Application Of Fluorescence Correlation Spectroscopy Research Articles

Temperature elevation and fluid convection under optical trapping condition as revealed by fluorescence correlation spectroscopy

Temperature of matter increases under intense photoirradiation owing to photothermal conversion. The photothermal effect is sometimes a significant issue in optical manipulation usually requiring intense optical fields. Quantitative evaluation of local temperature under photoirradiation can, therefore, provide indispensable information for optical manipulation. In a previous work, we have applied fluorescence correlation spectroscopy (FCS) to monitor the temperature under the optical trapping condition in water, ethanol, and ethylene glycol. We pointed out that analyses of diffusion time of fluorescent dyes could provide information about temperature on the basis of temperature-dependent viscosities of the solvents. In the present work, the FCS thermometry was applied to seven solvents including primary aliphatic alcohols, to examine universal applicability of the method. To verify the experimental results, numerical simulations were performed on the basis of two-dimensional heat conduction at a stationary state. The numerical results on the temperature field satisfactorily reproduced the experimental data, proving that the FCS thermometry is applicable to ordinary solvents. In addition, we also performed numerical simulations on velocity fields in the solvent, to evaluate contribution of natural convection under typical optical trapping condition at light intensity of ∼MW cm − 2. It was revealed that the contribution of the natural convection is not negligible for mass transfer in the solvents.

A Fluorescence Correlation Spectrometer for Measurements in Cuvettes

We have developed a fluorescence correlation spectroscopy (FCS) setup for performing single-molecule measurements on samples inside regular cuvettes. The cuvette FCS uses a horizontally mounted extra-long working distance, 0.7 NA, air objective with a working distance of >1.8 mm instead of a high NA water or oil immersion objective. The performance of the cuvette FCS is found to be highly sensitive to the quality and alignment of the cuvette. The radial resolution and effective observation volume obtained using the optimized setup are ∼340 nm and 1.8 fL, respectively. The highest molecular brightness and the signal/noise ratio in the autocorrelation data achieved using an aqueous solution of rhodamine B are greater than 44 kHz and 110, respectively. Here, we demonstrate two major advantages of cuvette FCS. For example, the cuvette FCS can be used for measurements over a wide range of temperatures that is beyond the range permitted in the microscope-based FCS. Furthermore, cuvette FCS can be coupled to automatic titrators to study urea-dependent unfolding of proteins with unprecedented accuracy. The ease of use and compatibility with various accessories will enable applications of cuvette FCS in the experiments that are regularly performed in spectrofluorometers but are generally avoided in microscope-based FCS.

Scanning fluorescence correlation spectroscopy comes full circle.

In this article, we review the application of fluorescence correlation spectroscopy (FCS) methods to studies on live cells. We begin with a brief overview of the theory underlying FCS, highlighting the type of information obtainable. We then focus on circular scanning FCS. Specifically, we discuss instrumentation and data analysis and offer some considerations regarding sample preparation. Two examples from the literature are discussed in detail. First, we show how this method, coupled with the photon counting histogram analysis, can provide information on yeast ribosomal structures in live cells. The combination of scanning FCS with dual channel detection in the study of lipid domains in live cells is also illustrated.

Abstract LB-B09: Application of Fluorescence Correlation Spectroscopy as a novel tool to quantify target occupancy in cells and tumor tissue

