Fluorescence Correlation Spectroscopy Method Research Articles

Review of contemporary fluorescence correlation spectroscopy method in diverse solution studies.

Fluorescence correlation spectroscopy (FCS) is a well-known and established non-invasive method for quantification of physical parameters that preside over molecular mechanisms and dynamics. It combines maximum sensitivity and statistical confidence for the analysis of speed, size, and number of fluorescent molecules and interactions with surrounding molecules by time-averaging fluctuation analysis in a well-defined volume element. The narrow compass of this study is to acquaint the basic principle of diffusion and the FCS method in general regarding variable magnitudes and standardization adjustment. In this review, we give a theoretical introduction, examples of experimental applications, and utensils in solution systems with future perspectives.

Objective scanning-based fluorescence cross-correlation spectroscopy (Scan-FCCS) for studying the fusion dynamics of protein phase separation.

Protein phase separation plays a very important role in many biological processes and is closely related to the occurrence and development of some serious diseases. So far, the fluorescence imaging method and fluorescence correlation spectroscopy (FCS) have been frequently used to study the phase separation behavior of proteins. Due to the wide size distribution of protein condensates in phase separation from nano-scale to micro-scale in solution and living cells, it is difficult for the fluorescence imaging method and conventional FCS to fully reflect the real state of protein phase separation in the solution due to the low spatio-temporal resolution of the conventional fluorescence imaging method and the limited detection area of FCS. Here, we proposed a novel method for studying the protein phase separation process by objective scanning-based fluorescence cross-correlation spectroscopy (Scan-FCCS). In this study, CRDBP proteins were used as a model and respectively fused with fluorescent proteins (EGFP and mCherry). We first compared conventional FCS and Scan-FCS methods for characterizing the CRDBP protein phase separation behaviors and found that the reproducibility of Scan-FCS is significantly improved by the scanning mode. We studied the self-fusion process of mCherry-CRDBP and EGFP-CRDBP and observed that the phase change concentration of CRDBP was 25 nM and the fusion of mCherry-CRDBP and EGFP-CRDBP at 500 nM was completed within 70 min. We studied the effects of salt concentration and molecular crowding agents on the phase separation of CRDBP and found that salt can prevent the self-fusion of CRDBP and molecular crowding agents can improve the self-fusion of CRDBP. Furthermore, we found the recruitment behavior of CRDBP to β-catenin proteins and studied their recruitment dynamics. Compared to conventional FCS, Scan-FCCS can significantly improve the reproducibility of measurements due to the dramatic increase of detection zone, and more importantly, this method can provide information about self-fusion and recruitment dynamics in protein phase separation.

CRDBP Protein Phase Separation and Its Recruitment to β-Catenin Protein in a Single Living Cell.

The Coding Region Determinant-Binding Protein (CRDBP) is a carcinoembryonic protein, and it is overexpressed in various cancer cells in the form of granules. We speculated the formation of CRDBP granules possibly through liquid-liquid phase separation (LLPS) processes due to the existence of intrinsically disordered regions (IDRs) in CRDBP. So far, we did not know whether or how phase separation processes of CRDBP occur in single living cells due to the lack of in vivo methods for studying intracellular protein phase separation. Therefore, to develop an in situ method for studying protein phase separation in living cells is a very urgent task. In this work, we proposed an efficient method for studying phase separation behavior of CRDBP in a single living cell by combining in situ fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) with a fluorescence protein fusion technique. We first predicted and confirmed that CRDBP has phase separation in solution by conventional fluorescence imaging and FCS methods. And then, we in situ studied the phase separation behaviors of CRDBP in living cells and observed three states of CRDBP phase separation such as monomer state, cluster state, and granule state. We studied the effects of CRDBP truncated forms and its inhibitor on the CRDBP phase separation. Furthermore, we discovered the recruitment of CRDBP to β-catenin protein in living cells and investigated the effects of CRDBP structures and inhibitor on CRDBP recruitment behavior. This finding may help us to further understand the mechanism of CRDBP protein for regulating Wnt signaling pathway. Additionally, our results documented that FCS/FCCS is an efficient and alternative method for studying protein phase separation in situ in living cells.

