Fluorescence Correlation Spectroscopy

Hitherto accredited prion tests use the PK resistance of PrP Sc , the pathogenic isoform of the prion protein, as a marker for the disease. Because of variations in the amount of disease-related aggregated PrP, which is not PK-resistant, these prion tests offer only limited sensitivity. Therefore, a prion detection method that does not rely on PK digestion would allow for the detection of both PK-resistant as well as PK-sensitive PrP Sc . Furthermore, single particle counting is more sensitive than methods measuring an integrated signal. Our new test system is based on dual-colour fluorescence correlation spectroscopy (FCS). This method quantifies the number of protein aggregates that have been simultaneously labelled with two different antibodies using dual-colour fluorescence intensity distribution analysis (2D-FIDA). This only counts PrP aggregates, and not PrP monomers. To increase the sensitivity, PrP Sc was concentrated in a two-dimensional space by immobilizing it so that the antibodies could be captured on the surface of the slide (surface-FIDA). When the surface was systematically scanned, even single prion particles were detected. Using this new technique, the sensitivity to identify samples from scrapie-infected hamster as well as BSE-infected cattle can be dramatically increased in comparison with identification using FIDA in solution.

Fluorescence (auto)correlation spectroscopy (FCS) has developed into a widely used method for investigating molecular dynamics and mobility of molecules in vitro and in vivo. Dual-color cross-correlation, an extension of this technique, also assesses the concomitant movement of two spectrally distinguishable fluorescent molecules and has therefore proven superior to autocorrelation analysis to study interactions between different molecular species in solution. Here we explore the benefits of cross-correlation analysis when applied to live cells, by demonstrating its potential in analyzing endocytic processes. Bacterial cholera toxin (CTX) was labeled with Cy2 and Cy5 dyes on different subunits of the same holotoxin. Along the endocytic pathway, positive cross-correlation between the A and B subunits was first preserved, later followed by a loss in cross-correlation upon their separation in the Golgi. Furthermore, endocytosis of a mixture of only Cy2-and only Cy5-labeled holotoxins also gave rise to cross-correlation. Our results suggest that cross-correlation may be used to recognize whether different cargoes use the same endocytic pathway. Additionally, we show that cross-correlation is applicable to two-dimensional membrane diffusion. CTX bound to GM1-containing artificial giant unilamellar vesicles was diffusible, whereas CTX bound to the plasma membrane was immobile on the FCS time-scale, possibly because of raft-association of GM1.

Poly(vinyl acetate-co-ethylene) latex dispersions are prepared and their films investigated with a focus on the effect of composition upon redispersion. Films of dispersions containing sufficient amounts of poly(vinyl alcohol) (PVA) can be redispersed in water. This property is lost in the presence of surfactant, a fact which suggests a procedure to control film formation. It is demonstrated that redispersion is due to a PVA-membrane which separates the particles. Loss of redispersibility in the presence of surfactant proceeds with the breakup of the membranes and a corresponding change of film properties. Experimental data is provided by light microscopy, mechanical testing, and TEM in conjunction with a staining method new to the field. The hypothesis is developed that interaction with surfactant leads to imperfect PVA-membranes that are no longer able to prevent latex polymer interdiffusion. Fluorescence correlation spectroscopy demonstrates the formation of surfactant micelles, as well as the simultaneous adsorption and aggregation of PVA onto the micelles. It is concluded that the competing surface of the surfactant micelles traps enough PVA to cause thinning and fragmentation of the membranes surrounding the particles, which enables interdiffusion of latex polymer. This effect can be used to convert the system from one forming a redispersible coating to one forming a nonredispersible (permanent) film.

Fluorescence correlation spectroscopy (FCS) can provide information about diffusion coefficients and rate constants of chemical reactions in small systems of interacting molecules. However, the interpretation of FCS experiments depends crucially on the model of the autocorrelation function for the fluorescence intensity fluctuations. In this theoretical work, we consider a system of fluorescent molecules that diffuse and interact with massive particles, e.g. surfactant micelles. Using the general formalism of FCS, we derive a new analytical approximation of the autocorrelation function for systems in which both diffusion and a binary reaction occur. This approximation provides a smooth interpolation between the limit of fast reaction (much faster than diffusion), and the opposite limit of slow reaction. Our studies of noncovalent interactions of micelles with dyes by FCS provided an experimental case to which the approximate autocorrelation function was successfully applied [X. Zhang, A.

Nanoviscosity of the cytoplasm is a key factor affecting diffusion of biomolecules and – as a consequence – rates of biochemical reactions in a cell. Nanoviscosity is an outcome of variable chemical and structural factors, which can temporarily change with cell-cycle associated changes of intracellular architecture. Thus, the question arises, whether rates of biochemical reactions depend on the point of cell cycle. In this paper we address this topic by constant observation of nanoviscosity of HeLa cells cytoplasm during S, G2 and G1 phases after Aphidicolin synchronization. For this purpose we measured diffusion rates of EGFP molecules using fluorescence correlation spectroscopy (FCS). To our surprise, a counter-intuitive stability of cytoplasmic viscosity was observed during the cell cycle. Our results hint at possible existence of robust mechanism maintaining stable physiological viscosity of the cytoplasm, despite huge structural changes during cell cycle.

