Combination Of Fluorescence Correlation Spectroscopy Research Articles

Solution-phase sample-averaged single-particle spectroscopy of quantum emitters with femtosecond resolution.

The development of many quantum optical technologies depends on the availability of single quantum emitters with near-perfect coherence. Systematic improvement is limited by a lack of understanding of the microscopic energy flow at the single-emitter level and ultrafast timescales. Here we utilize a combination of fluorescence correlation spectroscopy and ultrafast spectroscopy to capture the sample-averaged dynamics of defects with single-particle sensitivity. We employ this approach to study heterogeneous emitters in two-dimensional hexagonal boron nitride. From milliseconds to nanoseconds, the translational, shelving, rotational and antibunching features are disentangled in time, which quantifies the normalized two-photon emission quantum yield. Leveraging the femtosecond resolution of this technique, we visualize electron-phonon coupling and discover the acceleration of polaronic formation on multi-electron excitation. Corroborated with theory, this translates to the photon fidelity characterization of cascaded emission efficiency and decoherence time. Our work provides a framework for ultrafast spectroscopy in heterogeneous emitters, opening new avenues of extreme-scale characterization for quantum applications.

Photoisomerization of Heptamethine Cyanine Dyes Results in Red-Emissive Species: Implications for Near-IR, Single-Molecule, and Super-Resolution Fluorescence Spectroscopy and Imaging.

Photoisomerization kinetics of the near-infrared (NIR) fluorophore Sulfo-Cyanine7 (SCy7) was studied by a combination of fluorescence correlation spectroscopy (FCS) and transient state (TRAST) excitation modulation spectroscopy. A photoisomerized state with redshifted emission was identified, with kinetics consistent with a three-state photoisomerization model. Combining TRAST excitation modulation with spectrofluorimetry (spectral-TRAST) further confirmed an excitation-induced redshift in the emission spectrum of SCy7. We show how this red-emissive photoisomerized state contributes to the blinking kinetics in different emission bands of NIR cyanine dyes, and how it can influence single-molecule, super-resolution, as well as Förster resonance energy transfer (FRET) and multicolor readouts. Since this state can also be populated at moderate excitation intensities, it can also more broadly influence fluorescence readouts, also readouts not relying on high excitation conditions. However, this additional red-emissive state and its photodynamics, as identified and characterized in this work, can also be used as a strategy to push the emission of NIR cyanine dyes further into the NIR and to enhance photosensitization of nanoparticles with absorption spectra further into the NIR. Finally, we show that the photoisomerization kinetics of SCy7 and the formation of its redshifted photoisomer depend strongly on local environmental conditions, such as viscosity, polarity, and steric constraints, which suggests the use of SCy7 and other NIR cyanine dyes as environmental sensors. Such environmental information can be monitored by TRAST, in the NIR, with low autofluorescence and scattering conditions and on a broad range of samples and experimental conditions.

Combining fluorescence correlation spectroscopy and lifetime imaging to study the dynamics of drug delivery nanoparticles in mucus.

Fluorescence Correlation Spectroscopy (FCS) is a well-established technique to study dynamic processes like molecular diffusion. Alterations of the diffusional behavior of a given molecule, caused by molecular interactions, changes in local viscosity, etc., can be investigated by FCS. Here, we apply FCS to evaluate the stability of different nanoparticles used for drug delivery under in-vivo mimicking conditions. Nanoparticles are a powerful drug-delivery approach with enhanced permeability and precise targeting. Nevertheless, they need to overcome biological barriers such as nasal and pulmonary mucus to deliver their cargo. As mucus viscosity and adhesiveness constitute a major challenge for nanoparticle delivery, studying the stability and mobility of nanoparticles in mucus is crucial. With a fluorescently labelled cargo, FCS allows us to discriminate between properly formed nanoparticles and cargo molecules released in solution, which can be used as a readout of stability. However, we observed that mucus can induce aggregation of the released cargo hampering the possibility of addressing the nanoparticle behavior via their diffusion, as particles and cargo-aggregates are not distinguishable. Hence, we incorporated other photophysical information into the analysis. We show that, when the fluorescence lifetime of the nanoparticles and of the aggregated cargo remains different, Fluorescence Lifetime Imaging Microscopy (FLIM) and Single Particle Tracking (SPT) can be combined to tackle this issue. Our results indicate that tracking single particles/aggregates and extracting their particle-wise lifetime allows us to discriminate between properly formed nanoparticle and mere cargo-aggregates. With this method, nanoparticles movements and stability can be monitored over time independently of the aggregates. In summary, FCS, FLIM and SPT are powerful and complementary techniques and their combination makes it possible to investigate molecular behavior in complex environments.