Combined SPT and FCS methods reveal a mechanism of RNAP II oversampling in cell nuclei

Gene expression orchestration is a key question in fundamental and applied research. Different models for transcription regulation were proposed, yet the dynamic regulation of RNA polymerase II (RNAP II) activity remains a matter of debate. To improve our knowledge of this topic, we investigated RNAP II motility in eukaryotic cells by combining single particle tracking (SPT) and fluorescence correlation spectroscopy (FCS) techniques, to take advantage of their different sensitivities in order to analyze together slow and fast molecular movements. Thanks to calibrated samples, we developed a benchmark for quantitative analysis of molecular dynamics, to eliminate the main potential instrumental biases. We applied this workflow to study the diffusion of RPB1, the catalytic subunit of RNAP II. By a cross-analysis of FCS and SPT, we could highlight different RPB1 motility states and identifyed a stationary state, a slow diffusion state, and two different modes of subdiffusion. Interestingly, our analysis also unveiled the oversampling by RPB1 of nuclear subdomains. Based on these data, we propose a novel model of spatio-temporal transcription regulation. Altogether, our results highlight the importance of combining microscopy approaches at different time scales to get a full insight into the real complexity of molecular kinetics in cells.

Investigation of Molecular Diffusion at Block Copolymer Thin Films Using Maximum Entropy Method-Based Fluorescence Correlation Spectroscopy and Single Molecule Tracking.

Fluorescence correlation spectroscopy (FCS) has been widely used to investigate molecular diffusion behavior in various samples. The use of the maximum entropy method (MEM) for FCS data analysis provides a unique means to determine multiple distinct diffusion coefficients without a priori assumption of their number. Comparison of the MEM-based FCS method (MEM-FCS) with another method will reveal its utility and advantage as an analytical tool to investigate diffusion dynamics. Herein, we measured diffusion of fluorescent probes doped into nanostructured thin films using MEM-FCS, and validated the results with single molecule tracking (SMT) data. The efficacy of the MEM code employed was first demonstrated by analyzing simulated FCS data for systems incorporating one and two diffusion modes with broadly distributed diffusion coefficients. The MEM analysis accurately afforded the number of distinct diffusion modes and their mean diffusion coefficients. These results contrasted with those obtained by fitting the simulated data to conventional two-component and anomalous diffusion models, which yielded inaccurate estimates of the diffusion coefficients. Subsequently, the MEM analysis was applied to FCS data acquired from hydrophilic dye molecules incorporated into microphase-separated polystyrene-block-poly(ethylene oxide) (PS-b-PEO) thin films characterized under a water-saturated N2atmosphere. The MEM analysis revealed distinct fast and slow diffusion components attributable to molecules diffusing on the film surface and inside the film, respectively. SMT studies of the same materials yielded trajectories for mobile molecules that appear to follow the curved PEO microdomains. Diffusion coefficients obtained from the SMT data were consistent with those obtained for the slow diffusion component detected by MEM-FCS. These results highlight the utility of MEM-FCS and SMT for gaining complementary information on molecular diffusion processes in heterogeneous material systems.

Size and Fluorescence Properties of Algal Photosynthetic Antenna Proteins Estimated by Microscopy.

Antenna proteins play a major role in the regulation of light-harvesting in photosynthesis. However, less is known about a possible link between their sizes (oligomerization state) and fluorescence intensity (number of photons emitted). Here, we used a microscopy-based method, Fluorescence Correlation Spectroscopy (FCS), to analyze different antenna proteins at the particle level. The direct comparison indicated that Chromera Light Harvesting (CLH) antenna particles (isolated from Chromera velia) behaved as the monomeric Light Harvesting Complex II (LHCII) (from higher plants), in terms of their radius (based on the diffusion time) and fluorescence yields. FCS data thus indicated a monomeric oligomerization state of algal CLH antenna (at our experimental conditions) that was later confirmed also by biochemical experiments. Additionally, our data provide a proof of concept that the FCS method is well suited to measure proteins sizes (oligomerization state) and fluorescence intensities (photon counts) of antenna proteins per single particle (monomers and oligomers). We proved that antenna monomers (CLH and LHCIIm) are more “quenched” than the corresponding trimers. The FCS measurement thus represents a useful experimental approach that allows studying the role of antenna oligomerization in the mechanism of photoprotection.