The equilibrium and rate constants of molecular complex formation are of great interest both in the field of chemistry and biology. Here, we use fluorescence correlation spectroscopy (FCS), supplemented by dynamic light scattering (DLS) and Taylor dispersion analysis (TDA), to study the complex formation in model systems of dye-micelle interactions. In our case, dyes rhodamine 110 and ATTO-488 interact with three differently charged surfactant micelles: octaethylene glycol monododecyl ether C 12 E 8 (neutral), cetyltrimethylammonium chloride CTAC (positive) and sodium dodecyl sulfate SDS (negative). To determine the rate constants for the dye-micelle complex formation we fit the experimental data obtained by FCS with a new form of the autocorrelation function, derived in the accompanying paper. Our results show that the association rate constants for the model systems are roughly two orders of magnitude smaller than those in the case of the diffusion-controlled limit. Because the complex stability is determined by the dissociation rate constant, a two-step reaction mechanism, including the diffusion-controlled and reaction-controlled rates, is used to explain the dye-micelle interaction. In the limit of fast reaction, we apply FCS to determine the equilibrium constant from the effective diffusion coefficient of the fluorescent components. Depending on the value of the equilibrium constant, we distinguish three types of interaction in the studied systems: weak, intermediate and strong. The values of the equilibrium constant obtained from the FCS and TDA experiments are very close to each other, which supports the theoretical model used to interpret the FCS data.

Incorporation of membrane proteins into reconstituted lipid membranes is a common approach for studying their structure and function relationship in a native-like environment. In this work, we investigated fluorescence properties of liposome-reconstituted LHCII. By utilizing liposome labelling with the fluorescent dye molecules and single-molecule microscopy techniques, we were able to study truly liposome-reconstituted LHCII and compare them with bulk measurements and liposome-free LHCII aggregates on bound surface. Our results showed that fluorescence lifetime in bulk and of that for single liposome measurements were correlated. The fluorescence lifetimes of LHCII were shorter for liposome-free LHCII than for reconstituted LHCII. In the case of liposome-reconstituted LHCII, fluorescence lifetime showed dependence on the protein density reminiscent to concentration quenching. The dependence of fluorescence lifetime of LHCII on the liposome size was not significant. Our results demo...

Fluorescence correlation spectroscopy (FCS) is a sophisticated and an accurate analytical technique used to study the diffusion of molecules in a solution at the single-molecule level. FCS is strongly affected by many factors such as the stability of the excitation power, photochemical processes, mismatch between the refractive indices, and variations in the cover glass thickness. We have studied FCS near the surface of a cover glass by using rhodamine 123 as a fluorescent probe and have observed that the surface has a strong influence on the measurements. The temporal autocorrelation of FCS decays with two characteristic times when the confocal detection volume is positioned near the surface of the cover glass. As the position of the detection volume is moved away from the surface, the FCS autocorrelation becomes one-component decaying; the characteristic time of the decay is the same as the faster-decaying component in the FCS autocorrelation near the surface. This observation suggests that the faster component can be attributed to the free diffusion of the probe molecules in the solution, while the slow component has its origin from the interaction between the probe molecules and the surface. We have characterized the surface contribution to the FCS measurements near the surface by changing the position of the detection volume relative to the surface. The influence of the surface on the diffusion of the probe molecules was monitored by changing the chemical properties of the surface. The surface contribution to the temporal autocorrelation of the FCS strongly depends on the chemical nature of the surface. The hydrophobicity of the surface is a major factor determining the surface influence on the free diffusion of the probe molecules near the surface.

The papers included in this volume were part of the technical conference cited on the cover and title page. Papers were selected and subject to review by the editors and conference program committee. Some conference presentations may not be available for publication. The papers published in these proceedings reflect the work and thoughts of the authors and are published herein as submitted. The publisher is not responsible for the validity of the information or for any outcomes resulting from reliance thereon.

stimulation. We recently demonstrated the phasor approach to biosensor FRET detection by FLIM as a method that is robust towards biosensor design (single and dual chain) as well as the fluorescence artifacts inherent to the cellular environment. Using a frame mode acquisition we were able to map the spatial localization and quantify the fractional contribution of the free and bound state of a dual chain biosensor or the low and high FRET species of a single chain biosensor in each pixel of an image. To increase temporal resolution we find that line acquisition of FLIM data increases the total pixel integration and allows us to probe millisecond to second dynamics of RhoA and Rac1 activity across the cell. Given that this timescale is comparable to the diffusive rate at which Rac1 and RhoA traverse the cell upon activation we concomitantly perform pair correlation function (pCF) analysis along the line scan and investigate the molecular flow pattern of RhoA and Rac1 upon growth factor stimulation. We find for RhoA and Rac1 there are distinct gradients of activation from back to front (FLIM data) and a molecular flow pattern (pCF) that explains the observed polarized GTPase activity. Numerical Methods for Improving the Reliability of Number and Brightness (N&B) Analysis

Fluorescence Lifetime Correlation Spectroscopy (FLCS) is a variant of fluorescence correlation spectroscopy (FCS), which uses differences in fluorescence intensity decays to separate contributions of different fluorophore populations to FCS signal. Besides which, FLCS is a powerful tool to improve quality of FCS data by removing noise and distortion caused by scattered excitation light, detector thermal noise and detector afterpulsing. We are providing an overview of, to our knowledge, all published applications of FLCS. Although these are not numerous so far, they illustrate possibilities for the technique and the research topics in which FLCS has the potential to become widespread. Furthermore, we are addressing some questions which may be asked by a beginner user of FLCS. The last part of the text reviews other techniques closely related to FLCS. The generalization of the idea of FLCS paves the way for further promising application of the principle of statistical filtering of signals. Specifically, the idea of fluorescence spectral correlation spectroscopy is here outlined.

