even at temperatures well below the LCST. We conclude that, even far below LCST, different solutes can experience different environments in binary liquid mixtures depending on both the solute and solvent properties, all of which impact their reactivity.

A marker's dark triplet state is of great importance in fluorescence microscopy: It serves as a means to switch off fluorescent markers and is thus the enabling element for several super-resolution methods. On the other hand, intersystem-crossing to the electronic dark triplet state strongly reduces the fluorescence yield in conventional fluorescence microscopy. The ability to determine the kinetic parameters of transitions into the triplet state is thus of great importance and because fluorescence correlation spectroscopy (FCS) can be applied without disturbing the system under study, it is one of the preferred methods to do so. However, conventional FCS observations of triplet dynamics suffer from bias due to the spatially inhomogeneous irradiance profile of the excitation laser. Herein, we present a novel method to correct this bias and verify it by analyzing both Monte Carlo simulated and experimental data of the organic dye Rhodamine 110 in aqueous solution for both continuous-wave and pulsed excitation. Importantly, our approach can be readily generalized to most other FCS experiments that determine intensity dependent kinetic parameters. Microsc. Res. Tech. 77:528–536, 2014. © 2014 Wiley Periodicals, Inc.

Over the last decade, we have witnessed an outburst of many different optical techniques aimed at breaking the diffraction limit of light, providing super-resolution imaging on intact fixed cells. In parallel, single-molecule detection by means of fluorescence has become a common tool to investigate biological interactions at the molecular level both in vitro and in living cells. Despite these advances, visualization of dynamic events at relevant physiological concentrations at the nanometer scale remains challenging. In this review, we focus on recent advancements in the field of nanophotonics toward nanoimaging and single-molecule detection at ultrahigh sample concentrations. These approaches rely on the use of metal nanostructures known as optical antennas to localize and manipulate optical fields at the nanometer scale. We highlight examples on how different optical antenna geometries are being implemented for nanoscale imaging of cell membrane components. We also discuss different implementations of self-standing and two-dimensional antenna arrays for studying nanoscale dynamics in living cell membranes as well as detection of individual biomolecular interactions in the µM range for sensing applications. Microsc. Res. Tech. 77:537–545, 2014. © 2014 Wiley Periodicals, Inc.

The dynamic behaviour of gold nanoparticles functionalised with glucose (Glc-Au NPs) has been studied here by means of fluorescence correlation spectroscopy (FCS). Meaningful data on the state of aggregation and dynamics of Glc-Au NPs fluorescently-labelled with HiLyte Fluor647 (Glc-Au-Hi NPs) in the intracellular environment were obtained. Moreover, the work presented here shows that FCS can be used to visualise the presence of single NPs or NP aggregates following uptake and to estimate, locally, NP concentrations within the cell. FCS measurements become possible after applying a "prebleaching" methodology, when the immobile NP fraction has been effectively removed and thus significant FCS data has been recorded. In this study, Glc-Au-Hi NPs have been incubated with HepG2 cells and their diffusion time in the intracellular environment has been measured and compared with their diffusion value in water and cell media.

Single-molecule approaches to biology offer a powerful new vision to elucidate the mechanisms that underpin the functioning of living cells. However, conventional optical single molecule spectroscopy techniques such as Forster fluorescence resonance energy transfer (FRET) or fluorescence correlation spectroscopy (FCS) are limited by diffraction to the nanomolar concentration range, far below the physiological micromolar concentration range where most biological reaction occur. To breach the diffraction limit, zero-mode waveguides (ZMW) and plasmonic antennas exploit the surface plasmon resonances to confine and enhance light down to the nanometer scale. The ability of plasmonics to achieve extreme light concentration unlocks an enormous potential to enhance fluorescence detection, FRET, and FCS. Single molecule spectroscopy techniques greatly benefit from ZMW and plasmonic antennas to enter a new dimension of molecular concentration reaching physiological conditions. The application of nano-optics to biological problems with FRET and FCS is an emerging and exciting field, and is promising to reveal new insights on biological functions and dynamics. For further resources related to this article, please visit the WIREs website. Conflict of interest: The authors have declared no conflicts of interest for this article.