Abstract The quantification of Target Occupancy (TO) in cells after application of an inhibitor is an important parameter to correlate the biochemical features of a drug with its in vivo efficacy. Experimental evidence of TO is a robust support of the target hypothesis and helps to identify optimal dosing of the drug. The sensitive correlation spectroscopy methods (FCS and FCCS) have been used successfully in the past to analyze drug- target interactions. The technology is fast, specific and allows detection of drug target interactions in smallest sample volumes. Here we adopted the FCCS method to quantify the occupancy of the XPO1 target in cells and tumors treated with the Selective Inhibitor of Nuclear Export (SINE) compound Selinexor (KPT-330), currently in advanced clinical trials in patients with hematological and solid cancers. We used a fluorescently labeled tracer (Leptomycin B -Cy5; LMB-Cy5) to quantify the loading of XPO1 with Selinexor and confirmed target specificity of the signal using a fluorescent labeled antibody detecting native XPO1 protein. The amount of Antibody-XPO1-tracer complex was determined by FCCS. Occupancy of XPO1 target with Selinexor decreases the amount of tracer-target complexes in a dose and time dependent manner. Target occupancy studies of XPO1 were carried out in different cancer relevant cell lines and xenograft tumors retrieved from treated animals. As such the approach can be applied to study target occupancy in vitro and in vivo. Citation Format: Frank Becker, Kerrin Hansen, Marsha Crochiere, Yosef Landesmann, Stefan Hannus. Application of Fluorescence Correlation Spectroscopy as a novel tool to quantify target occupancy in cells and tumor tissue [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-B09.

Reconceptualizing Fluorescence Correlation Spectroscopy for Monitoring and Analyzing Periodically Passing Objects.

Fluorescence correlation spectroscopy (FCS) is a sensitive technique commonly applied for studying the dynamics of nanoscale-labeled objects in solution. Current analysis of FCS data is largely based on the assumption that the labeled objects are stochastically displaced due to Brownian motion. However, this assumption is often invalid for microscale objects, since the motion of these objects is dominated by Stokes drag and settling or rising effects, rather than stochastic Brownian motion. To utilize the power of FCS for systems with nonstochastic displacements of objects, the collection and analysis of FCS data have to be reconceptualized. Here, we extended the applicability of FCS for the detection and analysis of periodically passing objects. Toward this end, we implemented droplet-based microfluidics, in which monodispersed droplets containing fluorescent marker are flowing equally spaced within microchannels. We show by simulations and experiments that FCS can sensitively quantify the flow-rates, variability, and content of rapidly passing droplets. This information can be derived at high temporal resolution, based on the intensity fluctuations generated by only 5–10 passing droplets. Moreover, by utilizing the periodicity of the flowing droplets for noise reduction by averaging, FCS can monitor accurately the droplets flow even if their fluorescence intensity is negligible. Hence, extending FCS for periodically passing objects converts it into a powerful analytical tool for high-throughput droplet-based microfluidics. Moreover, based on the principles described here, FCS can be straightforwardly applied for a variety of systems in which the passing of objects is periodic rather than stochastic.

Fluorescence correlation spectroscopy reveals a cooperative unfolding of monomeric amyloid-\u03b2 42 with a low Gibbs free energy

The amyloid-beta peptide (Aβ) plays a major role in the progression of Alzheimer’s disease. Due to its high toxicity, the 42 amino acid long isoform Aβ42 has become of considerable interest. The Aβ42 monomer is prone to aggregation down to the nanomolar range which makes conventional structural methods such as NMR or X-ray crystallography infeasible. Conformational information, however, will be helpful to understand the different aggregation pathways reported in the literature and will allow to identify potential conditions that favour aggregation-incompetent conformations. In this study, we applied fluorescence correlation spectroscopy (FCS) to investigate the unfolding of Alexa Fluor 488 labelled monomeric Aβ42 using guanidine hydrochloride as a denaturant. We show that our Aβ42 pre-treatment and the low-nanomolar concentrations, typically used for FCS measurements, strongly favour the presence of monomers. Our results reveal that there is an unfolding/folding behaviour of monomeric Aβ42. The existence of a cooperative unfolding curve suggests the presence of structural elements with a Gibbs free energy of unfolding of about 2.8 kcal/mol.