FAP-fluorogen system is ideal for generating a renewable, photostable signal and providing high photon flux. Since the localization accuracy is in principle limited by the number of photons collected from fluorescent probes, our module holds great promise for achieving super-resolution imaging (See abstract by Lidke, K. A. et al.). In this study, we characterize the blinking behavior of FAPfluorogen pairs at equilibrium. We show that the on-time is highly dependent on the excitation power density and the off-time is controlled by the free dye concentration and therefore, is limited by diffusion. By adjusting the dye concentration in solution and the intensity of incident power, the on and off rates of the binding reaction are controllable and could be optimized to resolve the objects in time and space. Clones of the FAP with point mutations show different blinking behaviors, which suggests that these amino-acid residues could play an important role in FAP-fluorogen binding and indicates that structural differences of the FAP at the binding interface can change the photochemical and photophysical properties of fluorogen.

The two-dimensional diffusion of isolated molecular tracers at the water-n-alkane interface was studied with fluorescence correlation spectroscopy. The interfacial diffusion coefficients of larger tracers with a hydrodynamic radius of 4.0 nm agreed well with the values calculated from the macroscopic viscosities of the two bulk phases. However, for small molecule tracers with hydrodynamic radii of only 1.0 and 0.6 nm, notable deviations were observed, indicating the existence of an interfacial region with reduced effective viscosity and increased mobility.

The photophysical properties of a new water-soluble fluorescent dye based on the perylene diimide chromophore are investigated at the ensemble and single molecule level. Water solubility was obtained by attaching hydrophilic substituents in the bay region. The fluorophore has an absorption maximum at 567 nm and a high quantum yield of fluorescence (0.6). In addition, the two-photon absorption cross section has higher or comparable values with the ones reported in the literature for other water-soluble dyes used in biological studies. The most important finding is its improved photostability in single molecule experiments when compared to other water-soluble dyes. This is demonstrated by the increased observation time of immobilized single molecules, which is almost 10 times higher than for commonly studied fluorophores. A possible reason is the lower intersystem crossing rate constant of perylene diimide in comparison, for example, to rhodamine derivatives. A similar conclusion can be drawn for this chromophore in an aqueous solution, because single molecule measurements using fluorescence correlation spectroscopy demonstrate a very low contribution of the triplet state in the autocorrelation function. The possibility of imaging the new molecule in living cells is demonstrated by one-photon confocal microscopy and by fluorescence lifetime microscopy with two-photon excitation.

Giant unilamellar vesicles (GUVs) have been widely used as a model membrane system to study membrane organization, dynamics, and protein-membrane interactions. Most recent studies have relied on imaging methods, which require good contrast for image resolution. Multiple sequential image processing only detects slow components of membrane dynamics. We have developed a new fluorescence correlation spectroscopy (FCS) technique, termed scanning FCS (i.e., SFCS), which performs multiple FCS measurements simultaneously by rapidly directing the excitation laser beam in a uniform (circular) scan across the bilayer of the GUVs in a repetitive fashion. The scan rate is fast compared to the diffusion of the membrane proteins and even small molecules in the GUVs. Scanning FCS outputs a ''carpet'' of timed fluorescence intensity fluctuations at specific points along the scan. In this study, GUVs were assembled from rat kidney brush border membranes, which included the integral membrane proteins. Scanning FCS measurements on GUVs allowed for a straightforward detection of spatial-temporal interactions between the protein and the membrane based on the diffusion rate of the protein. To test for protein incorporation into the bilayers of the GUVs, antibodies against one specific membrane protein (NaPi II cotransporter) were labeled with ALEXA-488. Fluorescence images of the GUVs in the presence of the labeled antibody showed marginal fluorescence enhancement on the GUV membrane bilayers (poor image contrast and resolution). With the application of scanning FCS, the binding of the antibody to the GUVs was detected directly from the analysis of diffusion rates of the fluorescent antibody. The diffusion coefficient of the antibody bound to NaPi II in the GUVs was ;200-fold smaller than that in solution. Scanning FCS provided a simple, quantitative, yet highly sensitive method to study protein-membrane interactions.

Structural and optical characterization of water soluble, thermo-responsive quantum dot/poly(N-isopropyl acrylamide) (QD/PNIPAM) hybrid particles using fluorescence correlation spectroscopy (FCS) and time-correlated single photon counting (TCSPC) measurements performed at temperatures below and above the lower critical solution temperature (LCST) of PNIPAM is reported. By increasing the temperature above the LCST, the signature of the PNIPAM chain collapse covering the QDs is revealed by FCS measurements. Despite the significant structural change, the TCSPC measurements show that the fluorescence lifetimes remain of the same order of magnitude at T > LCST. Such QD/PNIPAM hybrid particles with water solubility and robust thermo-responsive behavior at physiologically relevant temperatures are potentially useful for (bio)molecular sensing and separation applications.

The diffusion of rhodamine-labeled poly(ethylene glycol) (r-PEG) within surface-grafted poly(ethylene glycol) (s-PEG) layers in aqueous solution at 18 °C was measured by fluorescence correlation spectroscopy. The diffusion coefficient of r-PEG within s-PEG was controlled by the grafting density, σ, and scaled as σ. It is proposed that a characteristic blob size associated with the grafted (brush) layer defines the region through which the r-PEG diffusion occurs. The diffusion coefficients for r-PEG in semidilute solution were found to be similar to those in the brushes.

ABSTRACTAlkylphospholipids are a novel class of antineoplastic drugs showing remarkable therapeutic potential. Among them, Erufosine (EPC3) is a promising drug for the treatment of several types of tumors which has not been sufficiently characterized. While EPC3 is supposed to exert its function by interacting with lipid membranes, the exact molecular mechanisms involved are not known yet. In this work, we applied a combination of several fluorescence microscopy techniques (i.e., scanning fluorescence correlation spectroscopy, line-scan fluorescence correlation spectroscopy and generalized polarization imaging) to quantify the effect of EPC3 in biophysical models of the plasma membrane, as well as in cancer cell lines. Our results indicate that EPC3 affects lipid-lipid interactions in cellular membranes by decreasing lipid packing and increasing membrane disorder and fluidity. As a consequence of these alterations in the lateral organization of lipid bilayers, the diffusive dynamics...

