Fluorescence correlation spectroscopy

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Gutenberg Open Science: Characterization of 
structure and dynamics of submicrometric systems via fluorescence correlation spectroscopy

Gutenberg Open Science: Characterization of structure and dynamics of submicrometric systems via fluorescence correlation spectroscopy

Since a large use of hydrogels is being made in biological environments, however, deeper knowledge of the system’s behavior in water is needed. In addition to the basis of a structure theory, the systematic engineering of hydrogels for sensing applications also has the potential to hugely benefit from a systematic understanding of the structure, dynamics and duration limits of the gels themselves. To this end, the present fluorescence correlation spectroscopy (FCS) study revolves around PNIPAAm anchored hydrogels, with multiple goals. These include primarily probing the gels’ behavior in aqueous environments at different temperatures, encompassing the thermal-dependence, and providing a more coherent picture of the structure-dynamics relationship at the nanometer scale. Hence, the present work focus on the analysis of the swelling ratio R S of and tracer diffusion in PNIPAAm hydrogels under different physicochemical conditions. These two quantities, which are obtained from the same experimental technique under equivalent conditions, represent space averaged quantities in the micrometer and submicrometer scales.
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Fluorescence correlation spectroscopy reveals a cooperative unfolding of monomeric amyloid-β 42 with a low Gibbs free energy

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

1-focus fluorescence correlation spectroscopy measurements. 1-focus fluorescence correlation spectroscopy (1fFCS) measurements were performed on a home-built confocal fluorescence detection setup equipped with a pulsed laser diode (λ = 470 nm, LDH-P-C-470, PicoQuant, Berlin, Germany). The laser light is forwarded into the microscope via a dichroic mirror (dichroic mirror z470/635, AHF Analysentechnik, Tübingen, Germany). A water-immersion objective (UplanApo 60 × 1.2 W, Olympus, Melville, NY, USA) focuses the laser beam into the sample solution where fluorescent molecules are excited. Fluorescence light is collected by the same objective in reverse direction, passing through the dichroic mirror and focused onto a pinhole of 100 µm diameter by a tube lens. A polarising beam splitter separates photons according to their polarisation. Each beam is subsequently focused onto a single-photon avalanche detector (SPAD) detector (PDM, MPD, Bolzano, Italy). For fluorescence correlation spectroscopy (FCS) analysis, the signals from both detectors were cross-correlated to remove detector artifacts such as afterpulsing 8 .
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Gutenberg Open Science: Diffusion in heterogeneous systems studied by laser scanning confocal microscopy and fluorescence correlation spectroscopy

Gutenberg Open Science: Diffusion in heterogeneous systems studied by laser scanning confocal microscopy and fluorescence correlation spectroscopy

In this thesis I used FCS to study tracer diffusion in heterogeneous systems of synthetic nature. In the first part of my thesis using the combination of the laser scanning confocal microscopy (LSCM) and fluorescence correlation spectroscopy (FCS) I studied in situ the dynamics of phase separation in the polymer blend polystyrene/poly(methyl phenyl siloxane) (PS/PMPS) at the macroscopic and microscopic length scales, respectively. LSCM was used to monitor the process of phase separation in real time providing images of droplet coalescence and growth during the intermediate and late stages. Measuring the small tracer (terrylene dye) diffusion in the PMPS phase of the phase separated blend and by comparing the diffusion of the same tracers in pure bulk PMPS, additional information on the purity of phases was obtained using FCS.
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Coagulation protein FVIII binding to phospholipid membranes investigated by Fluorescence Correlation Spectroscopy

Coagulation protein FVIII binding to phospholipid membranes investigated by Fluorescence Correlation Spectroscopy