Combined FCS and PCH Analysis to Quantify Protein Dimerization in Living Cells.

Protein dimerization plays a crucial role in the regulation of numerous biological processes. However, detecting protein dimers in a cellular environment is still a challenge. Here we present a methodology to measure the extent of dimerization of GFP-tagged proteins in living cells, using a combination of fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analysis of single-color fluorescence fluctuation data. We named this analysis method brightness and diffusion global analysis (BDGA) and adapted it for biological purposes. Using cell lysates containing different ratios of GFP and tandem-dimer GFP (diGFP), we show that the average brightness per particle is proportional to the fraction of dimer present. We further adapted this methodology for its application in living cells, and we were able to distinguish GFP, diGFP, as well as ligand-induced dimerization of FKBP12 (FK506 binding protein 12)-GFP. While other analysis methods have only sporadically been used to study dimerization in living cells and may be prone to errors, this paper provides a robust approach for the investigation of any cytosolic protein using single-color fluorescence fluctuation spectroscopy.

Quantitative analyses reveal extracellular dynamics of Wnt ligands in Xenopus embryos.

The mechanism of intercellular transport of Wnt ligands is still a matter of debate. To better understand this issue, we examined the distribution and dynamics of Wnt8 in Xenopus embryos. While Venus-tagged Wnt8 was found on the surfaces of cells close to Wnt-producing cells, we also detected its dispersal over distances of 15 cell diameters. A combination of fluorescence correlation spectroscopy and quantitative imaging suggested that only a small proportion of Wnt8 ligands diffuses freely, whereas most Wnt8 molecules are bound to cell surfaces. Fluorescence decay after photoconversion showed that Wnt8 ligands bound on cell surfaces decrease exponentially, suggesting a dynamic exchange of bound forms of Wnt ligands. Mathematical modeling based on this exchange recapitulates a graded distribution of bound, but not free, Wnt ligands. Based on these results, we propose that Wnt distribution in tissues is controlled by a dynamic exchange of its abundant bound and rare free populations.

Selective analysis of newly synthesized proteins by combining fluorescence correlation spectroscopy with bioorthogonal non-canonical amino acid tagging.

Protein expression is closely related to many biological processes including cell growth, differentiation and signaling. It is a challenge to selectively monitor newly synthesized proteins under both physiological and pathological conditions due to shortage of efficient analytical methods. Here, we proposed a new strategy to selectively monitor newly synthesized proteins in cells by combining fluorescence correlation spectroscopy (FCS) with bioorthogonal noncanonical amino acid tagging (BONCAT) technique. Firstly, homopropargylglycine (HPG), an alkyne surrogate of methionine, was metabolically incorporated into newly synthesized proteins in living cells, and the proteins containing the alkyne functional group were subsequently labeled with chemoselective fluorescence reporters using the Cu(i)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. Then, FCS was used to analyze the newly synthesized proteins based on the difference in the characteristic diffusion times of labeled proteins and free fluorescent dyes. We optimized the conditions of HPG metabolic incorporation and the CuAAC click reaction and applied this new method to study autophagic protein degradation and in situ monitor secreted proteins in cells. Compared to current methods, our method is simple, fast, and without separation, and it may become a promising approach for in situ studying protein expression in living cells.