Vesicle membrane fluctuations at nm resolution KEJIA CHEN, SUNG CHUL BAE, CHANG-KI MIN, STEVE GRANICK, University of Illinois at Urbana-Champaign, GRANICK GROUP TEAM -We measure membrane thermal fluctuations with nanometer spatial resolution and microsecond time resolution, extending a scattering technique used at the Curie Institute to study red blood cell dynamics (Timo Betz et al., Proc. Nat. Acad. Sci. USA 106, 15320, 2009). A laser beam is focused at the leading edge of a phospholipid vesicle membrane and the forward scattered light is detected by a quadrant photodiode. The measurements over 4 orders of magnitude of frequency allow quantification of more complete fluctuation spectra than competing methods, and therefore fuller understanding of the vesicle membrane mechanics. As a proof of concept, we quantify how adsorbed nanoparticles stiffen giant unilamellar vesicles (GUVs).

Fluorescent double-stranded DNA (dsDNA) molecules labeled at both ends are commonly produced by annealing of complementary single-stranded DNA (ssDNA) molecules, labeled with fluorescent dyes at the same (3′ or 5′) end. Because the labeling efficiency of ssDNA is smaller than 100%, the resulting dsDNA have two, one or are without a dye. Existing methods are insufficient to measure the percentage of the doubly-labeled dsDNA component in the fluorescent DNA sample and it is even difficult to distinguish the doubly-labeled DNA component from the singly-labeled component. Accurate measurement of the percentage of such doubly labeled dsDNA component is a critical prerequisite for quantitative biochemical measurements, which has puzzled scientists for decades. We established a fluorescence correlation spectroscopy (FCS) system to measure the percentage of doubly labeled dsDNA (PDL) in the total fluorescent dsDNA pool. The method is based on comparative analysis of the given sample and a reference dsDNA sample prepared by adding certain amount of unlabeled ssDNA into the original ssDNA solution. From FCS autocorrelation functions, we obtain the number of fluorescent dsDNA molecules in the focal volume of the confocal microscope and PDL. We also calculate the labeling efficiency of ssDNA. The method requires minimal amount of material. The samples have the concentration of DNA in the nano-molar/L range and the volume of tens of microliters. We verify our method by using restriction enzyme Hind III to cleave the fluorescent dsDNA. The kinetics of the reaction depends strongly on PDL, a critical parameter for quantitative biochemical measurements.

The width and shape of photon burst histograms pose significant limitations to the identification of single molecules in micro/nano-fluidic channels, and the nature of these histograms is not fully understood. To reach a deeper understanding, we performed computer simulations based on a Gaussian beam intensity profile with various fluidic channel diameters and assuming (i) a deterministic (noise-free) case, (ii) photon emission/absorption noise, and (iii) photon noise with diffusion. Photon noise in narrow channels yields a Gaussian burst distribution while additional strong diffusion produces skewed histograms. We use the fluctuating residence time picture [Phys. Rev. Lett. 80, 2386-2388 (1998)] and conclude that the skewness of the photon number distribution is caused by the longitudinal diffusive component of the motion of the molecules as they traverse the laser beam. In the case of strong diffusion in narrow channels, this effect leads to a log-normal distribution. We show that the same effect can transform the separate peaks of the photon burst histograms of multiple molecule mixtures into a single log-normal shape.

The objective of the project is to use the RICS analysis technique in complement with confocal microscopy to determine the diffusion coefficient of the selectively labeled green fluorescent protein (GFP), GFP-EGFR and GFP-p53 in cervical cancer cells. This is a collaboration work with MD Anderson Cancer Center. The application of the study is to lay the foundation for further study in understanding the cell metabolism, subcellular morphologic and dynamic biochemical processes to aid in the diagnosis and to potentially screen cancers. Fluorescence microscopy techniques have been developed for the study of cellular processes and molecular signal pathway. However, the spatial resolution to distinguish and resolve the interactions of single molecular complexes or molecule in cells is limited by the wavelength. Hence, indirect image correlation methods complementary to the imaging techniques were developed to obtain the dynamic information within the cell. RICS is one such mathematical i...

The width and shape of photon burst histograms pose significant limitations to the identification of single molecules in micro/nano-fluidic channels, and the nature of these histograms is not fully understood. To reach a deeper understanding, we performed computer simulations based on a Gaussian beam intensity profile with various fluidic channel diameters and assuming (1) a deterministic (noise-free) case, (2) photon emission/absorption noise, and (3) photon noise with diffusion. Photon noise in narrow channels yields a Gaussian burst distribution while additional strong diffusion produces skewed histograms. We use the fluctuating residence time picture [J. Söderlund et al., Phys. Rev. Lett. 80, 2386 (1998)] and conclude that the skewness of the photon number distribution is caused by the longitudinal diffusive component of the motion of the molecules as they traverse the laser beam. In the case of strong diffusion in narrow channels, this effect leads to a log-normal distribution....

Fluorescence lifetime sensing enables researchers to probe the physicochemical environment of a fluorophore providing a window through which we can observe the complex molecular make-up of the cell. Fluorescence lifetime imaging microscopy (FLIM) quantifies and maps cell biochemistry, a complex ensemble of dynamic processes. Unfortunately, typical high-resolution FLIM systems exhibit rather limited acquisition speeds, often insufficient to capture the time evolution of biochemical processes in living cells. Here, we describe the theoretical background that justifies the developments of high-speed single photon counting systems. We show that systems with low dead-times not only result in faster acquisition throughputs but also improved dynamic range and spatial resolution. We also share the implementation of hardware and software as an open platform, show applications of fast FLIM biochemical imaging on living cells and discuss strategies to balance precision and accuracy in FLIM. Th...