This work focusses on protein-membrane interactions within the coagulation cascade, particularly, the binding of FVIII to membranes. Investigations on PS-vesicles as model systems for platelets showed that binding of inactivated FVIII to membranes is strongly associated with electrostatic interactions and therefore increases with the concentration of charge (i.e. phosphatidylserine) in the membrane [6]. For activated FVIII however, binding and particularly, its dependence on the PS-content, has not yet been investi- gated. Likewise, there is not yet quantitative support for regulatory processes such as the scenario of competitive binding of FVIII and annexin as depicted in the coverfigure. Fluorescence Correlation Spectroscopy is a powerful tool for the investigation of protein-membrane interactions. It measures diffusion times and can discriminate freely diffusing proteins from proteins bound to vesicles as model membranes based on the size-dependence of the diffusion time [7, 8]. Hence, binding isotherms can be obtained from consecutive titration experiments. Compared to other conventional techniques used for binding experiments, such as ELISA, which all rely on one component bound to a surface, an advantage of Fluorescence Correlation Spectroscopy is that it allows for mea- surements in solution. However, if desired, experiments can be performed on supported lipid bilayers as described in chapter 12. In this thesis protein-membrane binding within coagulation was investigated with FCS using both, experiments in solution as well as on supported membranes.
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Surface-Integrated Fluorescence Correlation Spectroscopy (SI-FCS) for the quantification of transient membrane and surface binding

Surface-Integrated Fluorescence Correlation Spectroscopy (SI-FCS) for the quantification of transient membrane and surface binding

Following the concept of fluorescence correlation spectroscopy (FCS) [Magde et al., 1972], the autocorrelation function of the number and brightness of fluorescently labeled molecules within a small observation volume is determined by the dynamics that govern the fluorescence fluctuations. In the following years, FCS developed into an increasingly versatile tool to study not only molecular mobility [Aragón and Pecora, 1976,Koppel et al., 1976,Fahey et al., 1977], but also bi-molecular interactions [Kinjo and Rigler, 1995,Schwille et al., 1996, Rauer et al., 1996, Schwille et al., 1997]. Thus, kinetic parameters governing biochemical reactions can be extracted from the autocorrelation function. Focused laser excitation and tightly confined confocal detection allowed the study of individual molecules with FCS [Rigler et al., 1993, Eigen and Rigler, 1994]. More advanced FCS variants that scan the confocal detection in the sample [Berland et al., 1996] proved useful to elucidate not only mobility in the membrane [Benda et al., 2003, Ries and Schwille, 2006, Petrášek and Schwille, 2008], but also the partitioning into different membrane phases [Ries et al., 2009a] and the binding to membrane-localized receptors [Ries et al., 2009b]. Simultaneous to the advent of wide-field total internal reflection fluorescence (TIRF) microscopy [Ax- elrod, 1981], the pioneering works of Thompson and colleagues highlighted the potential of total internal reflection fluorescence correlation spectroscopy (TIR-FCS) to investigate surface binding [Thompson et al., 1981, Thompson, 1982, Thompson and Axelrod, 1983]. Studies on lipid mobility of the basal membrane of cells and of supported lipid bilay- ers (SLBs) [Tamm and McConnell, 1985] profited from the surface-selectivity of TIRF microscopy in combination with FCS [Thompson et al., 1993, Ohsugi et al., 2006]. Mod- ern electron-multiplying charge-coupled device (EMCCD) camera technology enabled the multiplexing of FCS acquisition in multiple detection modalities [Krieger et al., 2015], including TIRF microscopy [Burkhardt and Schwille, 2006, Kannan et al., 2006, Kannan et al., 2007, Sankaran et al., 2009]. Image correlation spectroscopy (ICS) [Petersen et al., 1993,Wiseman, 2013], a closely related method, extracts molecular mobilities and has been used to investigate the binding of membrane-diffusing receptors to larger protein complexes as well as of ligands to microtubules [Brandão et al., 2014].
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Binding of proteins to nanoparticles in complex fluids probed by fluorescence correlation spectroscopy

