Spelling suggestions: "subject:"fluorescence correlation"" "subject:"afluorescence correlation""
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Photo-induced dark states influorescence spectroscopy – investigations & applicationsChmyrov, Andriy January 2010 (has links)
This thesis focuses on investigations of transient dark states of fluorescentmolecules using spectroscopic techniques. The main purpose is to show andconvince the reader that transient dark states are not always a nuisance, butalso represent an additional source of information. Several studies with fluorescencecorrelation spectroscopy were performed, all related to non-fluorescentstates such as triplet state or isomerized states.Photobleaching is one of the main problems in virtually all of the fluorescencetechniques. In this thesis, mechanisms that retard photobleaching arecharacterized. Several compounds, antioxidants and triplet state quenchers,which decrease photobleaching, are studied, and guidelines for achieving optimalfluorescence brightness using these compounds are presented.Triplet state quenching by several compounds was studied. Detailed investigationsof the fluorescence quencher potassium iodide demonstratedthat for some of fluorophores, except of quenching, there is fluorescence enhancementmechanism present. In agreement with the first publication inthis thesis, antioxidative properties were found to play an important role inthe fluorescence enhancement. Quenching of the triplet state is proposedas a tool for monitoring diffusion mediated reactions over a wide range offrequencies.Specially designed fluorophores combining high triplet yields with reasonablefluorescence brightness and photostability were characterized forpossible applications in novel super-resolution imaging techniques based onfluorescence photoswitching. Except of benefits for imaging techniques, photoinducedswitching to non-fluorescent states could be used for monitoringmolecular diffusion, which was also demonstrated in this thesis.Studies of the triplet state kinetics of fluorophores close to dielectric interfaceswere performed using fluorescence spectroscopy. The analysis of thetriplet state kinetic can provide information about the local microenvironmentand electrostatic interactions near dielectric interfaces. / QC 20100414
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Synthesis and Tracking of Fluorescent and Polymerization-Propelled Single-Molecule NanomachinesGodoy Vargas, Jazmin 24 July 2013 (has links)
This dissertation describes the synthesis of molecular machines designed to operate on surfaces (nanocars) or in the solution phase (nanosubmarines), and the study of their diffusion using fluorescence techniques. The design of these molecular machines is aimed to facilitate monitoring of their movement and incorporation of a source of energy for propulsion.
To complement previous scanning tunneling microscopy studies of the translation of nanocars on surfaces, chapter 1 describes the synthesis of a family of fluorescently tagged nanocars. The nanocars were functionalized with a tetramethylrhodamine isothiocyanate (TRITC) fluorescent dye. Single-molecule fluorescence microscopy (SMFM) studies of one of these nanocars revealed that 25% of the nanocars moved on glass. The SMFM results also suggested that the dye hindered the mobility of the nanocars.
Seeking to improve the mobility, chapter 2 presents the synthesis of a new set of fluorescent nanocars, featuring a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) dye embedded in their axles. The mobility of these inherently fluorescent nanocars on glass was nearly double than that of their TRITC-tagged predecessors. Their diffusion was also studied on reactive-ion-etched glass, and amino-functionalized glass. The results showed that the mobility is affected by the substrate.
To equip the nanocars with an energy input for propulsion, two nanocars functionalized with an olefin metathesis catalyst were synthesized, as described in chapter 3. The catalytic activity of these nanocars toward ring-opening metathesis polymerization (ROMP) in solution was similar to that of their parent catalysts.
As an alternative approach to investigate if chemical propulsion through a ROMP process can be achieved at the molecular level, chapter 4 presents the synthesis of a fluorescent ROMP catalyst, termed a nanosubmarine, and the study of its diffusion using fluorescence correlation spectroscopy (FCS). FCS results showed an increase of 20 ± 7% in the diffusion constant of this nanosubmarine in presence of its fuel, cis,cis-1,5-cyclooctadiene.
Overall, the work accomplished in this dissertation constitutes a step forward toward development of easily tracked and highly mobile nanocars, and paves the way for the synthesis of truly nanosized chemically propelled molecular machines that operate in the solution phase.
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Quantifying the diffusion of membrane proteins and peptidesWeiß, Kerstin 05 August 2013 (has links)
No description available.
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Spatial-temporal actin dynamics during synaptic plasticity of single dendritic spine investigated by two- photon fluorescence correlation spectroscopyChen, Jian Hua 24 June 2013 (has links)
No description available.