ABSTRACTExtracellular vesicles (EV) convey biological information by transmitting macromolecules between cells and tissues and are of great promise as pharmaceutical nanocarriers, and as therapeutic per se. Strategies for customizing the EV surface and cargo are being developed to enable their tracking, visualization, loading with pharmaceutical agents and decoration of the surface with tissue targeting ligands. While much progress has been made in the engineering of EVs, an exhaustive comparative analysis of the most commonly exploited EV‐associated proteins, as well as a quantification at the molecular level are lacking. Here, we selected 12 EV‐related proteins based on MS‐proteomics data for comparative quantification of their EV engineering potential. All proteins were expressed with fluorescent protein (FP) tags in EV‐producing cells; both parent cells as well as the recovered vesicles were characterized biochemically and biophysically. Using Fluorescence Correlation Spectrosco...

A challenging topic in cancer research is to create drug system that can deliver in a specific and non-cytotoxic manner a therapeutic compound. Usually, tumor targeting requires very specific compounds. Currently, peptide analogues like somatostatin, neurotensin, or bombesin are used to target G-coupled receptors, which are over-expressed on tumor cells. We have recently designed a novel type of nanoparticles with regular icosahedral symmetry and a diameter of about 16 nm, which self-assemble from single polypeptide chains [1]. Here we propose an innovative drug targeting and delivery system based on such nanoparticles functionalized with somatostatin or bombesin as targeting units. Moreover, these nanoparticles can be radiolabeled with Technetium-99m or other radionuclides for diagnosis and treatment of cancer.

The role of actin in transcription and RNA processing is now widely accepted but the form of nuclear actin remains enigmatic. Monomeric, oligomeric or polymeric forms of actin seem to be involved in nuclear functions. Moreover, uncommon forms of actin such as the "lower dimer" have been observed in vitro. Antibodies have been pivotal in revealing the presence and distribution of different forms of actin in different cellular locations. Because of its high degree of conservation, actin is a poor immunogen and only few specific actin antibodies are available. To unravel the mystery of less common forms of actin, in particular those in the nucleus, we chose to tailor monoclonal antibodies to recognize distinct forms of actin. To increase the immune response, we used a new approach based on peptide nanoparticles, which are designed to mimic an icosahedral virus capsid and allow the repetitive, ordered display of a specific epitope on their surface. Actin sequences representing the highly conserved "hydrophobic loop," which is buried in the filamentous actin filament, were grafted onto the surface of nanoparticles by genetic engineering. After immunization with "loop nanoparticles," a number of monoclonal antibodies were established that bind to the hydrophobic loop both in vitro and in situ. Immunofluorescence studies on cells revealed that filamentous actin filaments were only labeled once the epitope had been exposed. Our studies indicate that self-assembling peptide nanoparticles represent a versatile platform that can easily be customized to present antigenic determinants in repetitive, ordered arrays and elicit an immune response against poor antigens.

Recoverin is a neuronal calcium sensor protein (NCS-protein) from the vertebrate photoreceptor which is involved in light adaptation. Recoverin changes its conformation upon sequential binding of two calcium ions. This conformational change induces the extrusion of a covalently attached myristoyl residue from a hydrophobic binding pocket which enables Recoverin to interact with lipid membranes (calcium-myristoyl switch). In this thesis, I report on my investigation of Recoverin's calcium myristoyl switch, using a recently developed modification of fluorescence correlation spectroscopy (FCS) that is called dual-focus FCS (2fFCS). This method allows for measuring absolute diffusion coefficients with an accuracy of better than a few percent. Recoverin, and the Recoverin mutants E85Q and E121Q which bind only one or no Ca 2+ , respectively, were labeled with the fluorescent dye Alexa647. Differences in the hydrodynamic radius due to conformational changes of Recoverin and its mutants upon calcium binding were monitored by measuring the diffusion coefficient of these molecules as a function of free calcium concentration. The calcium dependent interaction of Recoverin with lipid membranes was measured in solutions of small unilamellar vesicles (SUVs) of different lipid composition. Again, diffusion measurements were used to determine the fraction of free and lipid-bound Recoverin using the strongly different diffusion coefficients of both fractions. To account for the fluorescence brightness difference of the dye label when in solution and close to a lipid membrane, a new three-photon correlation analysis was developed and tested. Recoverin ist ein neuronales Kalzium Sensor Protein (NCS-Protein) aus dem Photorezeptor der Wirbeltiere, das an der Lichtadaptation beteiligt ist. Recoverin ändert seine Konformation durch die sequenzielle Bindung von zwei Kalzium-Ionen. Diese Konformationsänderung induziert die Freisetzung eines kovalent gebundenen Myristoylrestes aus einer hydrophoben Tasche des Proteins und erlaubt Recoverin, an Lipidmembranen zu binden (Kalzium-Myristoyl-Switch). In dieser Arbeit beschreibe ich die Untersuchung des Kalzium-Myristoyl-Switches von Recoverin mit einer vor kurzem entwickelten Modifikation der Fluoreszenz Korrelations Spektroskopie (FCS), der Zwei-Fokus FCS (2fFCS). Mit dieser Methode können absolute Diffusionskoeffizienten mit höchster Genauigkeit gemessen werden. Recoverin und die beiden Recoverin-Mutanten E85Q und E121Q, die nur ein bzw. kein Kalzium-Ion binden können, wurden mit dem Fluoreszenzfarbstoff Alexa647 markiert. Änderungen des hydrodynamischen Radius von Recoverin, die durch kalziuminduzierte Konformationsänderungen verursacht werden, können durch die Messung des Diffusionskoeffizienten als Funktion der Kalziumkonzentration beobachtet werden. Die kalziumabhängige Wechselwirkung von Recoverin und Lipidmembranen wurde mit Suspensionen kleiner, unilamellarer Vesikel (SUV) unterschiedlicher Zusammensetzung untersucht. Auch hier wurde das Diffusionsverhalten gemessen. Die sehr unterschiedlichen Diffusionskoeffizienten der beiden Fraktionen wurden benutzt, um den Anteil von freiem und lipidgebundenem Recoverin zu bestimmen. Eine neue Drei-Photonen Korrelationsanalyse, mit der die unterschiedlichen Fluoreszenzhelligkeiten der Farbstoffmarkierung in Lösung und nahe der Membran berücksichtigt werden können, wurde entwickelt und getestet.