A role of the SAM domain in EphA2 receptor activation

Among the 20 subfamilies of protein receptor tyrosine kinases (RTKs), Eph receptors are unique in possessing a sterile alpha motif (SAM domain) at their C-terminal ends. However, the functions of SAM domains in Eph receptors remain elusive. Here we report on a combined cell biology and quantitative fluorescence study to investigate the role of the SAM domain in EphA2 function. We observed elevated tyrosine autophosphorylation levels upon deletion of the EphA2 SAM domain (EphA2ΔS) in DU145 and PC3 prostate cancer cells and a skin tumor cell line derived from EphA1/A2 knockout mice. These results suggest that SAM domain deletion induced constitutive activation of EphA2 kinase activity. In order to explain these effects, we applied fluorescence correlation spectroscopy to investigate the lateral molecular organization of EphA2. Our results indicate that SAM domain deletion (EphA2ΔS-GFP) increases oligomerization compared to the full length receptor (EphA2FL-GFP). Stimulation with ephrinA1, a ligand for EphA2, induced further oligomerization and activation of EphA2FL-GFP. The SAM domain deletion mutant, EphA2ΔS-GFP, also underwent further oligomerization upon ephrinA1 stimulation, but the oligomers were larger than those observed for EphA2FL-GFP. Based on these results, we conclude that the EphA2 SAM domain inhibits kinase activity by reducing receptor oligomerization.

Applying Fluorescence Correlation Spectroscopy to Investigate Peptide-Induced Membrane Disruption.

There is considerable interest in understanding the interactions of antimicrobial peptides with phospholipid membranes. Fluorescence correlation spectroscopy (FCS) is a powerful experimental technique that can be used to gain insight into these interactions. Specifically, FCS can be used to quantify leakage of fluorescent molecules of different sizes from large unilamellar lipid vesicles, thereby providing a tool for estimating the size of peptide-induced membrane disruptions. If fluorescently labeled lipids are incorporated into the membranes of the vesicles, FCS can also be used to obtain information about whether leakage occurs due to localized membrane perturbations or global membrane destabilization. Here, we outline a detailed step-by-step protocol on how to optimally implement an FCS-based leakage assay. To make the protocol easily accessible to other researchers, it has been supplemented with a number of practical tips and tricks.

241 - Application of fluorescence correlation spectroscopy as a novel tool to quantify target occupancy in cells and tumor tissue

241 - Application of fluorescence correlation spectroscopy as a novel tool to quantify target occupancy in cells and tumor tissue

What information is contained in the fluorescence correlation spectroscopy curves, and where.

We discuss the application of fluorescence correlation spectroscopy (FCS) for characterization of anomalous diffusion of tracer particles in crowded environments. While the fact of anomaly may be detected by the standard fitting procedure, the value of the exponent α of anomalous diffusion may be not reproduced correctly for non-Gaussian anomalous diffusion processes. The important information is however contained in the asymptotic behavior of the fluorescence autocorrelation function at long and at short times. Thus, analysis of the short-time behavior gives reliable values of α and of lower moments of the distribution of particles' displacement, which allows us to confirm or reject its Gaussian nature. The method proposed was tested on the FCS data obtained in artificial crowded fluids and in living cells.

Fluorescence Correlation Spectroscopic Studies of Particle Properties for Colloidal Ceria Abrasives Used in Chemical-Mechanical Planarization

The application of fluorescence correlation spectroscopy (FCS) in the characterization of ceria abrasives used in chemical-mechanical planarization (CMP) slurries is described. A fluorescent dye, Rhodamine 110 (R110), is used for this purpose. R110 adsorption on ceria particles is found to be pH-dependent and colloidal ceria dispersions differing in mean hydrodynamic diameter are analyzed. A Langmuir isotherm is generated for R110 adsorption on ceria, and the magnitude of the Langmuir constant is found to be four-times smaller than the corresponding constant for R110 adsorption on colloidal silica CMP abrasives. R110-labeled ceria particles are generated to quantitatively determine the particle size distribution (PSD) of abrasive ceria dispersions. The Maximum Entropy Method is used reduce the FCS autocorrelation function of R110-labeled ceria particle dispersions thereby producing a PSD. These findings indicate that ceria size distributions containing particles as small as 5 to 10 nm in diameter can be readily characterized using FCS.