In Situ Study of the Drug-Target Protein Interaction in Single Living Cells by Combining Fluorescence Correlation Spectroscopy with Affinity Probes.

The drug-target protein interaction is the basis of drug screening and precise therapy in modern clinical medicine. How to acquire the information about the drug-target protein interaction in single living cell is a great challenge due to the shortage of efficient methods. Here we propose a new strategy for in situ studying the drug-target protein interaction in single living cells based on the competition of candidate drugs to the fluorescent probe-target complexes and fluorescence correlation spectroscopy (FCS) with a microfluidic chip. In this study, we used ABL kinase (target) as a model and synthesized a fluorescent probe (Cy3-dasatinib) with an affinity to the target using ABL inhibitor dasatinib as a precursor. We systematically investigated the association of the probe with targets and the dissociation of the drug-target complexes in the presence of candidate drug. We presented a new parameter IC50 (τD) to assess the inhibitory effect of drugs on the basis of the changes in the characteristic diffusion time (τD) and the binding ratio (y) of fluorescent probes during the drug competition process in living cells. We found a remarkable difference of IC50 (τD) values in living cells and in solutions, suggesting it is quite necessary to evaluate the drug-target interactions in living cells. Compared with current methods, our approach can be used to in situ and real-time study the drug-target interaction in living cells, and it may become a promising and universal tool for in situ drug research at molecular level.

Experimental Dissection of Excluded Volume Effects from Quinary Interactions in Macromolecular Crowding

Current models of diffusion are, at best, approximations of what happens in vivo. Fick's Law and Stokes-Einstein approximate dilute solution behavior, but they do not account for in vivo environmental factors that modulate particle motion such as electrostatic forces, hydrophobic interactions, and excluded volume. Such factors represent the contributions from macromolecular crowding that lead to diffusional anomalies seen in living cells. In the past, separation of excluded volume effects from the quinary interactions created by weak electrostatic and hydrophobic forces has been challenging. We are using a combination of fluorescence correlation spectroscopy (FCS) and microfluidic devices to tease apart these contributions such that they may be differentially quantified. FCS allows precise measurement of single molecule fluorescence thereby permitting assessment of local mobility in the presence and absence of crowders. By modulating the crowder surface area under conditions of constant excluded volume, quinary forces may be assessed and compared to the diffusional impact of excluded volume alone. Our long-term goal is to derive a more precise mathematical model for how a particle will diffuse in an intracellular environment. ∗EJB and VJA contributed equally to this work.

From a Protein's Perspective: Elution at the Single-Molecule Level.

Column chromatography is a widely used analytical technique capable of identifying and isolating a desired chemical species from a more complicated mixture. Despite the method's prevalence, theoretical descriptions have not advanced to accommodate today's common analyte, proteins. Proteins are increasingly used as biologics, a term that refers to biological pharmaceuticals, and present new complexities for chromatographic separation. Large variations in surface charge, chemistry, and structure among protein analytes expose the limits in the current theoretical framework's ability to predict the efficiency of a column without empirical data. The bottleneck created by empirical optimization is a strong motivation for a renewed effort to achieve an in-depth understanding of the range of interactions that occur between a protein analyte and the stationary phase that together enable its selective separation from other constituents of a mixture. The physical and chemical processes that dictate the amount of time an analyte spends in the column are often abstracted by theory and treated as statistical distributions. Until recently, these distributions could not be mapped experimentally as traditional experimental techniques could not reveal underlying heterogeneity in structure, charge, and dynamics. Aligning the latest experimental and theoretical advances is thus a hurdle to be overcome so that significant progress can be made toward a predictive chromatographic theory. In this Account, we detail the work of the Landes Lab in developing single-molecule techniques that refine the stochastic theory of chromatography as a first step toward predictive chromatographic column design. We provide a brief review of the development of stochastic theory and establish a mathematical framework to put the discussed physical chemistry in context. We describe our investigations of three pertinent phenomena: mobile/stationary phase exchange, adsorption/desorption kinetics, and hindered diffusion. We highlight experimental evidence that points to nonuniform behavior. Then, we describe our work in developing single-molecule techniques that can evaluate these effects on a protein-by-protein basis. We highlight two developments: fast imaging via super temporal-resolved microscopy (STReM) and visualizing diffusion within pores via a combination of fluorescence correlation spectroscopy and super-resolution optical fluctuation imaging (fcsSOFI). Both methods offer new ways to study chromatographic elution at the single-protein level. Such methods can identify the rare heterogeneities that prevent efficient separations and advance the field closer to predictively optimized protein separations.