Recoverin is a neuronal calcium sensor protein (NCS-protein) from the vertebrate photoreceptor which is involved in light adaptation. Recoverin changes its conformation upon sequential binding of two calcium ions. This conformational change induces the extrusion of a covalently attached myristoyl residue from a hydrophobic binding pocket which enables Recoverin to interact with lipid membranes (calcium-myristoyl switch). In this thesis, I report on my investigation of Recoverin's calcium myristoyl switch, using a recently developed modification of fluorescence correlation spectroscopy (FCS) that is called dual-focus FCS (2fFCS). This method allows for measuring absolute diffusion coefficients with an accuracy of better than a few percent. Recoverin, and the Recoverin mutants E85Q and E121Q which bind only one or no Ca 2+ , respectively, were labeled with the fluorescent dye Alexa647. Differences in the hydrodynamic radius due to conformational changes of Recoverin and its mutants upon calcium binding were monitored by measuring the diffusion coefficient of these molecules as a function of free calcium concentration. The calcium dependent interaction of Recoverin with lipid membranes was measured in solutions of small unilamellar vesicles (SUVs) of different lipid composition. Again, diffusion measurements were used to determine the fraction of free and lipid-bound Recoverin using the strongly different diffusion coefficients of both fractions. To account for the fluorescence brightness difference of the dye label when in solution and close to a lipid membrane, a new three-photon correlation analysis was developed and tested. Recoverin ist ein neuronales Kalzium Sensor Protein (NCS-Protein) aus dem Photorezeptor der Wirbeltiere, das an der Lichtadaptation beteiligt ist. Recoverin ändert seine Konformation durch die sequenzielle Bindung von zwei Kalzium-Ionen. Diese Konformationsänderung induziert die Freisetzung eines kovalent gebundenen Myristoylrestes aus einer hydrophoben Tasche des Proteins und erlaubt Recoverin, an Lipidmembranen zu binden (Kalzium-Myristoyl-Switch). In dieser Arbeit beschreibe ich die Untersuchung des Kalzium-Myristoyl-Switches von Recoverin mit einer vor kurzem entwickelten Modifikation der Fluoreszenz Korrelations Spektroskopie (FCS), der Zwei-Fokus FCS (2fFCS). Mit dieser Methode können absolute Diffusionskoeffizienten mit höchster Genauigkeit gemessen werden. Recoverin und die beiden Recoverin-Mutanten E85Q und E121Q, die nur ein bzw. kein Kalzium-Ion binden können, wurden mit dem Fluoreszenzfarbstoff Alexa647 markiert. Änderungen des hydrodynamischen Radius von Recoverin, die durch kalziuminduzierte Konformationsänderungen verursacht werden, können durch die Messung des Diffusionskoeffizienten als Funktion der Kalziumkonzentration beobachtet werden. Die kalziumabhängige Wechselwirkung von Recoverin und Lipidmembranen wurde mit Suspensionen kleiner, unilamellarer Vesikel (SUV) unterschiedlicher Zusammensetzung untersucht. Auch hier wurde das Diffusionsverhalten gemessen. Die sehr unterschiedlichen Diffusionskoeffizienten der beiden Fraktionen wurden benutzt, um den Anteil von freiem und lipidgebundenem Recoverin zu bestimmen. Eine neue Drei-Photonen Korrelationsanalyse, mit der die unterschiedlichen Fluoreszenzhelligkeiten der Farbstoffmarkierung in Lösung und nahe der Membran berücksichtigt werden können, wurde entwickelt und getestet.

The cover picture shows the two focal regions of a two‐focus fluorescence correlation spectroscopy set‐up with molecules diffusing through these regions. The depicted curves are the fluorescence autocorrelation curves for the first and the second focus, respectively, as well as the fluorescence cross‐correlation between both foci. In their Article on page 433 Dertinger et al. introduce an external ruler into a fluorescence correlation measurement by having two focal regions with a fixed and well‐defined distance. This allows for absolute and precise determination of molecular diffusion.

We present a new method to measure absolute diffusion coefficients at nanomolar concentrations with high precision. Based on a modified fluorescence correlation spectroscopy (FCS)‐setup, this method is improved by introducing an external ruler for measuring the diffusion time by generating two laterally shifted and overlapping laser foci at a fixed and known distance. Data fitting is facilitated by a new two‐parameter model to describe the molecule detection function (MDF). We present a recorded MDF and show the excellent agreement with the fitting model. We measure the diffusion coefficient of the red fluorescent dye Atto655 under various conditions and compare these values with a value achieved by gradient pulsed field NMR (GPF NMR). From these measurements we conclude, that the new measurement scheme is robust against optical and photophysical artefacts which are inherent to standard FCS. With two‐focus‐FCS, the diffusion coefficient of 4.26×10−6 cm2 s−1 for Atto655 in water at 2...

