Application Of Fluorescence Correlation Spectroscopy Research Articles

Addressing spatiotemporal signal variations in pair correlation function analysis

Fluorescence correlation spectroscopy (FCS) is a cornerstone technique in optical microscopy to measure, for example, the concentration and diffusivity of fluorescent emitters and biomolecules in solution. The application of FCS to complex biological systems, however, is fraught with inherent intricacies that impair the interpretation of correlation patterns. Critical among these intricacies are temporal variations beyond diffusion in the quantity, intensity, and spatial distribution of fluorescent emitters. These variations introduce distortions into correlated intensity data, thus compromising the accuracy and reproducibility of the analysis. This issue is accentuated in imaging-based approaches such as pair correlation function (pCF) analysis due to their broader regions of interest compared with point-detector-based approaches. Despite ongoing developments in FCS, attention to systems characterized by a spatiotemporal-dependent probability distribution function (ST-PDF) has been lacking. To address this knowledge gap, we developed a new analytical framework for ST-PDF systems that introduces a dual-timescale model function within the conventional pCF analysis. Our approach selectively differentiates the signals associated with rapid processes, such as particle diffusion, from signals stemming from spatiotemporal variations in the distribution of fluorescent emitters occurring at extended delay timescales. To corroborate our approach, we conducted proof-of-concept experiments on an ST-PDF system, wherein the, initially, uniform distribution of fluorescent microspheres within a microfluidic channel changes into a localized accumulation of microspheres over time. Our framework is offering a comprehensive solution for investigating various phenomena such as biomolecular binding, sedimentation, and particle accumulation.

Correlative Effects on Nanoplastic Aggregation in Model Extracellular Biofilm Substances Investigated with Fluorescence Correlation Spectroscopy.

Recent studies show that biofilm substances in contact with nanoplastics play an important role in the aggregation and sedimentation of nanoplastics. Consequences of these processes are changes in biofilm formation and stability and changes in the transport and fate of pollutants in the environment. Having a deeper understanding of the nanoplastics-biofilm interaction would help to evaluate the risks posed by uncontrolled nanoplastic pollution. These interactions are impacted by environmental changes due to climate change, such as, e.g., the acidification of surface waters. We apply fluorescence correlation spectroscopy (FCS) to investigate the pH-dependent aggregation tendency of non-functionalized polystyrene (PS) nanoparticles (NPs) due to intermolecular forces with model extracellular biofilm substances. Our biofilm model consists of bovine serum albumin (BSA), which serves as a representative for globular proteins, and the polysaccharide alginate, which is a main component in many biofilms, in solutions containing Na+ with an ionic strength being realistic for fresh-water conditions. Biomolecule concentrations ranging from 0.5 g/L up to at maximum 21 g/L are considered. We use non-functionalized PS NPs as representative for mostly negatively charged nanoplastics. BSA promotes NP aggregation through adsorption onto the NPs and BSA-mediated bridging. In BSA-alginate mixtures, the alginate hampers this interaction, most likely due to alginate-BSA complex formation. In most BSA-alginate mixtures as in alginate alone, NP aggregation is predominantly driven by weaker, pH-independent depletion forces. The stabilizing effect of alginate is only weakened at high BSA contents, when the electrostatic BSA-BSA attraction is not sufficiently screened by the alginate. This study clearly shows that it is crucial to consider correlative effects between multiple biofilm components to better understand the NP aggregation in the presence of complex biofilm substances. Single-component biofilm model systems based on comparing the total organic carbon (TOC) content of the extracellular biofilm substances, as usually considered, would have led to a misjudgment of the stability towards aggregation.

RNA Double-Helix Hybridization Measured by Fluorescence Correlation Spectroscopy.

RNA double-strand hybridization is a key player in gene expression regulation. Single-stranded RNA of up to 300 nucleotides forms Watson-Crick base pairs with complementary messenger RNA. Fluorescence-based single-molecule methods allow to study RNA-RNA interaction under physiological conditions. Here is described, how the dissociation constant of RNA double strands can be determined by applying fluorescence correlation spectroscopy.

Regulation of Ebola GP conformation and membrane binding by the chemical environment of the late endosome.