Quantifying spatial and temporal variations of the cell membrane ultra-structure by bimFCS.

It has been long recognized that the cell membrane is heterogeneous on scales ranging from a couple of molecules to micrometers in size and hence diffusion of receptors is length scale dependent. This heterogeneity modulates many cell-membrane-associated processes requiring transient spatiotemporal separation of components. The transient increase in local concentration of interacting signal components enables robust signaling in an otherwise thermally noisy system. Understanding how lipids and proteins self-organize and interact with the cell cortex requires quantifying the motion of the components. Multi-length scale diffusion measurements by single particle tracking, fluorescence correlation spectroscopy (FCS) or related techniques are able to identify components being transiently trapped in nanodomains, from freely moving one and from ones with reduced long-scale diffusion due to interaction with the cell cortex. One particular implementation of multi-length scale diffusion measurements is the combination of FCS with a spatially resolved detector, such as a camera and two-dimensional extended excitation profile. The main advantages of this approach are that all length scales are interrogated simultaneously, uniquely permits quantifying changes to the membrane structure caused by extrenal or internal perturbations. Here, we review how combining total internal reflection microscopy (TIRF) with FC resolves the membrane organization in living cells. We show how to implement the method, which requires only a few seconds of data acquisition to quantify membrane nanodomains, or the spacing of membrane fences caused by the actin cortex. The choice of diffusing fluorescent probe determines which membrane heterogeneity is detected. We review the instrument, sample preparation, experimental and computational requirements to perform such measurements, and discuss the potential and limitations. The discussion includes examples of spatial and temporal comparisons of the membrane structure in response to perturbations demonstrating the complex cell physiology.

Designing Ligands for Structure-Less Proteins

Disordered proteins can play physiologically important roles, but can also lead to major diseases especially when they aggregate. Designing small molecule / peptide ligands for disordered proteins is a difficult challenge, since structure cannot be used as a guide. Here we demonstrate effective approaches for three major cases: 1) for partially disordered proteins, we identify key structured portions and design ligands for disrupting those, 2) for mostly disordered proteins, we measure spontaneous fluctuations and use that as an assay to quantify transient interactions with candidate ligands, and 3) for unfolded states of well-folded proteins, we use the folding nucleus to design an artificially stabilized exo-nucleus which can induce folding. We demonstrate the first strategy with amyloid beta (Aβ), whose aggregation is associated with Alzheimer's disease. Using a combination of fluorescence correlation spectroscopy (FCS), Forster resonance Energy transfer (FRET) and solid state NMR, we show that a small peptide designed to specifically disrupt a distal intra-molecular contact can drastically alter the membrane affinity of the Aβ oligomers. We demonstrate the second strategy with IAPP (amylin), whose aggregation is associated with Type II diabetes. We show that ns-timescale spontaneous fluctuations of IAPP, measured using FRET and FCS, can provide a sensitive assay for interactions of IAPP oligomers with small molecule drug candidates. The third case is demonstrated by designing small peptides which can specifically perturb the folding kinetics of GdnHCl-unfolded ubiquitin. We construct a chemically stabilized peptide fragment that mimics the folding nucleus of ubiquitin. We show that this peptide can accelerate the folding of ubiquitin. Overall, here we demonstrate “dynamics” based methods which can help in designing drugs for disordered proteins.

Quantitative Determination of Telomerase Activity by Combining Fluorescence Correlation Spectroscopy with Telomerase Repeat Amplification Protocol.