In order to monitor membrane–protein binding in lipid bilayers at physiological protein concentrations, we employed the recently developed dual‐focus fluorescence correlation spectroscopy (2fFCS) technique. In a case study on a photoreceptor consisting of seven transmembrane helices and its cognate transducer (two transmembrane helices), the lateral diffusion for these integral membrane proteins was analyzed in giant unilamellar vesicles (GUVs). The two‐dimensional diffusion coefficients of both separately diffusing proteins differ significantly, with D=2.2×10−8 cm2 s−1 for the photoreceptor and with D=4.1×10−8 cm2 s−1 for the transducer. In GUVs with both membrane proteins present together, we observed significantly smaller diffusion coefficients for labelled transducer molecules; this indicates the presence of larger diffusing units and therefore intermolecular protein binding. Based on the phenomenological dependence of diffusion coefficients on the molecule's cylindrical rad...

Incorporation of membrane proteins into reconstituted lipid membranes is a common approach for studying their structure and function relationship in a native-like environment. In this work, we investigated fluorescence properties of liposome-reconstituted LHCII. By utilizing liposome labelling with the fluorescent dye molecules and single-molecule microscopy techniques, we were able to study truly liposome-reconstituted LHCII and compare them with bulk measurements and liposome-free LHCII aggregates on bound surface. Our results showed that fluorescence lifetime in bulk and of that for single liposome measurements were correlated. The fluorescence lifetimes of LHCII were shorter for liposome-free LHCII than for reconstituted LHCII. In the case of liposomereconstituted LHCII, fluorescence lifetime showed dependence on the protein density reminiscent to concentration quenching. The dependence of fluorescence lifetime of LHCII on the liposome size was not significant. Our results demonstrated that fluorescence quenching can be induced by LHCII-LHCII interactions in reconstituted membranes, most likely occurring via the same mechanism as photoprotective non-photochemical quenching in vivo.

Linear arrays of single photon avalanche detectors (SPADs), fabricated using a novel planar process that is compatible with CMOS technology, are presented here. Their suitability for application in fluorescence correlation spectroscopy (FCS) is investigated by examining characteristics such as the dark counting rate, breakdown voltage and quantum efficiency. The problem of optical crosstalk between pixels in the array is investigated and a trench isolation process is proposed to eliminate crosstalk between adjacent pixels in the array.

Single wavelength fluorescence cross-correlation spectroscopy (SW-FCCS), introduced to study biomolecular interactions, has recently been reported to monitor enzyme activity by using a newly developed fluorescent protein variant together with cyan fluorescent protein. Here, for the first time to our knowledge, SW-FCCS is applied to detect interactions between membrane receptors in vivo by using the widely used enhanced green fluorescent protein and monomeric red fluorescent protein. The biological system studied here is the epidermal growth factor/ErbB receptor family, which plays pivotal roles in the development of organisms ranging from worms to humans. It is widely thought that a ligand binds to the monomeric form of the receptor and induces its dimeric form for activation. By using SW-FCCS and Fo ¨rster resonance energy transfer, we show that the epidermal growth factor receptor and ErbB2 have preformed homo-and heterodimeric structures on the cell surface and quantitation of dimer fractions is performed by SW-FCCS. These receptors are major targets of anti-cancer drug development, and the receptors' homo-and heterodimeric structures are relevant for such developments.

Imaging fluorescence correlation spectroscopy (imaging FCS), or the acquisition of fluorescence correlation functions at contiguous points in an imaging format, is a recent addition to quantitative bioimaging. Imaging FCS has been implemented in various modalities. These techniques provide excellent time resolution, have single molecules sensitivity and can be combined with super-resolution techniques, thus combining high spatial and temporal resolution. Although still at its beginning it has been applied in different forms to biological problems. This review looks at applications of imaging FCS in the last two years with the aim to give the reader an overview of the capabilities of these new techniques.

Background: Protein aggregation is implicated in numerous diseases. Results: Polyglutamine oligomers maintain a heterogeneous distribution that reaches an equilibrium during aging and can be altered by genetic modifiers that suppress aggregation. Conclusion: Shifts in populations of oligomers do not correlate with toxicity. Significance: The dynamics of oligomerization and aggregation to inert species is essential for protein misfolding and toxicity. Protein misfolding and aggregation are exacerbated by aging and diseases of protein conformation including neurodegeneration, metabolic diseases, and cancer. In the cellular environment, aggregates can exist as discrete entities, or heterogeneous complexes of diverse solubility and conformational state. In this study, we have examined the in vivo dynamics of aggregation using imaging methods including fluorescence microscopy, fluorescence recovery after photobleaching (FRAP), and fluorescence correlation spectroscopy (FCS), to monitor the diverse biophysical states of expanded polyglutamine (polyQ) proteins expressed in Caenorhabditis elegans. We show that monomers, oligomers and aggregates co-exist at different concentrations in young and aged animals expressing different polyQ-lengths. During aging, when aggregation and toxicity are exacerbated, FCS-based burst analysis and purified single molecule FCS detected a populational shift toward an increase in the frequency of brighter and larger oligomeric species. Regardless of age or polyQ-length, oligomers were maintained in a heterogeneous distribution that spans multiple orders of magnitude in brightness. We employed genetic suppressors that prevent polyQ aggregation and observed a reduction in visible immobile species with the persistence of heterogeneous oligomers, yet our analysis did not detect the appearance of any discrete oligomeric states associated with toxicity. These studies reveal that the reversible transition from monomers to immobile aggregates is not represented by discrete oligomeric states, but rather suggests that the process of aggregation involves a more complex pattern of molecular interactions of diverse intermediate species that can appear in vivo and contribute to aggregate formation and toxicity.