Fluorescence Correlation Spectroscopic Studies of Particle Properties for Colloidal Ceria Abrasives Used in Chemical-Mechanical Planarization

Fluorescence correlation spectroscopy (FCS) has been demonstrated as a versatile technique for the analysis of the smallest size fraction (diameter < 20 nm) of an abrasive silica particle dispersion and for small molecule adsorption on these particles (1-3). The technique employs the adsorption of a fluorophore at an abrasive particle’s surface, followed by single-particle detection of the dye-bound abrasive particle. Subsequent evaluation of the diffusion coefficients for all fluorescent species in the abrasive dispersion enables the quantitative determination of the fraction of adsorbed and non-adsorbed dye molecules. In the present study, the FCS method is extended to the analysis of particle size distributions of smaller-sized colloidal ceria CMP abrasives and the quantitative characterization of dye adsorption on ceria particles. This application of FCS reflects the growing commercial importance of ceria-based slurries in CMP processes, such as shallow trench isolation (STI), which rely on both mechanical and chemical mechanisms in the CMP process4. Adsorption of the high quantum yield dye, Rhodamine 110 (R110), on ceria abrasive particles is shown to render the particles fluorescent for abrasives as small as 10 nm in hydrodynamic diameter. The sizing of particles in this diameter range is often problematic for a number of commonly employed techniques, such as static laser light differential centrifugal sedimentation. The FCS approach is demonstrated to have the additional advantage of being a single-particle technique which enables sizing determinations at nanomolar concentrations of fluorescently tagged ceria particle dispersions. Because dye molecules adsorb at specific sites on the particles, the technique provides a window on the surface chemistry of abrasive ceria particles. This characterization is discussed in conjunction with the FCS analysis of competitive adsorption of R110 and a widely employed CMP slurry additive, picolinic acid, which is used in STI processes. Further enhancements of the FCS technique for the characterization abrasive ceria dispersions, such as particle size distribution determination via the method of maximum entropy (MEM-FCS) and the use of the photon counting histogram (PCH) technique for the determination of molecular brightness of dye-bound particles as a metric for quantitative adsorption analysis are discussed. References Moinpour, M.; Wayman, A.; Rawat, R.; Carver, C. T.; Remsen, E. E. Surface Adsorption of CMP Slurry Additives on Abrasive Particles. ECS Trans. 2013, 52,489-494.Jacobson, L.M.; Turner, D.K.; Rawat, A.; Carver, C.T.; Tripathi, A.; Moinpour, M.; Remsen, E.E. Application of Fluorescence Correlation Spectroscopy in the Characterization of Particle Size Distributions of Colloidal Silica Abrasives Used in Chemical-Mechanical Planarization. ECS Trans. 2014, 61, 55-63.Jacobson, L.M.; Turner, D.K.; Wayman, A.; Rawat, A.; Carver, C.T..; Moinpour, M.; Remsen, E.E. Characterization of Particle Size and Surface Adsorption for SiO2 Abrasives Used in Chemical Mechanical Planarization via Fluorescence Correlation Spectroscopy. ECS J. Solid State Sci. Tech. 2015 , 4, P5053-P5057.Carter, P. W.; Johns, T. P. Interfacial Reactivity between Ceria and Silicon Dioxide and Silicon Nitride Surfaces: Organic Additive Effects. Electrochem. Solid-State Lett. 2005, 8, G218–G221.

Structure and Dimensions of Core-Shell Nanoparticles Comparable to the Confocal Volume Studied by Means of Fluorescence Correlation Spectroscopy.

In some applications the dye distribution within fluorescently labeled nanoparticles and its stability over long periods of time are important issues. In this article we study numerically and experimentally the applicability of fluorescence correlation spectroscopy (FCS) to resolve such questions. When the size of fluorescently labeled particles is comparable to or larger than the confocal volume, the effective confocal volume seen in FCS experiments is increasing. Such an effect has already been studied for uniformly labeled spherical particles. In this work we analyze the form of the FCS correlation functions (CFs) for core-labeled and shell-labeled core-shell particles. For shell-labeled particles an additional fast decay was found both in simulations and in experiments on custom-made surface-labeled particles. Universal scaling of FCS correlation times based on the squared ratio of the labeled part radius of gyration to the Gaussian radius of the beam profile was found. Recipes based on the analysis of simulated CFs, proposed for interpretation of experimental results, were successfully applied to the FCS results on suspensions of large core-labeled and surface-labeled particles.