Subcellular compartmentalization of cells, a defining characteristic of eukaryotes, is fundamental for the fine tuning of internal processes and the responding to external stimuli. Reproducing and controlling such compartmentalized hierarchical organization, responsiveness, and communication is important toward understanding biological systems and applying them to smart materials. Herein, a cellular signal transduction strategy (triggered release from subcompartments) is leveraged to develop responsive, purely artificial, polymeric multicompartment assemblies. Incorporation of responsive nanoparticles-loaded with enzymatic substrate or ion channels-as subcompartments inside micrometer-sized polymeric vesicles (polymersomes) allowed to conduct bioinspired signaling cascades. Response of these subcompartments to an externally added stimulus is achieved and studied by using confocal laser scanning microscopy (CLSM) coupled with in situ fluorescence correlation spectroscopy (FCS). Signal triggered activity of an enzymatic reaction is demonstrated in multicompartments through recombination of compartmentalized substrate and enzyme. In parallel, a two-step signaling cascade is achieved by triggering the recruitment of ion channels from inner subcompartments to the vesicles' membrane, inducing ion permeability, mimicking endosome-mediated insertion of internally stored channels. This design shows remarkable versatility, robustness, and controllability, demonstrating that multicompartment polymer-based assemblies offer an ideal scaffold for the development of complex cell-inspired responsive systems for applications in biosensing, catalysis, and medicine.

To quantify more precisely and more reliably diffusion and reaction properties of biomolecules in living cells, a novel closed description in 3D of both the bleach and the post-bleach segment of fluorescence recovery after photobleaching (FRAP) data acquired at a point, i.e., a diffraction-limited observation area, termed point FRAP, is presented. It covers a complete coupled reaction-diffusion scheme for mobile molecules undergoing transient or long-term immobilization because of binding. We assess and confirm the feasibility with numerical solutions of the differential equations. By applying this model to free EYFP expressed in HeLa cells using a customized confocal laser scanning microscope that integrates point FRAP and fluorescence correlation spectroscopy (FCS), the applicability is validated by comparison with results from FCS. We show that by taking diffusion during bleaching into consideration and/or by employing a global analysis of series of bleach times, the results can be improved significantly. As the point FRAP approach allows to obtain data with diffraction-limited positioning accuracy, diffusion and binding properties of the exon-exon junction complex (EJC) components REF2-II and Magoh are obtained at different localizations in the nucleus of MCF7 cells and refine our view on the position-dependent association of the EJC factors with a maturating mRNP complex. Our findings corroborate the concept of combining point FRAP and FCS for a better understanding of the underlying diffusion and binding processes. (c) 2013 International Society for Advancement of Cytometry

Nanolipoprotein particles (NLPs) are nanometer-scale discoidal particles that feature a phospholipid bilayer confined within an apolipoprotein “scaffold,” which are useful for solubilizing hydrophobic molecules such as drugs and membrane proteins. NLPs are synthesized either by mixing the purified apolipoprotein with phospholipids and other cofactors or by cell-free protein synthesis followed by self-assembly of the nanoparticles in the reaction mixture. Either method can be problematic regarding the production of homogeneous and monodispersed populations of NLPs, which also currently requires multiple synthesis and purification steps. Telodendrimers (TD) are branched polymers made up of a dendritic oligo-lysine core that is conjugated to linear polyethylene glycol (PEG) on one end, and the lysine “branches” are terminated with cholic acid moieties that enable the formation of nanomicelles in aqueous solution. We report herein that the addition of TD during cell-free synthesis of NLPs produces unique hybrid nanoparticles that have drastically reduced polydispersity as compared to NLPs made in the absence of TD. This finding was supported by dynamic light scattering, fluorescence correlation spectroscopy, and cryo transmission electron microscopy (Cryo-EM). These techniques demonstrate the ability of TDs to modulate both the NLP size (6–30 nm) and polydispersity. The telodendrimer NLPs (TD-NLPs) also showed 80% less aggregation as compared to NLPs alone. Furthermore, the versatility of these novel nanoparticles was shown through direct conjugation of small molecules such as fluorescent dyes directly to the TD as well as the insertion of a functional membrane protein.

Determining the structure of a protein and its transformation under different conditions is key to understanding its activity. The structural stability and activity of proteins in aqueous-organic solvent mixtures, which is an intriguing topic of research in biochemistry, is dependent on the nature of the protein and the properties of the medium. Herein, the effect of a commonly used cosolvent, dimethyl sulfoxide (DMSO), on the structure and conformational dynamics of bovine serum albumin (BSA) protein is studied by fluorescence correlation spectroscopy (FCS) measurements on fluorescein isothiocyanate (FITC)-labeled BSA. The FCS study reveals a change of the hydrodynamic radius of BSA from 3.7 nm in the native state to 7.0 nm in the presence of 40% DMSO, which suggests complete unfolding of the protein under these conditions. Fluorescence self-quenching of FITC has been exploited to understand the conformational dynamics of BSA. The time constant of the conformational dynamics of BSA is found to change from 35 μs in its native state to 50 μs as the protein unfolds with increasing DMSO concentration. The FCS results are corroborated by the near-UV circular dichroism spectra of the protein, which suggest a loss of its tertiary structure with increasing concentration of DMSO. The intrinsic fluorescence of BSA and the fluorescence response of 1-anilinonaphthalene-8-sulfonic acid, used as a probe molecule, provide information that is consistent with the FCS measurements, except that aggregation of BSA is observed in the presence of 40% DMSO in the ensemble measurements.