Telomerase is a key enzyme for maintaining the telomere length and is regarded as a versatile cancer biomarker and a potential drug target due to its important role in cancer and aging. It is necessary to develop a sensitive and reliable method for detection of telomerase activity due to its very low level in cells. In this Article, we propose an ultrasensitive and robust method for quantitative determination of telomerase activity by combining single molecule fluorescence correlation spectroscopy (FCS) with telomerase repeat amplification protocol (TRAP). The principle of this new method (FCS-TRAP) is based on measurement of the change in characteristic diffusion time and molecule number of TRAP products by FCS. The characteristic diffusion time is related to the length of TRAP products, and the molecule number represents the concentration of TRAP products. We optimized the conditions of TRAP procedure and FCS measurements. We observed that the telomerase activities are positively correlated to characteristic diffusion time and molecule number of TRAP products at optimal conditions. This method was successfully used for determination of telomerase activity of different cells, and detection of a single cell was realized. Meanwhile, this method was used to evaluate the inhibition efficiency of inhibitors, and the IC50 values obtained were in good agreement with the references. Compared to current TRAP methods, this method shows reliable quantification, ultrahigh sensitivity, and short detection time and is without separation. We believe that the FCS-TRAP method has a potential application in clinical diagnosis and screening of telomerase inhibitors.

Determination of Caspase-3 Activity and Its Inhibition Constant by Combination of Fluorescence Correlation Spectroscopy with a Microwell Chip.

Caspase-3 is a key enzyme executing apoptosis during ontogenesis and homeostasis of multicellular organisms, and is a very important and potential drug target in treatment of apoptosis disturbance. So far, no commercial drugs for caspase-3 are available, and it is urgently necessitated to develop an effective method for caspase-3 activity assay and its inhibitor screening. In this paper, we propose a new method for determination of caspase-3 activity and its inhibition constant by combining single molecule fluorescence correlation spectroscopy (FCS) with a microwell chip. Its principle is based on measurement of the enzyme reaction kinetics and homogeneous detection of the reaction product by FCS. This system can reduce the requirement sample volume to 1 μL level. The caspase-3 substrates are doubly labeled with fluorophore and biotin, the enzyme reaction can be quickly terminated in the presence of streptavidin, and the reaction products can be selectively detected by FCS. We established the model of caspase-3 inhibitor screening by combining the dynamics of enzyme reaction with FCS theory. This new method was successfully used for determination of inhibition constants of certain inhibitors and assay of drug-induced apoptosis. Compared to current methods, this method shows high sensitivity, small reagent dosage and short analysis time. We believe that this method will become an efficient platform for screening of caspase-3 inhibitors and detection of apoptosis.

Size distribution of extracellular vesicles by optical correlation techniques

Understanding the colloidal properties of extracellular vesicles (EVs) is key to advance fundamental knowledge in this field and to develop effective EV-based diagnostics, therapeutics and devices. Determination of size distribution and of colloidal stability of purified EVs resuspended in buffered media is a complex and challenging issue – because of the wide range of EV diameters (from 30 to 2000nm), concentrations of interest and membrane properties, and the possible presence of co-isolated contaminants with similar size and densities, such as protein aggregates and fat globules – which is still waiting to be fully addressed. We report here a fully detailed protocol for accurate and robust determination of the size distribution and stability of EV samples which leverages a dedicated combination of Fluorescence Correlation Spectroscopy (FCS) and Dynamic Light Scattering (DLS). The theoretical background, critical experimental steps and data analysis procedures are thoroughly presented and finally illustrated through the representative case study of EV formulations obtained from culture media of B16 melanoma cells, a murine tumor cell line used as a model for human skin cancers.

α-Synuclein's Uniquely Long Amphipathic Helix Enhances its Membrane Binding and Remodeling Capacity.