Interaction between the Ebola virus envelope glycoprotein (GP) and the endosomal membrane is an essential step during virus entry into the cell. Acidic pH and Ca2+ have been implicated in mediating the GP-membrane interaction. However, the molecular mechanism by which these environmental factors regulate the conformational changes that enable engagement of GP with the target membrane is unknown. Here, we apply fluorescence correlation spectroscopy (FCS) and single-molecule Förster resonance energy transfer (smFRET) imaging to elucidate how the acidic pH, Ca2+ and anionic phospholipids in the late endosome promote GP-membrane interaction, thereby facilitating virus entry. We find that bis(monoacylglycero)phosphate (BMP), which is specific to the late endosome, is especially critical in determining the Ca2+-dependence of the GP-membrane interaction. Molecular dynamics (MD) simulations suggested residues in GP that sense pH and induce conformational changes that make the fusion loop available for insertion into the membrane. We similarly confirm residues in the fusion loop that mediate GP's interaction with Ca2+, which likely promotes local conformational changes in the fusion loop and mediates electrostatic interactions with the anionic phospholipids. Collectively, our results provide a mechanistic understanding of how the environment of the late endosome regulates the timing and efficiency of virus entry.

Cellular Uptake of Bevacizumab in Cervical and Breast Cancer Cells Revealed by Single-Molecule Spectroscopy

Bevacizumab is a biological drug that is now extensively studied in clinics against various types of cancer. Although bevacizumab's action is preferably extracellular, there are reports suggesting its internalization into cancer cells, consequently decreasing its therapeutic potential. Here we are solving this issue by applying fluorescence correlation spectroscopy in living cells. We tracked single molecules of fluorescent bevacizumab in MDA-MB-231 and HeLa cells and proved that mobility measurements bring significant added value to standard imaging techniques. We confirmed the presence of the drug in intracellular vesicles. Additionally, we explicitly excluded the presence of a free cytosolic fraction of bevacizumab in both studied cell types. Extracellular and intracellular concentrations of the drug were measured, giving a partition coefficient on the order of 10-5, comparable with the spontaneous uptake of biologically inert nanoparticles. Our work presents how techniques and models developed for physics can answer biological questions.

Quantitative characterization of membrane-protein reversible association using FCS

Functionally meaningful reversible protein-membrane interactions mediate many biological events. Fluorescence correlation spectroscopy (FCS) is increasingly used to quantitatively study the non-reversible binding of proteins to membranes using lipid vesicles in solution. However, the lack of a complete description of the phase and statistical equilibria in the case of reversible protein-membrane partitioning has hampered the application of FCS to quantify the partition coefficient (Kx). In this work, we further extend the theory that describes membrane-protein partitioning to account for spontaneous protein-membrane dissociation and reassociation to the same or a different lipid vesicle. We derive the probability distribution of proteins on lipid vesicles for reversible binding and demonstrate that FCS is a suitable technique for accurate Kx quantification of membrane-protein reversible association. We also establish the limits to Kx determination by FCS studying the Cramer-Rao bound on the variance of the retrieved parameters. We validate the mathematical formulation against reaction-diffusion simulations to study phase and statistical equilibria and compare the Kx obtained from a computational FCS titration experiment with the experimental ground truth. Finally, we demonstrate the application of our methodology studying the association of anti-HIV broadly neutralizing antibody (10E8-3R) to the membrane.

Phase-separating pyrenoid proteins form complexes in the dilute phase

While most studies of biomolecular phase separation have focused on the condensed phase, relatively little is known about the dilute phase. Theory suggests that stable complexes form in the dilute phase of two-component phase-separating systems, impacting phase separation; however, these complexes have not been interrogated experimentally. We show that such complexes indeed exist, using an in vitro reconstitution system of a phase-separated organelle, the algal pyrenoid, consisting of purified proteins Rubisco and EPYC1. Applying fluorescence correlation spectroscopy (FCS) to measure diffusion coefficients, we found that complexes form in the dilute phase with or without condensates present. The majority of these complexes contain exactly one Rubisco molecule. Additionally, we developed a simple analytical model which recapitulates experimental findings and provides molecular insights into the dilute phase organization. Thus, our results demonstrate the existence of protein complexes in the dilute phase, which could play important roles in the stability, dynamics, and regulation of condensates.

RNA double strand hybridization measured at the single molecule level

RNA double strand hybridization is a hallmark for gene expression regulation. In this function, single stranded regulatory RNA forms Watson-Crick base pairs with complementary messenger RNA. In the presented work the dissociation constants of complementary equally sized RNA single strands were measured at the single molecule level applying fluorescence correlation spectroscopy (FCS). Dissociation constants of 3.2 nM, 1.4 nM and 1.0 nM were determined for 26 bp, 41 bp and 54 bp dsRNA, respectively. The translational diffusion coefficients of RNA, measured at infinite dilution, could be accurately predicted applying the model D = 4.58 × 10−10 N−0.39 m2s−1.

Application of fluorescence correlation spectroscopy to investigate the dynamics of a ribosome-associated trigger factor in Escherichia coli.