Binding of proteins to nanoparticles in complex fluids probed by fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) is a sensitive and versatile technique allowing the diffusion of fluorescently labeled nano- and meso-scale objects in solution to be measured down to picomolar concentrations (1). The technique was first described in the early 1970s (2-4). FCS monitors the intensity fluctuations of a fluorescently labeled particle as it passes through a fixed volume (Fig. 1a and 1b). The use of a confocal microscope with FCS reduces the measurement volume to less than 1 femtolitre, allowing for single molecule detection (5, 6). By time averaging these fluorescence intensity fluctuations, an autocorrelation function is calculated (Fig. 1c), which contains information about the time taken for the labeled particle to pass through the measurement volume and the concentration of labeled particles in solution. This information in turn can be used to determine the particle size. One (a single label), two or more color FCS can be performed when the wavelengths of emission maxima of the different dyes are sufficiently separated (or that filters are used) to eliminate cross-talk. If two labeled components (of different colors) bind together, this can be detected by measuring a cross –correlation (7, 8). Hence, the variety of ways in which measurements can be performed has allowed the technique to be used for a diverse range of measurements including the hybridization of DNA (6), the binding of peptides to lipid membranes (9) and the assembly of virus capsids (10).
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Fluorescence correlation spectroscopy of autofluorescent proteins and its applications in live cell membranes

Fluorescence correlation spectroscopy of autofluorescent proteins and its applications in live cell membranes

The two amino acid substitutions F64L and S65T are responsible for convenient use of EGFP in biological applications. The S65T and F64L mutations result a 35 fold increase in fluorescence over wt-GFP when excited at 488 nm and this proves that the second mutation is critical for the maximal fluorescence (Cormack et al., 1996). EYFP contains substitutions such as S65G, V68L, S72A and T203Y and is the most red shifted avGFP. An improved version of YFP, Venus have been presented recently (Nagai et al., 2000). As a result of well known folding mutations, F64L/M153T/V163A/S175G, the folding of Venus is dramatically increased. Venus also contains a novel mutation F46L which greatly facilitated the maturation of YFP by accelerating the oxidation step of the chromophore formation. Oxidation of chromophore is considered to be a rate limiting step of maturation. Beyond that, the protein became more tolerant to acid-sensitivity and quenching by chloride ion (Nagai et al., 2000). In this aspect, we focus on F64L mutation which is an important constituent of EGFP that gives maximal fluorescence. So we introduced super-EYFP (SEYFP) in fluorescence correlation spectroscopy measurements together with EYFP and EGFP. SEYFP was created with the help of multiple site mutagenesis by the introduction of four folding mutations simultaneously into EYFP (EYFP - F64L/M153T/V163A/S175G) which facilitates the folding of YFP at 37 0 C (Nagai et al., 2000).
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Effect of the supramolecular interactions on the nanostructure of halloysite/biopolymer hybrids: a comprehensive study by SANS, fluorescence correlation spectroscopy and electric birefringence

Effect of the supramolecular interactions on the nanostructure of halloysite/biopolymer hybrids: a comprehensive study by SANS, fluorescence correlation spectroscopy and electric birefringence

The structural properties of halloysite/biopolymer aqueous mixtures were firstly investigated by means of combining different techniques, including small-angle neutron scattering (SANS), electric birefringence (EBR) and fluorescence correlation spectroscopy (FCS). Among the biopolymers, non-ionic hydroxypropylcellulose and polyelectrolytes (anionic alginate and cationic chitosan) were selected. On this basis, the specific supramolecular interactions were correlated to the structural behavior of the halloysite/biopolymer mixtures. SANS data were analyzed in order to investigate the influence of the biopolymer adsorption on the halloysite gyration radius. In addition, a morphological description of the biopolymer-coated halloysite nanotubes (HNTs) was obtained by the simulation of SANS curves. EBR experiments evidenced that the orientation dynamics of the nanotubes in the electric field is influenced by the specific interactions with the polymers. Namely, both variations of the polymer charge and/or wrapping mechanisms strongly affected the HNT alignment process and, consequently, the rotational mobility of the nanotubes. FCS measurements with fluorescently labeled biopolymers allowed us to study the aqueous dynamic behavior of ionic biopolymers after their adsorption onto the HNT surfaces. The combination of EBR and FCS results revealed that the adsorption process reduces the mobility in water of both components. These effects are strongly enhanced by HNT/polyelectrolyte electrostatic interactions and wrapping processes occurring in the halloysite/chitosan mixture. The attained findings can be useful for designing halloysite/polymer hybrids with controlled structural properties.
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Gutenberg Open Science: Diffusion in polymer systems studied by fluorescence correlation spectroscopy