Owing to the highly efficient two-photon fluorescence of gold nanorods and very short fluorescence lifetime compared with the rotational correlation time, the rotation and diffusion of a single gold nanorod can be easily observed by two-photon fluorescence correlation spectroscopy (TP-FCS). This property, along with the previous successful use as a contrast agent in two-photon flu- orescence imaging, suggests a potential application in TP-FCS as well. Although the FCS measure- ment becomes highly efficient with gold nanorods as probes, the amplitude and temporal decay of the measured correlation functions depend critically on excitation power. Here, we investigate various photophysical processes of gold nanorods to determine the cause of such a sensitive power depend- ency. This understanding provides a basis for choosing appropriate FCS models to recover reasonable physical parameters. Although the correlation function amplitude G(0) is 32 times lower when the excitation power increases from 20 mW to 1.12 mW, the application of a saturation-modified FCS model yields very good fit to each data set and the fitted concentration of 0.64 nM is comparable to the 0.7 nM given by the inductively coupled plasma mass spectrometry measurement. The FCS assay appears to be an efficient method for the quantification of gold nanorods when correctly interpreted. However, even with the saturation considered in the fitting model, the fitted rotational and transla- tional diffusion rates are getting faster as the power increases. This indicates that other effects such as photothermal effects may raise the local temperature, and thus increasing the rotational and translational diffusion rate. Microsc. Res. Tech. 76:882-889, 2013. V C 2013 Wiley Periodicals, Inc.

The bioconjugation of fluorescent quantum dots (QDs) and purification of QDs bioconjugates are of vital importance in bioapplications. In this paper, we systematically investigated the purification efficiency of QDs bioconjugates and their stability during separation process by using size exclusion chromatography (SEC) technique, fluorescence spectroscopy, and fluorescence correlation spectroscopy (FCS). In this study, commercial QDs and fluorescein isothiocyanate (FITC) were used as labeling probes, and bovine serum albumin (BSA) and antibody (Erbitux) were used as mode samples. The covalently linkage and the electrostatic interaction were used in bioconjugation procedures. We systematically studied the effects of certain factors such as the scales of column, loading volume, elution buffer, and separation media on the purification efficiency of QDs bioconjugates by a home-built SEC device. And highly pure and monodisperse QDs bioconjugates could be obtained by SEC purification twice under the optimized conditions. Furthermore, we investigated the stability of QDs bioconjugates in different conjugation ways and purification conditions by FCS, and found that the stability of bioconjugates were poor when electrostatic binding mode was used. We also observed that Sephacryl S300 HR (separation range for globular proteins: 1 × 10(4) -1.5 × 10(6) Da) was the best choice for purifying the vast majority of QDs-bioconjugates. Our work described here will be constructive to popularization and applications of QDs in life science.

Nanoparticles are increasingly used in medical applications such as drug delivery, imaging, and biodiagnostics, particularly for cancer. The design of nanoparticles for tumor delivery has been largely empirical, owing to a lack of quantitative data on angiogenic tissue sequestration. Using fluorescence correlation spectroscopy, the deposition rate constants of nanoparticles into angiogenic blood vessel tissue are determined. It is shown that deposition is dependent on surface charge. Moreover, the size dependency strongly suggests that nanoparticles are taken up by a passive mechanism that depends largely on geometry. These findings imply that it is possible to tune nanoparticle pharmacokinetics simply by adjusting nanoparticle size.

Fluorescence spectroscopy and imaging have been widely used for in vivo cancer diagnosis and therapy monitoring in preclinical models, as well as clinical translation. Great attempts have been made to develop novel fluorescence techniques and improve on existing ones, which can now be used in conjunction with newly developed fluorescent probes for specific cancer imaging. In this review, a broad overview of fluorescence techniques is provided, including photodynamic diagnosis, laser confocal endomicroscopy and fluorescence lifetime imaging, coupled with endogenous and exogenous fluorophores. In particular, endogenous fluorophores, such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), are highlighted as they are linked to cellular metabolism in precancer growth. The use of near-infrared dyes, such as indocynanine green (ICG), for imaging deep-tissue regions is also reviewed. In addition, diagnostic algorithms used for tissue classification and cancer detection will be discussed. Lastly, emerging technologies in fluorescence diagnosis will also be included.