Nanodrug Transmembrane Transport Research Based on Fluorescence Correlation Spectroscopy.

Although conventional fluorescence intensity imaging can be used to qualitatively study the drug toxicity of nanodrug carrier systems at the single-cell level, it has limitations for studying nanodrug transport across membranes. Fluorescence correlation spectroscopy (FCS) can provide quantitative information on nanodrug concentration and diffusion in a small area of the cell membrane; thus, it is an ideal tool for studying drug transport across the membrane. In this paper, the FCS method was used to measure the diffusion coefficients and concentrations of carbon dots (CDs), doxorubicin (DOX) and CDs-DOX composites in living cells (COS7 and U2OS) for the first time. The drug concentration and diffusion coefficient in living cells determined by FCS measurements indicated that the CDs-DOX composite distinctively improved the transmembrane efficiency and rate of drug molecules, in accordance with the conclusions drawn from the fluorescence imaging results. Furthermore, the effects of pH values and ATP concentrations on drug transport across the membrane were also studied. Compared with free DOX under acidic conditions, the CDs-DOX complex has higher cellular uptake and better transmembrane efficacy in U2OS cells. Additionally, high concentrations of ATP will cause negative changes in cell membrane permeability, which will hinder the transmembrane transport of CDs and DOX and delay the rapid diffusion of CDs-DOX. The results of this study show that the FCS method can be utilized as a powerful tool for studying the expansion and transport of nanodrugs in living cells, and might provide a new drug exploitation strategy for cancer treatment in vivo.

The Mean Single Molecule Rate (mSMR) in the Analysis of Fluorescence Fluctuations: Measurements on DNA Mixtures of Defined Composition

We present a method for the evaluation of fluorescence fluctuations on the basis of Mandel’s Q parameter, using sampling time-dependent factorial cumulants. By relating the Q parameter to the sampling time, we obtain the mean single molecule rate (mSMR), an easy to interpret expression that provides both brightness and diffusion information. The model is suitable for the widely used confocal setups with single photon excitation and a single detection channel. We present a way to correct the mSMR for afterpulsing, dead time and background noise. To account for photokinetic effects at short sampling times, we expand the model by a simple on/off isomerization term, which is similar to the well-known triplet model. The functionality of the mSMR is shown using Monte Carlo simulations. The correction mechanisms and the experimental applicability of the model are then demonstrated by DNA measurements of defined composition. By systematically analyzing DNA mixtures, we can show that at large sampling times, the mSMR correctly describes the single molecule brightness rates and the diffusive properties of DNA molecules. At short sampling times, the photokinetic effects of isomerization are accurately described by the mSMR model. Since additionally the mSMR can easily be corrected for measurement artefacts such as detector dead time, afterpulsing and background noise, this is a valuable advantage over the standard method of fluorescence correlation spectroscopy.

In situ determination of secretory kinase Fam20C from living cells using fluorescence correlation spectroscopy

Secretory proteins constitute a biologically crucial subset of proteins for regulation of some pathological and physiological processes, and they have become very important biomarkers in clinical diagnosis and therapeutic targets. So far, secretory protein functions and mechanisms have not been fully understood due to methodological limitations in detection of low-abundance proteins against medium background. Here, we propose a strategy to determine secretory protein from living cells in situ using fluorescence correlation spectroscopy (FCS). In this study, the recombinant protein Fam20C with SNAP-tag was used as a model protein, and O6-benzylguanine (BG) derivatives bearing fluorescent dye as probes. We synthesized three fluorescent probes and investigated their fluorescent properties and diffusion behaviors in solution, and found the probe BG-Bodipy-561 more suitable for in situ labeling of Fam20C. We confirmed the specific binding of the probe to the target protein by combining FCS and in-gel fluorescence scanning methods. We studied the effects of some factors of the secretory Fam20C, and found that RNA interference significantly inhibited the synthesis of secretory fused Fam20C, and myriocin had no significant effect on the expression of secretory Fam20C, which indirectly illustrated that sphingolipid signaling can regulate the Fam20C activity. We believe that FCS is a very promising method to analyze secretory proteins from living cells in situ.