Gutenberg Open Science: Diffusion in polymer systems studied by fluorescence correlation spectroscopy

In the recent years, the fluorescence correlation spectroscopy has emerged as a powerful tool for investigation of the diffusion of fluorescent molecules, macromolecules or nanoparticles in various environments. FCS is a single molecule spectroscopic technique able to address extremely low concentrations (nano molar) of fluorescence species. Furthermore the method offers an extremely small observation volume (<1µm 3 ) that provides a great potential to locally access the systems which require high spatial resolution. Not surprisingly therefore, during the last decade, FCS has become one of the best tools to evaluate the diffusion and transport properties in complex systems. Mainly for traditional reasons, however, so far the utilization of the FCS has been limited mostly to biological studies, i.e. aqueous environments. [1, 10, 48, 49, 55-58] Only recently FCS was successfully applied to study the size and conformation of macromolecules in organic solvents [4, 9] , adsorbed polymers, [14-16] grafted gel, [8] diffusion in polymer solutions, [3, 5, 7, 11, 13] and thin polymer films. [2] Furthermore it was shown that FCS can follows the process of radical polymerization of styrene over an extensive conversion range [12] and address the swelling of crosslinked polymer microbeads in organic solvents. [17]
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Systematic characterization of extracellular vesicle sorting domains and quantification at the single molecule single vesicle level by fluorescence correlation spectroscopy and single particle imaging

Systematic characterization of extracellular vesicle sorting domains and quantification at the single molecule single vesicle level by fluorescence correlation spectroscopy and single particle imaging

Extracellular 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 Spectroscopy (FCS) we quantified the number of FP-tagged molecules per vesicle. We observed different loading efficiencies and specificities for the different proteins into EVs. For the candidates showing the highest loading efficiency in terms of engineering, the molecular levels in the vesicles did not exceed ca 40 –60 fluorescent proteins per vesicle upon transient overexpression in the cells. Some of the GFP- tagged EV reporters showed quenched fluorescence and were either non-vesicular, despite co- purification with EVs, or comprised a significant fraction of truncated GFP. The co-expression of each target protein with CD63 was further quantified by widefield and confocal imaging of single vesicles after double transfection of parent cells. In summary, we provide a quantitative comparison for the most commonly used sorting proteins for bioengineering of EVs and introduce a set of biophysical techniques for straightforward quantitative and qualitative characterization of fluores- cent EVs to link single vesicle analysis with single molecule quantification.
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Quantification of biomolecular binding dynamics by Fluorescence Correlation Spectroscopy

Quantification of biomolecular binding dynamics by Fluorescence Correlation Spectroscopy

Very shortly after the introduction of objective-type TIR-FCS, the group of Thorsten Wohland introduced electron-multiplying charge-coupled device (EMCCD) camera detec- tion instead of point detectors [Kannan et al., 2007]. The massive parallel detection boosts the multiplexing by exploiting the widefield excitation in TIRF microscopy, but comes at the cost of lower time resolution, and to date still lower quantum yield of the detec- tor, compared to point-detectors. In detail, the integrated signals from a set of region of interests (ROIs) are autocorrelated to sample the local dynamics. Thus, this approach has the potential to resolve maps of dynamics. Moreover, as the ROIs are defined dur- ing post-processing, their size can be systematically varied, based on which Bag et al. elegantly circumvented the need for calibration measurements before 2D diffusion mea- surements in SLBs [Bag et al., 2012]. To date, the potential of camera-based TIR-FCS was demonstrated in several studies [Guo et al., 2008, Sankaran et al., 2009, Bag et al., 2012,Lim et al., 2013,Bag et al., 2014,Huang et al., 2015]. It should be noted that in our view camera-based TIR-FCS is conceptually identical to the differently termed methods binned imaging FCS (bimFCS) [Lim et al., 2013,Huang et al., 2015], and temporal image correlation spectroscopy (TICS) using TIRF microscopy [Wiseman, 2013,Wiseman, 2015]. Camera-based TIR-FCS has developed into an powerful tool to measure lateral membrane diffusion, but except for the reported k d for doublecortin from surface immobilized micro- tubules [Brandão et al., 2014] has not been employed to study binding kinetics.
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Smart crowding in poly-Acrylamide/Poly(N-isopropylacrylamide) composite hydrogels, studied by two-focus fluorescence correlation spectroscopy