Co-translational protein folding is one of the central topics in molecular biology. In Escherichia coli, trigger factor (TF) is a primary chaperone that facilitates co-translational folding by directly interacting with nascent polypeptide chains on translating ribosomes. In this study, we applied fluorescence correlation spectroscopy (FCS), which can analyze the diffusion properties of fluorescent molecules by measuring the fluctuations of the fluorescent intensity, to investigate the interaction between TF and a nascent chain on translating ribosomes both in vitro and in vivo. The FCS analysis with a reconstituted cell-free translation system revealed that the interaction of fluorescently labeled TF with a nascent chain depended on the emergence of the nascent chain from the ribosome exit tunnel, and this interaction was not inhibited by excess amounts of other chaperones. Furthermore, the translation-dependent interaction between GFP-fused TFs and nascent chains was also observed in living E. coli cells. The FCS-based approach established here could be an effective method to investigate the dynamics of other ribosome-associated chaperones besides TF.

Cell cycle-dependent binding between Cyclin B1 and Cdk1 revealed by time-resolved fluorescence correlation spectroscopy.

Measuring the dynamics with which the regulatory complexes assemble and disassemble is a crucial barrier to our understanding of how the cell cycle is controlled that until now has been difficult to address. This considerable gap in our understanding is due to the difficulty of reconciling biochemical assays with single cell-based techniques, but recent advances in microscopy and gene editing techniques now enable the measurement of the kinetics of protein-protein interaction in living cells. Here, we apply fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy to study the dynamics of the cell cycle machinery, beginning with Cyclin B1 and its binding to its partner kinase Cdk1 that together form the major mitotic kinase. Although Cyclin B1 and Cdk1 are known to bind with high affinity, our results reveal that in living cells there is a pool of Cyclin B1 that is not bound to Cdk1. Furthermore, we provide evidence that the affinity of Cyclin B1 for Cdk1 increases during the cell cycle, indicating that the assembly of the complex is a regulated step. Our work lays the groundwork for studying the kinetics of protein complex assembly and disassembly during the cell cycle in living cells.

Advances in the application of fluorescence correlation spectroscopy to study detergent purified and encapsulated membrane proteins.

Fluorescence correlation spectroscopy (FCS) is a quantitative spectroscopy technique which could potentially increase throughput and sensitivity of screening for ligand, substrate and inhibitor binding to membrane proteins in solution. However, the purification of membrane proteins in their active forms is complex, as the lipid bilayer provides stability and its removal often causes the protein to become conformationally unstable. This has limited the application of biophysical techniques such as FCS to study the function of membrane proteins. The recent application of native extraction techniques such as styrene maleic acid lipid particles (SMALPs) has resolved this issue and FCS has emerged as a powerful option for studying proteins extracted in this way. This review will discuss the application of FCS to study purified membrane proteins in detergent micelles, nanodiscs and SMALPs and its potential to be used routinely in membrane protein drug discovery.

Role of C-Terminal Domain and Membrane Potential in the Mobility of Kv1.3 Channels in Immune Synapse Forming T Cells.

Voltage-gated Kv1.3 potassium channels are essential for maintaining negative membrane potential during T-cell activation. They interact with membrane-associated guanylate kinases (MAGUK-s) via their C-terminus and with TCR/CD3, leading to enrichment at the immunological synapse (IS). Molecular interactions and mobility may impact each other and the function of these proteins. We aimed to identify molecular determinants of Kv1.3 mobility, applying fluorescence correlation spectroscopy on human Jurkat T-cells expressing WT, C-terminally truncated (ΔC), and non-conducting mutants of mGFP-Kv1.3. ΔC cannot interact with MAGUK-s and is not enriched at the IS, whereas cells expressing the non-conducting mutant are depolarized. Here, we found that in standalone cells, mobility of ΔC increased relative to the WT, likely due to abrogation of interactions, whereas mobility of the non-conducting mutant decreased, similar to our previous observations on other membrane proteins in depolarized cells. At the IS formed with Raji B-cells, mobility of WT and non-conducting channels, unlike ΔC, was lower than outside the IS. The Kv1.3 variants possessing an intact C-terminus had lower mobility in standalone cells than in IS-engaged cells. This may be related to the observed segregation of F-actin into a ring-like structure at the periphery of the IS, leaving much of the cell almost void of F-actin. Upon depolarizing treatment, mobility of WT and ΔC channels decreased both in standalone and IS-engaged cells, contrary to non-conducting channels, which themselves caused depolarization. Our results support that Kv1.3 is enriched at the IS via its C-terminal region regardless of conductivity, and that depolarization decreases channel mobility.