Fast Diffusion of the Unassembled PetC1-GFP Protein in the Cyanobacterial Thylakoid Membrane.

Biological membranes were originally described as a fluid mosaic with uniform distribution of proteins and lipids. Later, heterogeneous membrane areas were found in many membrane systems including cyanobacterial thylakoids. In fact, cyanobacterial pigment–protein complexes (photosystems, phycobilisomes) form a heterogeneous mosaic of thylakoid membrane microdomains (MDs) restricting protein mobility. The trafficking of membrane proteins is one of the key factors for long-term survival under stress conditions, for instance during exposure to photoinhibitory light conditions. However, the mobility of unbound ‘free’ proteins in thylakoid membrane is poorly characterized. In this work, we assessed the maximal diffusional ability of a small, unbound thylakoid membrane protein by semi-single molecule FCS (fluorescence correlation spectroscopy) method in the cyanobacterium Synechocystis sp. PCC6803. We utilized a GFP-tagged variant of the cytochrome b6f subunit PetC1 (PetC1-GFP), which was not assembled in the b6f complex due to the presence of the tag. Subsequent FCS measurements have identified a very fast diffusion of the PetC1-GFP protein in the thylakoid membrane (D = 0.14 − 2.95 µm2s−1). This means that the mobility of PetC1-GFP was comparable with that of free lipids and was 50–500 times higher in comparison to the mobility of proteins (e.g., IsiA, LHCII—light-harvesting complexes of PSII) naturally associated with larger thylakoid membrane complexes like photosystems. Our results thus demonstrate the ability of free thylakoid-membrane proteins to move very fast, revealing the crucial role of protein–protein interactions in the mobility restrictions for large thylakoid protein complexes.

Direct Single Molecule Imaging of Enhanced Enzyme Diffusion.

Recent experimental results have shown that enzymes can diffuse faster when they are in the presence of their reactants (substrate). This faster diffusion has been termed enhanced diffusion. Fluorescence correlation spectroscopy (FCS), which has been employed as the only method to make these measurements, relies on analyzing the fluctuations in fluorescence intensity to measure the diffusion coefficient of particles. Recently, artifacts in FCS measurements due to its sensitivity to environmental conditions have been evaluated, calling prior enhanced diffusion results into question. It behooves us to adopt complementary and direct methods to measure the mobility of enzymes. Herein, we use a technique of direct single molecule imaging to observe the diffusion of individual enzymes in solution. This technique is less sensitive to intensity fluctuations and deduces the diffusion coefficient directly based on the trajectory of the enzyme. Our measurements recapitulate that enzyme diffusion is enhanced in the presence of its substrate and find that the relative increase in diffusion of a single enzyme is even higher than those previously reported using FCS. We also use this complementary method to test if the total enzyme concentration affects the relative increase in diffusion and if the enzyme oligomerization state changes during its catalytic turnover. We find that the diffusion increase is independent of the total concentration of enzymes and the presence of substrate does not change the oligomerization state of enzymes.

Measuring Mobility in Chromatin by Intensity-Sorted FCS

The architectural organization of chromatin can play an important role in genome regulation by affecting the mobility of molecules within its surroundings via binding interactions and molecular crowding. The diffusion of molecules at specific locations in the nucleus can be studied by fluorescence correlation spectroscopy (FCS), a well-established technique based on the analysis of fluorescence intensity fluctuations detected in a confocal observation volume. However, detecting subtle variations of mobility between different chromatin regions remains challenging with currently available FCS methods. Here, we introduce a method that samples multiple positions by slowly scanning the FCS observation volume across the nucleus. Analyzing the data in short time segments, we preserve the high temporal resolution of single-point FCS while probing different nuclear regions in the same cell. Using the intensity level of the probe (or a DNA marker) as a reference, we efficiently sort the FCS segments into different populations and obtain average correlation functions that are associated to different chromatin regions. This sorting and averaging strategy renders the method statistically robust while preserving the observation of intranuclear variations of mobility. Using this approach, we quantified diffusion of monomeric GFP in high versus low chromatin density regions. We found that GFP mobility was reduced in heterochromatin, especially within perinucleolar heterochromatin. Moreover, we found that modulation of chromatin compaction by ATP depletion, or treatment with solutions of different osmolarity, differentially affected the ratio of diffusion in both regions. Then, we used the approach to probe the mobility of estrogen receptor-α in the vicinity of an integrated multicopy prolactin gene array. Finally, we discussed the coupling of this method with stimulated emission depletion FCS for performing FCS at subdiffraction spatial scales.