Application of Fluorescence Correlation Spectroscopy to Study Dynamics of Proteins Involved in Neuronal Synapse-to-Nucleus Signaling

Activity-dependent gene transcription is required for the long-term stabilization of synaptic plasticity, a process that is key to learning and memory. We are studying the transcription factor, NFAT, which has been shown to be actuated by postsynaptic signaling. Ca2+ influx through voltage-gated L-type calcium channels plays an exclusive role in initiating NFAT signaling via the action of the Ca2+/calmodulin- phosphatase calcineurin (CaN). CaN dephosphorylates multiple residues on NFAT, resulting in the exposure of its nuclear localization sequence and subsequent translocation to the nucleus. A required component of this cascade is the A-kinase anchoring protein (AKAP) 79/150, which serves to recruit CaN to the channel. New evidence from our group indicates that NFAT is also tethered to this complex via a transient association with AKAP.This scenario suggests a model wherein NFAT signaling is instigated by local synaptic activity and the actuated transcription factor is then conveyed to the nucleus over long intracellular distances via diffusion or active transport. However, the molecular mechanisms of this process have yet to be elucidated. A potentially powerful approach to investigate the dynamics of the signaling molecules in this pathway is fluorescence correlation spectroscopy (FCS). This technique yields quantitative information on protein mobility in living cells by measuring intensity fluctuations produced by fluorescent (or fluorescently tagged) molecules diffusing through a confined detection volume.Here we present FCS measurements characterizing NFAT dynamics in response to induced synaptic activity in cultured hippocampal neurons, utilizing models that account for dendritic morphology. Additionally, we have found that there is a prominent component in the autocorrelation curve due to GFP photophysics (triplet state transitions and dynamic quenching) in FCS data from these cells, which requires further modifications to the standard fitting models.

Core–shell fluorinated methacrylate nanoparticles with Rhodamine-B for confocal microscopy and fluorescence correlation spectroscopy applications

Abstract There is a need to develop nanosized and submicron sized stable and efficient fluorescent particles for confocal microscopy and fluorescence correlation spectroscopy, which are useful methods to study dynamics and interactions in complex colloidal systems and living cells. Fluorescent core–transparent shell fluorinated polymer nanoparticles exhibit low refractive index, close to that of water, high fluorescence efficiency, high stability and no cytotoxicity which makes them excellent probes for water based and living systems. Fluorescent core–transparent shell submicron particles were synthesized using 1H,1H-heptafluoro- n -butyl methacrylate (HFBMA) as a monomer and Rhodamine-B-isothiocyanate as a dye. The fluorescent polymeric core was surrounded by a non-fluorescent shell obtained by the seeded growth synthesis. The spherical shape of the particles was confirmed by scanning electron microscopy and the particle size in suspension was characterized by dynamic light scattering (DLS) and fluorescence correlation spectroscopy (FCS). The effect of different reaction parameters, such as initiator concentration and polymerization time on the particle size and charge density as well as their toxicity were studied. The smallest nanoparticles, of diameter d = 240 nm for the core measured by DLS, were obtained with higher initiator concentration. Repeated confocal imaging, DLS and FCS measurements after a few months up to two years confirmed high fluorescence efficiency, stability and usefulness of prepared particles as versatile systems for confocal microscopy and fluorescence correlation spectroscopy studies in water-based colloidal suspensions. The cytotoxicity tests confirmed a possible use of these nanoparticles also in living cells.

Single Molecule Tools for Probing Protein Aggregation

Protein aggregation poses a fundamental problem in biophysics, whose solutions have enormous potential for societal benefits. Many devastating and incurable diseases, such as Alzheimer’’s, Parkinson’’s and Type II diabetes, are strongly linked to the misfolding and aggregation of specific proteins. The links between misfolding, aggregation and toxicity, with clues spread across fields from physics and physical chemistry to clinical science and epidemiology, have remained difficult to decipher. In addition, the transience of the aggregation intermediates makes it a difficult challenge. Optical spectroscopy and microscopy, providing unparalleled sensitivity and resolution, are two of the few tools which have been able to provide some effective leads in understanding the process. Here we present a short summary of the applications of fluorescence correlation spectroscopy and total internal reflection fluorescence microscopy to this problem, primarily focusing on the progress made in our laboratory.