Principles of fluorescence correlation spectroscopy applied to studies of biomolecular liquid-liquid phase separation.

Fluorescence correlation spectroscopy (FCS) investigates the temporal relationship of fluctuating fluorescence signals reflecting underlying molecular processes occurring in a solution sample or a single live cell. This review article introduces the principles of two basic and most used FCS techniques: fluorescence auto-correlation spectroscopy (FACS) and fluorescence cross-correlation spectroscopy (FCCS). Combined, FACS and FCCS techniques can quantitatively analyze multiple properties of molecule or nanoparticle samples, including molar concentration, diffusion coefficient and hydrodynamic radius, homo- or hetero-interaction, fluorescence brightness, etc. Not surprisingly, FCS techniques have long been used to investigate molecular mechanisms of biomolecular phase separation, first in the lipid bilayer and more recently in cell cytosol and nucleoplasm. The latter applications are especially exciting since a whole new class of membraneless cellular organelles have been discovered, which are proposed to be results of biomolecule liquid-liquid phase separation (LLPS). LLPS research can benefit significantly from the multifunctionality and single-molecule sensitivity of a variety of FCS techniques, particularly for live-cell studies. This review illustrates how FACS and FCCS techniques can be used to investigate multiple aspects of the molecular mechanisms of LLPS, and summarizes FCS applications to LLPS research in vivo and in vitro.

A Comprehensive Review of Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) is a powerful technique for quantification of molecular dynamics, and it has been widely applied in diverse fields, e.g., biomedicine, biophysics, and chemistry. By time-correlation of the fluorescence fluctuations induced by molecules diffusing through a focused light, FCS can quantitatively evaluate the concentration, diffusion coefficient, and interaction of the molecules in vitro or in vivo. In this review, the basic principle and implementation of FCS are introduced. Then, the advances of FCS variants are reviewed, covering dual-color FCCS, multi-focus FCS, pair correlation function (pCF), scanning FCS, focus-reduced FCS, SPIM-FCS, and inverse-FCS. Besides, the applications of FCS are demonstrated with the measurement of local concentration, hydrodynamic radius, diffusion coefficient, and the interaction of different molecules. Lastly, a discussion is given by summarizing the pros and cons of different FCS techniques, as well as the outlooks and perspectives of FCS.

The use of fluorescence correlation spectroscopy to monitor cell surface β2-adrenoceptors at low expression levels in human embryonic stem cell-derived cardiomyocytes and fibroblasts.

The importance of cell phenotype in determining the molecular mechanisms underlying β2 -adrenoceptor (β2AR) function has been noted previously when comparing responses in primary cells and recombinant model cell lines. Here, we have generated haplotype-specific SNAP-tagged β2AR human embryonic stem (ES) cell lines and applied fluorescence correlation spectroscopy (FCS) to study cell surface receptors in progenitor cells and in differentiated fibroblasts and cardiomyocytes. FCS was able to quantify SNAP-tagged β2AR number and diffusion in both ES-derived cardiomyocytes and CRISPR/Cas9 genome-edited HEK293T cells, where the expression level was too low to detect using standard confocal microscopy. These studies demonstrate the power of FCS in investigating cell surface β2ARs at the very low expression levels often seen in endogenously expressing cells. Furthermore, the use of ES cell technology in combination with FCS allowed us to demonstrate that cell surface β2ARs internalize in response to formoterol-stimulation in ES progenitor cells but not following their differentiation into ES-derived fibroblasts. This indicates that the process of agonist-induced receptor internalization is strongly influenced by cell phenotype and this may have important implications for drug treatment with long-acting β2AR agonists.

Fluorescence correlation spectroscopy to unravel the interactions between macromolecules in wine

In this work, the interaction of wine macromolecules with a bovine serum albumin (BSA) was investigated using fluorescence correlation spectroscopy (FCS). FCS offers the opportunity to study molecular and macromolecular aggregation without disturbing the wine by introducing only very small amounts of fluorescently labelled molecules to the system. It was observed that the diffusion coefficient of fluorescently labelled BSA varies with the addition of wine macromolecules, indicating changes in the protein conformation and the formation of complexes and aggregates. The addition of a wine polysaccharide rhamnogalacturonan II-enriched fraction led to aggregation, while addition of a mannoprotein-enriched fraction exhibited a protective effect on protein aggregation. Proteins strongly interacted with tannins, leading to the precipitation of protein-tannin complexes, while the presence of polysaccharides prevented this precipitation. Finally, the application of FCS was demonstrated in real wines, to investigate the problem of protein haze formation through live monitoring of heat-induced aggregation in wine.