Absolute Quantification of RNA Molecules Using Fluorescence Correlation Spectroscopy with Certified Reference Materials.

The accuracy and precision of quantification values of biomolecules, such as nucleic acids, are critical for the reliability of biomedical research and clinical examinations. To obtain an accurate quantitative value, it is necessary to use a measurement standard that has the same sequence and length as the target gene. The absence of an appropriate measurement standard leads to uncertain results. The development of a wide variety of different kinds of measurement standards, which have different sequences and lengths, is time-consuming and troublesome. We employed fluorescence correlation spectroscopy (FCS), which can be used to count the molecular number (absolute concentration) regardless of the molecular size and shape, without a standard curve. The confocal volume (i.e., the volume of excitation laser focus) of the FCS system was calibrated by measuring the primary standard of the fluorescent material. Furthermore, we investigated how to avoid artifacts originating from systematic aberrations or sample conditions. We validated the RNA concentration obtained from our FCS measurements using another primary standard RNA solution as a sample. Here, we describe an FCS calibration procedure with fluorescein solution standard reference material (SRM) 1932 as a primary standard and cross-validation of FCS values using RNA solutions certified reference material (CRM) 6204-a. The established method was applied to determine the concentrations of RNA samples that can be used as a laboratory working standards. The FCS method with a characterized SRM and CRM should serve as a universal method for absolute quantification of the number of biomolecules.

Distribution of Diffusion Times Determined by Fluorescence (Lifetime) Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) provides most commonly values of diffusion coefficients of fluorescently labeled species in a solution, especially biopolymers. A newly developed procedure for the determination of diffusion coefficient distributions applicable to polydisperse polymers or nanoparticles, based on the well-known CONTIN algorithm, is described and tested on both simulated FCS correlation functions and real experimental data. Good resolution of bimodal distributions is observed, and it is quantitatively established how the resolution depends on the level of experimental noise. Effects of incorrect calibration of the focal volume on the obtained diffusion coefficient and its distribution are described for single-focus FCS. With rapid expansion of the FCS method from biology and biochemistry into the polymer field, and recently into many other disciplines including, e.g., environmental sciences, such a technique was very much needed.

A study of the diffusion dynamics and concentration distribution of gold nanospheres (GNSs) without fluorescent labeling inside live cells using fluorescence single particle spectroscopy.

Colloidal gold nanospheres (GNSs) have become important nanomaterials in biomedical applications due to their special optical properties, good chemical stability, and biocompatibility. However, measuring the diffusion coefficients or concentration distribution of GNSs within live cells accurately without any extra fluorescent labeling in situ has still not been resolved. In this work, a single particle method is developed to study the concentration distribution of folic acid-modified GNSs (FA-GNSs) internalized via folate receptors, and investigates their diffusion dynamics within live cells using single particle fluorescence correlation spectroscopy (FCS). We optimized the experimental conditions and verified the feasibility of 30 nm GNSs without extra fluorescence labeling being used for single particle detection inside live cells. Then, the FCS characterization strategy was used to measure the concentration and diffusion coefficient distributions of GNSs inside live cells and the obtained results were basically in agreement with those obtained by TEM. The results demonstrate that our strategy is characterized as an in situ, nondestructive, rapid and dynamic method for the assay of live cells, and it may be widely used in the further design of GNP-based drug delivery and therapeutics.

Mapping the dynamical organization of the cell nucleus through fluorescence correlation spectroscopy.