Smart crowding in poly-Acrylamide/Poly(N-isopropylacrylamide) composite hydrogels, studied by two-focus fluorescence correlation spectroscopy

Figure 4.2 Confocal fluorescence microscopy images of a core–shell microgel particle as sketched in Scheme 1. The images are taken at the equator plan of the core of a core–shell particle lying on the lower cover slide of the sample cell. a) – d): particle in pure water at different temperatures; the core diameter of about 60  m is retained upon heating and cooling. e): particle in water/MeOH mixture with 40 mol% of MeOH. Adding MeOH lead to deswelling of the core and the shell, but the shell is more swollen than the core. The shell thickness influences the deswelling of the core and leads to a deformation of the core in regions where the shell is thin (lower-left part of the particle); vice versa, this deformation also deforms the shell in these regions. No effect of adhesion between particle and cover slide on the deswelling of the core is visible. The scale bar in Panel e) equally applies to all panels.
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Dynamics of tracer particles in complex environments investigated by dual-focus fluorescence correlation spectroscopy

Dynamics of tracer particles in complex environments investigated by dual-focus fluorescence correlation spectroscopy

Dynamic Light Scattering (DLS) DLS measurements were performed on a standard ALV setup equipped with HeNe laser (JDS Uniphase, 633 nm, 35 mW), digital hardware correlator (ALV 5000), two avalanche photo diodes (SPCM-CD2969, Perkin Elmer), goniometer (CGS-8F, ALV), and light scattering electronics (LSE-5003, ALV). Highly diluted samples were prepared to prevent multiple scattering. Before measurements, the samples were filtered several times through regenerated cellulose filters (Sartorius) with a pore size of 0.2 µm. Measurements were recorded in pseudo cross-correlation mode. The scattering angle was varied between 30° and 142° in steps of 4° for PHEMA-b-PGlcNAcEMA and between 34° and 124° in steps of 3° for GS-II. Measurement times were 90 s and 240 s, respectively. The temperature was set to 25 ◦ C. Data were evaluated in the following way: intensity autocorrelation functions were transformed to electric field autocorrelation functions by use of the Siegert relation. A second-order cumulant analysis was applied to obtain the decay rate Γ 2 . The cumulant fit range was limited between the lag time of 10 µs and the lag time where the electric field autocorrelation function amplitude had decayed to 30% of the experimental maximum value. Γ 2 was plotted against the square of the scattering vector q, so that the z-averaged translational diffusion coefficient ¯ D was obtained from the following equation:
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Smart crowding in poly-Acrylamide/Poly(N-isopropylacrylamide) composite hydrogels, studied by two-focus fluorescence correlation spectroscopy

Smart crowding in poly-Acrylamide/Poly(N-isopropylacrylamide) composite hydrogels, studied by two-focus fluorescence correlation spectroscopy