High photon count rates improve the quality of super-resolution fluorescence fluctuation spectroscopy

Probing the diffusion of molecules has become a routine measurement across the life sciences, chemistry and physics. It provides valuable insights into reaction dynamics, oligomerisation, molecular (re-)organisation or cellular heterogeneities. Fluorescence correlation spectroscopy (FCS) is one of the widely applied techniques to determine diffusion dynamics in two and three dimensions. This technique relies on the temporal autocorrelation of intensity fluctuations but recording these fluctuations has thus far been limited by the detection electronics, which could not efficiently and accurately time-tag photons at high count rates. This has until now restricted the range of measurable dye concentrations, as well as the data quality of the FCS recordings, especially in combination with super-resolution stimulated emission depletion (STED) nanoscopy.Here, we investigate the applicability and reliability of (STED-)FCS at high photon count rates (average intensities of more than 1 MHz) using novel detection equipment, namely hybrid detectors and real-time gigahertz sampling of the photon streams implemented on a commercial microscope. By measuring the diffusion of fluorophores in solution and cytoplasm of live cells, as well as in model and cellular membranes, we show that accurate diffusion and concentration measurements are possible in these previously inaccessible high photon count regimes. Specifically, it offers much greater flexibility of experiments with biological samples with highly variable intensity, e.g. due to a wide range of expression levels of fluorescent proteins. In this context, we highlight the independence of diffusion properties of cytosolic GFP in a concentration range of approx. 0.01–1 µm. We further show that higher photon count rates also allow for much shorter acquisition times, and improved data quality. Finally, this approach also pronouncedly increases the robustness of challenging live cell STED-FCS measurements of nanoscale diffusion dynamics, which we testify by confirming a free diffusion pattern for a fluorescent lipid analogue on the apical membrane of adherent cells.

Application of fluorescence correlation spectroscopy to study substrate binding in styrene maleic acid lipid copolymer encapsulated ABCG2

ABCG2 is one of a trio of human ATP binding cassette transporters that have the ability to bind and transport a diverse array of chemical substrates out of cells. This so-called “multidrug” transport has numerous physiological consequences including effects on how drugs are absorbed into and eliminated from the body. Understanding how ABCG2 is able to interact with multiple drug substrates remains an important goal in transporter biology. Most drugs are believed to interact with ABCG2 through the hydrophobic lipid bilayer and experimental systems for ABCG2 study need to incorporate this. We have exploited styrene maleic acid to solubilise ABCG2 from HEK293T cells overexpressing the transporter, and confirmed by dynamic light scattering and fluorescence correlation spectroscopy (FCS) that this results in the extraction of SMA lipid copolymer (SMALP) particles that are uniform in size and contain a dimer of ABCG2, which is the predominant physiological state. FCS was further employed to measure the diffusion of a fluorescent ABCG2 substrate (BODIPY-prazosin) in the presence and absence of SMALP particles of purified ABCG2. Autocorrelation analysis of FCS traces enabled the mathematical separation of free BODIPY-prazosin from drug bound to ABCG2 and allowed us to show that combining SMALP extraction with FCS can be used to study specific drug: transporter interactions.

Review of the Principles and the Applications of Fluorescence Correlation Spectroscopy

Review of the Principles and the Applications of Fluorescence Correlation Spectroscopy

Determining Metal Ion Complexation Kinetics with Fluorescent Ligands by Using Fluorescence Correlation Spectroscopy.

Fluorescence correlation spectroscopy (FCS) has been extensively used to measure equilibrium binding constants (K) or association and dissociation rates in many reversible chemical reactions across chemistry and biology. For the majority of investigated reactions, the binding constant was on the order of ∼100 M-1 , with dissociation constants faster or equal to 103 s-1 , which ensured that enough association/dissociation events occur during the typical diffusion-determined transition time of molecules through the FCS detection volume. However, complexation reactions involving metal ions and chelating ligands exhibit equilibrium constants exceeding 104 M-1 . In the present paper, we explore the applicability of FCS for measuring reaction rates of such complexation reactions, and apply it to binding of iron, europium and uranyl ions to a fluorescent chelating ligand, calcein. For this purpose, we exploit the fact that the ligand fluorescence becomes strongly quenched after binding a metal ion, which results in strong intensity fluctuations that lead to a partial correlation decay in FCS. We also present measurements for the strongly radioactive ions of 241 Am3+ , where the extreme sensitivity of FCS allows us to work with sample concentrations and volumes that exhibit close to negligible radioactivity levels. A general discussion of the applicability of FCS to the investigation of metal-ligand binding reactions concludes our paper.