The hierarchical organization of the cell nucleus into specialized open reservoirs and the nucleoplasm overcrowding impose restrictions to the mobility of biomolecules and their interactions with nuclear targets. These properties determine that many nuclear functions such as transcription, replication, splicing or DNA repair are regulated by complex, dynamical processes that do not follow simple rules. Advanced fluorescence microscopy tools and, in particular, fluorescence correlation spectroscopy (FCS) provide complementary and exquisite information on the dynamics of fluorescent labeled molecules moving through the nuclear space and are helping us to comprehend the complexity of the nuclear structure. Here, we describe how FCS methods can be applied to reveal the dynamical organization of the nucleus in live cells. Specifically, we provide instructions for the preparation of cellular samples with fluorescent tagged proteins and detail how FCS can be easily instrumented in commercial confocal microscopes. In addition, we describe general rules to set the parameters for one and two-color experiments and the required controls for these experiments. Finally, we review the statistical analysis of the FCS data and summarize the use of numerical simulations as a complementary approach that helps us to understand the complex matrix of molecular interactions network within the nucleus.

A study of the dynamics of PTEN proteins in living cells using in vivo fluorescence correlation spectroscopy

PTEN (phosphatase and tensin homolog on chromosome 10) is one of the most important tumor-suppressor proteins, which plays a key role in negative regulation of the PI3K/AKT pathway, and governs many cellular processes including growth, proliferation, survival and migration. The dynamics of PTEN proteins in single living cells is as yet unclear owing to a shortage of suitable in vivo approaches. Here, we report a single-molecule method for in vivo study of the dynamics of PTEN proteins in living cells using fluorescence correlation spectroscopy (FCS). First, we established a monoclonal H1299 stable cell line expressing enhanced green fluorescent protein (EGFP) and PTEN (EGFP-PTEN) fusion proteins; we then developed an in vivo FCS method to study the dynamics of EGFP-PTEN both in the nucleus and the cytoplasm. We investigated the diffusion behaviors of EGFP and EGFP-PTEN in solution, nucleus and cytosol, and observed that the motion of PTEN in living cells was restricted compared with EGFP. Finally, we investigated the protein dynamics in living cells under oxidative stress stimulation and a cellular ATP depletion treatment. Under oxidative stress stimulation, the EGFP-PTEN concentration increased in the nucleus, but slightly decreased in the cytoplasm. The diffusion coefficient and alpha value of EGFP-PTEN reduced significantly both in the nucleus and cytoplasm; the significantly decreased alpha parameter indicates a more restricted Brownian diffusion behavior. Under the cellular ATP depletion treatment, the concentration of EGFP-PTEN remained unchanged in the nucleus and decreased significantly in cytosol. The diffusion coefficient of EGFP-PTEN decreased significantly in cytosol, but showed no significant change in the nucleus; the alpha value decreased significantly in both the nucleus and cytoplasm. These results suggest that the concentration and mobility of PTEN in the nucleus and cytoplasm can be regulated by stimulation methods. Our approach provides a unique method for real-time monitoring of protein dynamics in different subcellular compartments under different stimulation treatments.

In Situ Characterization of Protein Adsorption onto Nanoparticles by Fluorescence Correlation Spectroscopy.