Background: Protein aggregation is implicated in numerous diseases. Results: Polyglutamine oligomers maintain a heterogeneous distribution that reaches an equilibrium during aging and can be altered by genetic modifiers that suppress aggregation. Conclusion: Shifts in populations of oligomers do not correlate with toxicity. Significance: The dynamics of oligomerization and aggregation to inert species is essential for protein misfolding and toxicity. Protein misfolding and aggregation are exacerbated by aging and diseases of protein conformation including neurodegeneration, metabolic diseases, and cancer. In the cellular environment, aggregates can exist as discrete entities, or heterogeneous complexes of diverse solubility and conformational state. In this study, we have examined the in vivo dynamics of aggregation using imaging methods including fluorescence microscopy, fluorescence recovery after photobleaching (FRAP), and fluorescence correlation spectroscopy (FCS), to monitor the diverse biophysical states of expanded polyglutamine (polyQ) proteins expressed in Caenorhabditis elegans. We show that monomers, oligomers and aggregates co-exist at different concentrations in young and aged animals expressing different polyQ-lengths. During aging, when aggregation and toxicity are exacerbated, FCS-based burst analysis and purified single molecule FCS detected a populational shift toward an increase in the frequency of brighter and larger oligomeric species. Regardless of age or polyQ-length, oligomers were maintained in a heterogeneous distribution that spans multiple orders of magnitude in brightness. We employed genetic suppressors that prevent polyQ aggregation and observed a reduction in visible immobile species with the persistence of heterogeneous oligomers, yet our analysis did not detect the appearance of any discrete oligomeric states associated with toxicity. These studies reveal that the reversible transition from monomers to immobile aggregates is not represented by discrete oligomeric states, but rather suggests that the process of aggregation involves a more complex pattern of molecular interactions of diverse intermediate species that can appear in vivo and contribute to aggregate formation and toxicity.

Real-time feedback-driven single-particle tracking is a technique that uses feedback control to enable single-molecule spectroscopy of freely diffusing particles in native or near-native environments. A number of different real-time feedback-driven single-particle tracking (RT-FD-SPT) approaches exist, and comparisons between methods based on experimental results are of limited use due to differences in samples and setups. In this study, we used statistical calculations and dynamical simulations to directly compare the performance of different methods. The methods considered were the orbital method, the knight‘s tour (grid scan) method, and MINFLUX, and we considered both fluorescence-based and interferometric scattering (iSCAT) approaches. There is a fundamental trade-off between precision and speed, with the knight’s tour method being able to track the fastest diffusion but with low precision, and MINFLUX being the most precise but only tracking slow diffusion. To compare iSCAT an...

We describe for the first time the combination between cross-pair correlation function analysis (pair correlation analysis or pCF) and stimulated emission depletion (STED) to obtain diffusion maps at spatial resolution below the optical diffraction limit (super-resolution). Our approach was tested in systems characterized by high and low signal to noise ratio, i.e. Capsid Like Particles (CLPs) bearing several (.100) active fluorescent proteins and monomeric fluorescent proteins transiently expressed in living Chinese Hamster Ovary cells, respectively. The latter system represents the usual condition encountered in living cell studies on fluorescent protein chimeras. Spatial resolution of STED-pCF was found to be about 110 nm, with a more than twofold improvement over conventional confocal acquisition. We successfully applied our method to highlight how the proximity to nuclear envelope affects the mobility features of proteins actively imported into the nucleus in living cells. Remarkably, STED-pCF unveiled the existence of local barriers to diffusion as well as the presence of a slow component at distances up to 500-700 nm from either sides of nuclear envelope. The mobility of this component is similar to that previously described for transport complexes. Remarkably, all these features were invisible in conventional confocal mode.

Hyperosmotic stress activates in live cells numerous processes the net result of which is readily recognized at the cellular level through morphological changes. It also promotes intracellular protein/RNA aggregation and phase separation, observable through the formation of stress granules in the cytoplasm. However, the time course and extent of these changes remain largely uncharacterized. To investigate in live cells dynamic changes in intracellular macromolecular crowding (MMC) induced by hyperosmotic stress, we used Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Lifetime Imaging Microscopy (FLIM) to quantify changes in the local environment by measuring the diffusion and the fluorescence lifetime of the monomeric enhanced Green Fluorescent Protein (eGFP), respectively. In addition, Confocal Laser Scanning Microscopy (CLSM) was used to map the acceptor/donor emission ratio (Y/C ratio) in cells expressing the glycine-inserted mutant Förster Resonance Energy Transfer (FRET) sensor (GimRET) that is indicative of MMC. Real-time monitoring of eGFP fluorescence lifetime showed that a faster response to environmental changes due to MMC is observed than when measuring the Y/C ratio using the MMC-sensitive GimRET sensor. This suggests that eGFP molecular electronic states and/or collision frequency are affected by changes in the immediate surroundings due to MMC without requiring conformational changes as is the case for the GimRET sensor. Furthermore, slower eGFP mobility and a lower anomalous diffusion parameter (α) are measured in cells exposed to hyperosmotic stress, indicating higher intracellular viscosity due to increased MMC. Our findings reveal that changes in eGFP fluorescence lifetime and diffusion are early indicators of elevated intracellular MMC. These variables can therefore be used for quantitative characterization of MMC under various stress/disease conditions in live cells.