α-Synuclein is the primary protein found in Lewy bodies, the protein and lipid aggregates associated with Parkinson's disease and Lewy body dementia. The protein folds into a uniquely long amphipathic α-helix (AH) when bound to a membrane, and at high enough concentrations, it induces large-scale remodeling of membranes (tubulation and vesiculation). By engineering a less hydrophobic variant of α-Synuclein, we previously showed that the energy associated with binding of α-Synuclein's AH correlates with the extent of membrane remodeling (Braun et al. in J Am Chem Soc 136:9962-9972, 2014). In this study, we combine fluorescence correlation spectroscopy, electron microscopy, and vesicle clearance assays with coarse-grained molecular dynamics simulations to test the impact of decreasing the length of the amphipathic helix on membrane binding energy and tubulation. We show that truncation of α-Synuclein's AH length by approximately 15% reduces both its membrane binding affinity (by fivefold) and membrane remodeling capacity (by nearly 50% on per mole of bound protein basis). Results from simulations correlate well with the experiments and lend support to the idea that at high protein density there is a stabilization of individual, protein-induced membrane curvature fields. The extent to which these curvature fields are stabilized, a function of binding energy, dictates the extent of tubulation. Somewhat surprisingly, we find that this stabilization does not correlate directly with the geometric distribution of the proteins on the membrane surface.

Interactions and Translational Dynamics of Phosphatidylinositol Bisphosphate (PIP2) Lipids in Asymmetric Lipid Bilayers.

Phosphatidylinositol phosphate (PIP) lipids are critical to many cell signaling pathways, in part by acting as molecular beacons that recruit peripheral membrane proteins to specific locations within the plasma membrane. Understanding the biophysics of PIP-protein interactions is critical to developing a chemically detailed model of cell communication. Resolving such interactions is challenging, even in model membrane systems, because of the difficulty in preparing PIP-containing membranes with high fluidity and integrity. Here we report on a simple, vesicle-based protocol for preparing asymmetric supported lipid bilayers in which fluorescent PIP lipid analogues are found only on the top leaflet of the supported membrane facing the bulk solution. With this asymmetric distribution of lipids between the leaflets, the fluorescent signal from the PIP lipid analogue reports directly on interactions between the peripheral molecules and the top leaflet of the membrane. Asymmetric PIP-containing bilayers are an ideal platform to investigate the interaction of PIP with peripheral membrane proteins using fluorescence-based imaging approaches. We demonstrate their usefulness here with a combined fluorescence correlation spectroscopy and single particle tracking study of the interaction between PIP2 lipids and a polycationic polymer, quaternized polyvinylpyridine (QPVP). With this approach we are able to quantify the microscopic features of the mobility coupling between PIP2 lipids and polybasic QPVP. With single particle tracking we observe individual PIP2 lipids switch from Brownian to intermittent motion as they become transiently trapped by QPVP.

Shear-Induced Unfolding and Enzymatic Cleavage of Full-Length VWF Multimers

Proteolysis of the multimeric blood coagulation protein von Willebrand Factor (VWF) by ADAMTS13 is crucial for prevention of microvascular thrombosis. ADAMTS13 cleaves VWF within the mechanosensitive A2 domain, which is believed to open under shear flow. In this study, we combine fluorescence correlation spectroscopy (FCS) and a microfluidic shear cell to monitor real-time kinetics of full-length VWF proteolysis as a function of shear stress. For comparison, we also measure the Michaelis-Menten kinetics of ADAMTS13 cleavage of wild-type VWF in the absence of shear but partially denaturing conditions. Under shear, ADAMTS13 activity on full-length VWF arises without denaturing agent as evidenced by FCS and gel-based multimer analysis. In agreement with Brownian hydrodynamics simulations, we find a sigmoidal increase of the enzymatic rate as a function of shear at a threshold shear rate γ˙1/2 = 5522/s. The same flow-rate dependence of ADAMTS13 activity we also observe in blood plasma, which is relevant to predict hemostatic dysfunction.