Figure 4.2 Confocal fluorescence microscopy images of a core ʹshell microgel particle as sketched in Scheme 1. The images are taken at the equator plan of the core of a coreʹshell particle lying on the lower cover slide of the sample cell. a) ʹ d): particle in pure water at different temperatures; the core diameter of about 60Pm is retained upon heating and cooling. e): particle in water/MeOH mixture with 40 mol% of MeOH. Adding MeOH lead to deswelling of the core and the shell, but the shell is more swollen than the core. The shell thickness influences the deswelling of the core and leads to a deformation of the core in regions where the shell is thin (lower-left part of the particle); vice versa, this deformation also deforms the shell in these regions. No effect of adhesion between particle and cover slide on the deswelling of the core is visible. The scale bar in Panel e) equally applies to all panels.
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Applications of two focus fluorescence correlation spectroscopy in colloid and polymer science

Applications of two focus fluorescence correlation spectroscopy in colloid and polymer science

a) In a first step it was necessary to invent an extended theoretical model, which took into account size and labelling effects in 2fFCS data analysis, because impact of non-negligible particle size with different fluorescent labelling could not be neglected in FCS and 2fFCS measurements. Various labelling distributions and their influence on correlation func- tions were modelled and discussed. Performed DLS and 2fFCS measurements on large uniformly labelled latex beads of different size gave excellent agreement between DLS and 2fFCS measurements when the extended model was applied to 2fFCS data analysis. The experimentally determined values for the hydrodynamic radius as calculated with the ex- tended model were in perfect agreement with the specifications of the bead manufacturer as well as with the results from the DLS measurements. The proposed models for differ- ent labelling geometries allow utilization of 2fFCS (and also standard FCS) as a powerful complementary method to investigate hydrodynamic radii of extended diffusing objects at very low concentrations in colloid and polymer science, e.g. Ref. [98–105] . Nevertheless,
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Gutenberg Open Science: Monitoring kinetic processes in polymeric colloids by fluorescence correlation spectroscopy

Gutenberg Open Science: Monitoring kinetic processes in polymeric colloids by fluorescence correlation spectroscopy

In summary, we have presented a new method for studying the chain exchange kinet- ics in diblock copolymer micelles by using dual color fluorescence cross-correlation spectroscopy (DC FCCS). This technique employs tabletop equipment and fluores- cent labeling that makes it accessible to large research community and applicable to broad range of copolymer systems. We applied the new method to measure the exchange kinetics of micelles formed by a linear-brush copolymer PS-POEGMA, as a model system with short and bulky corona block. By varying the temperature and comparing the results with a scaling theory reported earlier, [ 37 , 38 , 45 ] we were able to quantify the extent of swelling of the PS micelle’s core and explain the fast exchange that takes place at temperatures well below the nominal glass transition of PS. Furthermore, we showed that the addition of small amounts of either good or bad solvent for the PS core had a tremendous effect on the exchange kinetics.
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Dynamics of tracer particles in complex environments investigated by dual-focus fluorescence correlation spectroscopy

Dynamics of tracer particles in complex environments investigated by dual-focus fluorescence correlation spectroscopy

Despite sometimes challenging labeling procedures, fluorescence-based methods are very promising for diffusion measurements due to their unprecedented selectivity. Fluorescence recovery after photobleaching (FRAP) 2 is based on the irreversible photobleaching of fluorescently labeled particles inside a region of interest by a laser pulse of high intensity. Hereafter, the unbleached particles from the surrounding region diffuse into the photo- bleached region. The diffusion coefficient is determined from the rate of the fluorescence recovery process. Although the total amount of sample is very small, a relatively high concentration of fluorophores is needed. Another drawback is the large measured region of interest limiting the spatial resolution to length scales of 10–100 µm. In microscopy-based single-particle tracking (SPT) 3 the positions of individual particles are recorded within the sample by a series of images. The resulting trajectories are analyzed with respect to the mean-squared displacement (MSD) of the particles as a function of lag time. The MSD is directly related to the translational diffusion constant, but this technique requires a large number of tracked particles to obtain good statistics and allows the study of slowly diffusing species only.
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Gutenberg Open Science: Complex tracer mobilities in polymer networks revealed by fluorescence correlation spectroscopy