Nanotechnology holds great promise for applications in many fields including biology and medicine. Unfortunately, the processes occurring at the interface between nanomaterials and living systems are exceedingly complex and not yet well understood, which has significantly hampered the realization of many nanobiotechnology applications. Whenever nanoparticles (NPs) are incorporated by a living organism, a protein adsorption layer, also known as the "protein corona", forms on the NP surface. Accordingly, living organisms interact with protein-coated rather than bare NPs, and their biological responses depend on the nature of the protein corona. In recent years, a wide variety of biophysical techniques have been employed to elucidate mechanistic aspects of NP-protein interactions. In most studies, NPs are immersed in protein or biofluid (e.g., blood serum) solutions and then separated from the liquid for analysis. Because this approach may modify the composition and structure of the protein corona, our group has pioneered the use of fluorescence correlation spectroscopy (FCS) as an in situ technique, capable of examining NP-protein interactions while the NPs are suspended in biological fluids. FCS allows us to measure, with subnanometer precision and as a function of protein concentration, the increase in hydrodynamic radius of the NPs due to protein adsorption. This Account aims at reviewing recent progress in the exploration of NP-protein interactions by using FCS. In vitro FCS studies of the adsorption of important serum proteins onto water-solubilized luminescent NPs always showed a stepwise increase of the NP radius upon protein binding in the form of a binding isotherm, regardless of the type of NP and its specific surface functionalization. This observation indicates formation of a protein monolayer on the NP. Structure-based calculations of protein surface potentials revealed that positively charged patches on the proteins interact electrostatically with negatively charged NP surfaces, and the observed protein layer thickness always matched the known molecular dimensions of the proteins binding in certain orientations. Temperature and NP surface functionalization have also been identified as important parameters controlling protein corona formation. Notably, while the corona formed from a single type of serum protein was reversible, protein adsorption from complex biological media such as blood serum was entirely irreversible. These quantitative in vitro studies are of great relevance to the bio-nano community and especially to researchers developing engineered nanomaterials for biological and biomedical applications. Future efforts will be directed toward elucidating kinetic aspects of protein corona formation and the detailed structure of the adsorbed proteins at the molecular level. To better appreciate the biological responses triggered by NP exposure, more efforts will be devoted to the exploration of the biomolecular corona as it forms on NPs in contact with living cells, tissues, and even entire model organisms. These studies are challenging when performed in a well-controlled and quantitative fashion and rely on the availability of sophisticated analytical tools, particularly, quantitative optical imaging techniques including FCS and related fluctuation methods.

Synaptic Protein Dynamics Measured by Fluorescence Correlation Spectroscopy

The activity dependent adjustment of synaptic strength (synaptic plasticity) involves the reorganization of post-synaptic proteins. The fast diffusion of synaptic proteins has been shown to play an important role in such molecular rearrangements, but the dynamic events that lead to the formation of molecular complexes are still poorly understood. Taking advantages of Single Particle Tracking (SPT) and FRAP approaches, our group has recently demonstrated that during inhibitory long term potentiation stimuli (iLTP) Gephyrin and GABAA receptors are accumulated at post-synaptic inhibitory sites. In addition we have shown that the synaptic clustering of the scaffold protein gephyrin do not precede the synaptic inclusion of GABA receptors. However, the coordinated dynamics of gephyrin and GABAA receptors outside and inside the synapse remain to be elucidated. To investigate the diffusion dynamics of the main synaptic proteins we exploited here Fluorescence Correlation Spectroscopy (FCS), an established technique to study the mobility of molecules and to measure the protein diffusion parameters with high statistics in unperturbed live cell, in both the plasma membrane and the cytoplasmic space. We calibrated an FCS method (FCS) to study the coordinated multilevel organization of receptors and adhesion molecules that laterally diffuse on the plasma membrane and the scaffold proteins that diffuse in the cytoplasmic space. Our data suggest that, the scaffold protein freely diffuse in the extrasynaptic space and is suddenly immobilized at both synaptic site and extrasynaptic clusters. This approach may clarify the coordinated diffusion of membrane synaptic proteins and cytoplasmic scaffold proteins thus clarifying the mechanisms of synaptic clustering in basal conditions and during synaptic plasticity.

Quantitative deconvolution of autocorrelations and cross correlations from two-dimensional lifetime decay maps in fluorescence lifetime correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) is a widely used method for measuring molecular diffusion and chemical kinetics. However, when a mixture of fluorescent species is taken into account, the conventional FCS method has limitations in extracting autocorrelations for different species and cross correlations between different species. Recently developed fluorescence lifetime correlation spectroscopy (FLCS) based on time-tagged time-resolved (TTTR) photon recording, which can record the global and micro arrival time for each individual photon, has been used to discriminate different species according to fluorescence lifetime. Here, based on two-dimensional lifetime decay maps constructed from TTTR photon stream, we have developed a quantitative lifetime-deconvolution FCS model (LDFCS) to extract precise chemical rates for chemical conversions in multi-species systems. The key point of LDFCS model is separation of different species according to the global distribution of fluorescence lifetime and then deconvolution of autocorrelations and cross-correlations from the two-dimensional lifetime decay maps constructed by the micro arrival times of photon pairs at each delay time.