Tubulin Nanorings

ABSTRACTBiological systems routinely produce nanoscopic molecular structures with considerably less dispersion in size and shape than encountered in most manufactured materials. Indeed, Biological structures are frequently and essentially monodisperse. An example of this uniformity, combined with an intriguing geometry, is the nanometer-scale protein nanorings produced by interaction of the protein tubulin with certain hydrophobic tri-, tetra- and pentapeptides originally extracted as natural products from marine biosystems. Different peptides produce different sized nanorings, but we focus on those produced by binding to tubulin of the cyclic depsipeptide cryptophycin. The nanorings that form upon binding of this ligand show a sharp mass distribution indicating that the nanorings are made of 8 tubulin dimers of 100 kDa.In this submission, we demonstrate how a combination of fluorescence correlation spectroscopy, dynamic light scattering, electron microscopy, analytical ultracentrifugation, small-angle neutron scattering, and modeling is applied to reveal interactions of tubulin and cryptophycin in solution and to characterize their structures. We find that the cryptophycin-tubulin nanorings (∼25 nm diameter) are single-walled, appear rigid, are composed of 8 tubulin dimers in a single closed ring, and are stable upon dilution to nanomolar concentrations.Similar studies with a different peptide, the linear pentapeptide dolastatin 10, demonstrated that binding of this peptide to tubulin produces larger nanorings (14 tubulin dimers, ∼45 nm diameter rings), with slightly different properties. The ability to adjust the ring size with different peptides, and produce uniform nanorings with properties that differ slightly between size classes, makes the tubulin-peptide ring structures an appealing structural system.

Heterogeneity of crowded cellular fluids on the meso- and nanoscale.

Cellular fluids are complex media that are crowded with macromolecules and membrane-enclosed organelles on several length scales. Many studies have shown that crowding can significantly alter transport and reaction kinetics in biological but also in bio-mimetic fluids. Yet, experimental insights on how well bio-mimetic fluids can capture the complexity of cellular fluids are virtually missing. Therefore, we have combined fluorescence correlation spectroscopy (FCS) and fluorescence lifetime imaging microscopy (FLIM) to compare the spatial heterogeneities of biological and simple bio-mimetic crowded fluids. As a result, we find that these artificial fluids are capable of mimicking the average diffusion behavior but not the considerable heterogeneity of cellular fluids on the mesoscale (∼100 nm). On the nanoscale, not even the average properties are captured. Thus, cellular fluids feature a distinct, heterogeneous crowding state that differs from simple bio-mimetic fluids.

The Effect of a Fluorophore Photo-Physics on the Lipid Vesicle Diffusion Coefficient Studied by Fluorescence Correlation Spectroscopy.

Fluorescence Correlation Spectroscopy (FCS) is a technique, which allows determination of the diffusion coefficient and concentration of fluorescent objects suspended in the solution. The measured parameter is the fluctuation of the fluorescence signal emitted by diffusing molecules. When 100 nm DOPC vesicles labeled with various fluorescent dyes (Fluorescein-PE, NBD-PE, Atto488 DOPE or βBodipy FL) were measured, different values of diffusion coefficients have been obtained. These diffusion coefficients were different from the expected values measured using the dynamic light scattering method (DLS). The FCS was initially developed for solutions containing small fluorescent molecules therefore the observed inconsistency may result from the nature of vesicle suspension itself. The duration of the fluorescence signal may depend on the following factors: the exposure time of the labeled object to the excitation beam, the photo-physical properties (e.g., stability) of a fluorophore, the theoretical model used for the calculations of the diffusion coefficient and optical properties of the vesicle suspension. The diffusion coefficients determined for differently labeled liposomes show that its dependence on vesicle size and quantity of fluorescent probed used for labeling was significant demonstrating that the fluorescence properties of the fluorophore itself (bleaching and/or blinking) were critical factors for a correct outcome of FCS experiment. The new, based on combined FCS and DLS measurements, method for the determination of the focal volume prove itself to be useful for the evaluation of a fluorescence dye with respect to its applicability for FCS experiment.Electronic supplementary materialThe online version of this article (doi:10.1007/s10895-015-1752-5) contains supplementary material, which is available to authorized users.