Gutenberg Open Science: Complex tracer mobilities in polymer networks revealed by fluorescence correlation spectroscopy

was used (filtered through a MilliQ purification system, resistivity 18.2 MΩ∙cm) without any buffers. However, only for the study of antibody diffusion (4 th Chapter, section III) acetate buffer was used to swell the gels. The temperature-dependent swelling ratio, ( ), was determined as the ratio between the fully swollen thickness and the dry thickness. The dry thickness has been measured by a step profiler (KLA-Tencor Stylus P-16+ profilometer) as mentioned previously, while the thickness of the fully swollen hydrogels was determined using the FCS setup. By shifting the microscope objective, the position of the FCS observation volume was scanned in z-direction (normal to the film plane) with a step of 1 μm and the average fluorescence intensity signal that is proportional to the local density of the tracers was recorded as a function of the focus position (z-scan). A typical z-scan (Fig.3.1) depicts 2 transition regions, representing the hydrogel/water and glass/hydrogel interfaces. The distance between these regions represents the thickness of the fully swollen gel. The monomer concentrations (monomer volume fractions, ( ) ( )) for the studied hydrogels (HG) are shown in Tables 4.1 and 4.2(4 th
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Single-molecule multiparameter fluorescence spectroscopy reveals directional MutS binding to mismatched bases in DNA

Single-molecule multiparameter fluorescence spectroscopy reveals directional MutS binding to mismatched bases in DNA

with the absence of contact between MutS and the donor dye when the protein binds the mismatch with Phe-36 stacked on the T base in the lower strand ( Figure 1 C, Model II). The high r D population will be addressed further below. We were interested in determining if the unkinked G A :T D DNA is free or MutS-bound, and thus measured the DNA diffusion times by subensemble Fluorescence Correlation Spectroscopy (seFCS) of bursts selected for this FRET species from the data trace (see Supplementary Data Section 2.4 ). The obtained apparent diffusion times are displayed in the bar diagrams of Figure 4 A and B (left panel) for G A :T D DNA without and with MutS, respectively. The apparent diffusion times of both FRET species are clearly longer in the presence of MutS, which reveals that also unkinked DNA is in complex with MutS ( Supplementary Figure S1 ). This population corresponds to either a non-specific DNA–MutS complex (MutS bound to any position on DNA) or to a specific unkinked DNA–MutS complex (MutS bound at the mismatch site but without inducing a kink). The latter species has been proposed earlier, based on AFM and FRET analysis of immobilized DNA ( 36 , 37 ); however, it remains to be shown if MutS is in direct contact with the mismatch in the unkinked complex, and whether this complex is a bona fide intermediate in the mismatch rec- ognition pathway.
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Gutenberg Open Science: Grating coupled surface plasmon enhanced fluorescence spectroscopy

Gutenberg Open Science: Grating coupled surface plasmon enhanced fluorescence spectroscopy

Molecules which absorb radiation in the ultra-violet, the visible range or the near infra- red may re-emit light at longer wavelengths. This phenomenon is called fluorescence. Mole- cules exhibiting it are named fluorophores and are for example polyaromatic hydrocarbons or heterocycles. Fluorescence based detection methods are widely used in the fields of biology, chemistry and medicine and include fluorescence microscopy [Wan96], fluorescence correlation spectroscopy [Hes02], fluorescence recovery after photo bleaching (FRAP) [Joh96], flow cytom- etry [Giv01] and green fluorescent protein (GFP) expression studies [Tsi98]. One reason for this is the extraordinary sensitivity that can be achieved with fluorescent probes, even down to the single molecule level [Pla97]. The typical long time scale of 10 ns of the fluorescence cycle allows the study of a range of molecular processes that can occur during this period and alter the photophysical parameters of the fluorescence emission. Fluorescence based techniques have also become successful in the life sciences due to the fact that fluorophores are already present in many biological molecules (amino acids like tryptophan and tyrosine in proteins, GFP, co- factors like NADH, riboflavin, etc.) or synthetic dyes can readily be introduced via coupling chemistries acting on proteins, nucleotide bases, membrane lipids, antibodies and much more.
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