Characterization of Diblock Copolymer Order-Order Transitions in Semidilute Aqueous Solution Using Fluorescence Correlation Spectroscopy

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.

Monitoring the diffusion behavior of Na,K-ATPase by fluorescence correlation spectroscopy (FCS) upon fluorescence labelling with eGFP or Dreiklang

AbstractMeasurement of lateral mobility of membraneembedded proteins in living cells with high spatial and temporal precision is a challenging task of optofluidics. Biological membranes are complex structures, whose physico-chemical properties depend on the local lipid composition, cholesterol content and the presence of integral or peripheral membrane proteins, which may be involved in supramolecular complexes or are linked to cellular matrix proteins or the cytoskeleton. The high proteinto- lipid ratios in biomembranes indicate that membrane proteins are particularly subject to molecular crowding, making it difficult to follow the track of individual molecules carrying a fluorescence label. Novel switchable fluorescence proteins such as Dreiklang [1], are, in principle, promising tools to study the diffusion behavior of individual molecules in situations of molecular crowding due to excellent spectral control of the ON- and OFF-switching process. In this work, we expressed an integral membrane transport protein, the Na,K-ATPase comprising the human α2-subunit carrying an N-terminal eGFP or Dreiklang tag and human β1-subunit, in HEK293T cells and measured autocorrelation curves by fluorescence correlation spectroscopy (FCS). Furthermore,we measured diffusion times and diffusion constants of eGFP and Dreiklang by FCS, first, in aqueous solution after purification of the proteins upon expression in E. coli, and, second, upon expression as soluble proteins in the cytoplasm of HEK293T cells. Our data show that the diffusion behavior of the purified eGFP and Dreiklang in solution as well as the properties of the proteins expressed in the cytoplasm are very similar. However, the autocorrelation curves of eGFP- and Dreiklanglabeled Na,K-ATPase measured in the plasma membrane exhibit marked differences, with the Dreiklang-labeled construct showing shorter diffusion times. This may be related to an additional, as yet unrecognized quenching process that occurs on the same time scale as the diffusion of the labeled complexes through the detection volume (1– 100 ms). Since the origin of this quenching process is currently unclear, care has to be taken when the Dreiklang label is intended to be used in FCS applications.

Scanning fluorescence correlation spectroscopy on biomembranes.

Fluorescence correlation spectroscopy (FCS) is a powerful quantitative method to study dynamical properties of biophysical systems. It exploits the temporal autocorrelation of fluorescence intensity fluctuations originating from a tiny volume (~fL). A theoretical model function can be then fitted to the measured auto-correlation curve to obtain physical parameters such as local concentration and diffusion time. However, the application of FCS on membranes is coupled to several difficulties like accurate positioning and stability of the set-up. In this book chapter, we explain the theoretical framework of point FCS and Scanning FCS (SFCS), which is a variation especially suitable for membrane studies. We present a list of materials necessary for SFCS studies on Giant Unilamellar Vesicles (GUVs). Finally, we provide simple protocols for the preparation of GUVs, calibration of the microscope setup, and acquisition and analysis of SFCS data to determine diffusion coefficients and concentrations of fluorescent particles embedded in lipid membranes.

Application of FCS in studies of rhinovirus receptor binding and uncoating.

Fluorescence correlation spectroscopy (FCS) allows determining diffusion and relaxation properties of fluorescent molecules. It requires only extremely small amounts of sample, down to picomolar concentrations, in an effective analysis volume of a few femtoliters. In essence, FCS determines the autocorrelation of fluorescence fluctuations caused by single labeled molecules passing through the confocal volume of a microscope equipped with a suitable detector; it permits investigating interactions of (macro)molecules, even in single cells. We present an FCS protocol for exploring, under in vitro conditions, the dynamic processes that take place during the early steps of virus infection. We cover two important issues of rhinovirus research, the kinetics of directional RNA release, and virus-receptor interactions exemplified by using human rhinovirus type A2 (HRV-A2) as a model.