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Development and application of ultra-sensitive fluorescence spectroscopy and microscopy for biomolecular interaction studiesXu, Lei January 2014 (has links)
This thesis describes the development of sensitive and high-resolution fluorescence spectroscopic and microscopic techniques and their application to probe biomolecules and their interactions in solution, lipid membrane model systems and in cells. Paper I-IV are largely focused on methodological developments. In paper I, a new fluorescence method based on fluorescence correlation spectroscopy (FCS) for detecting single particles was realized, requiring no fluorescent labeling of the particles. The method can yield information both about the diffusion properties of the particles as well as about their volumes. In paper II, a modified fluorescence cross correlation spectroscopy procedure with well characterized instrumental calibration was developed and applied to study cis interactions between an inhibitory receptor and its Major Histocompatibility Complex class I ligand molecule, both within the same cellular membranes. The quantitative analysis brought new insights into the Nature killer cell’s self-regulating of tolerance and aggressiveness for immune responses. Paper III describes a multi-color STED (STimulated Emission Depletion) microscopy procedure, capable of imaging four different targets in the same cells at 40nm optical resolution, which was developed and successfully demonstrated on platelets. In paper IV, a modified co-localization algorithm for fluorescence images analysis was proposed, which is essentially insensitive to resolutions and molecule densities. Further, the performance of this algorithm and of using STED microscopy for co-localization analysis was evaluated using both simulated and experimentally acquired images. Papers V-VII have their main emphasis on the application side. In paper V, transient state imaging was demonstrated on live cells to image intracellular oxygen concentration and successfully differentiated different breast cancer cell lines and the different metabolic pathways they adopted to under different culturing conditions. Paper VI describes a FCS-based study of proton exchange at biological membranes, the size-dependence of the membrane proton collecting antenna effect as well as effects of external buffer solutions on the proton exchange, in a nanodisc lipid membrane model system. These findings provide insights for understanding proton transport at and across membranes of live cells, which has a central biological relevance. In paper VII, STED imaging and co-localization analysis was applied to analyze cell adhesion related protein interactions, which are believed to have an important modulating role for the proliferation, differentiation, survival and motility of the cells. The outcome of efforts taken to develop means for early cancer diagnosis are also presented. It is based on single cells extracted by fine needle aspiration and the use of multi-parameter fluorescence detection and STED imaging to detect protein interactions in the clinical samples. Taken together, detailed studies at a molecular level are critical to understand complex systems such as living organisms. It is the hope that the methodologies developed and applied in this thesis can contribute not only to the development of fundamental science, but also that they can be of benefit to mankind in the field of biomedicine, especially with an ultimate goal of developing novel techniques for cancer diagnosis. / <p>QC 20140609</p>
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Single molecule tracking studies of flow-aligned mesoporous silica monoliths: pore order and pore wall permeabilityPark, Seok Chan January 1900 (has links)
Doctor of Philosophy / Department of Chemistry / Daniel A. Higgins / This dissertation describes single-molecule tracking (SMT) studies for the quantitative characterization of one-dimensional (1D) nanostructures in surfactant-templated mesoporous silica monoliths prepared within microfluidic channels. Single molecule diffusion of fluorescent probe molecules within the cylindrical mesopores reflects microscopic morphologies and mass-transport properties of the materials with high temporal and spatial resolution. The pore organization and materials order are initially investigated as a function of sol aging prior to loading into the microfluidic channels. Mesopores in these materials are templated by Cetyltrimethylammonium bromide (CTAB). Wide-field fluorescence videos depict 1D motion of the dyes within the individual mesopores. Orthogonal regression analysis of these motions provides a measure of the mesopore orientation. Channels filled prior to gelation of the sol produce monoliths incorporating large monodomains with highly aligned mesopores. In contrast, channels filled close to or after gelation yield monoliths with misaligned pores that are also more disordered. Two-dimensional (2D) small angle X-ray scattering (SAXS) experiments support the results obtained by SMT. These studies help to identify conditions under which highly aligned mesoporous monoliths can be obtained and also demonstrate the utility of SMT for characterization of mesopore order.
The non-ionic surfactant Pluronic F127 is also utilized as the structural-directing agent. The diffusive motions of PDI dyes that are uncharged, cationic and anionic are explored by SMT and fluorescence correlation spectroscopy (FCS). The SMT studies for the uncharged dye show development of 1D diffusion along the flow direction while charged dyes exhibit predominant isotropic diffusion, with each of these behaviors becoming more prevalent as a function of aging time after filling of the microfluidic channels. SMT studies from silica-free F127 gels suggest that partitioning plays a important role in governing the diffusion behavior of the PDI dyes within the surfactant-filled mesopores. FCS results exhibit similar mean diffusion coefficients for all three dyes that suggest these dyes diffuse through similar sample regions. These studies demonstrate that the silica pore walls in the mesoporous silica monoliths remain permeable after gelation and that partitioning of solute species to different regions within the pores plays an important role in restricting the dimensionality of their diffusive motion
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Investigating Dynamics Using Three Systems: Cy3 on DNA, ME1 Heterodimers, and DNA Processivity ClampsJanuary 2015 (has links)
abstract: Biophysical techniques have been increasingly applied toward answering biological questions with more precision. Here, three different biological systems were studied with the goal of understanding their dynamic differences, either conformational dynamics within the system or oligomerization dynamics between monomers. With Cy3 on the 5' end of DNA, the effects of changing the terminal base pair were explored using temperature-dependent quantum yields. It was discovered, in combination with simulations, that a terminal thymine base has the weakest stacking interactions with the Cy3 dye compared to the other three bases. With ME1 heterodimers, the goal was to see if engineering a salt bridge at the dimerization interface could allow for control over dimerization in a pH-dependent manner. This was performed experimentally by measuring FRET between monomers containing either a Dap or an Asp mutation and comparing FRET efficiency at different pHs. It was demonstrated that the heterodimeric salt bridge would only form in a pH range near neutrality. Finally, with DNA processivity clamps, one aim was to compare the equilibrium dissociation constants, kinetic rate constants, and lifetimes of the closed rings for beta clamp and PCNA. This was done using a variety of biophysical techniques but with three as the main focus: fluorescence correlation spectroscopy, single-molecule experiments, and time-correlated single photon counting measurements. The stability of beta clamp was found to be three orders of magnitude higher when measuring solution stability but only one order of magnitude higher when measuring intrinsic stability, which is a result of salt bridge interactions in the interface of beta clamp. Ongoing work built upon the findings from this project by attempting to disrupt interface stability of different beta clamp mutants by adding salt or changing the pH of the solution. Lingering questions about the dynamics of different areas of the clamps has led to another project for which we have developed a control to demystify some unexpected similarities between beta clamp mutants. With that project, we show that single-labeled and double-labeled samples have similar autocorrelation decays in florescence correlation spectroscopy, allowing us to rule out the dyes themselves as causing fluctuations in the 10-100 microsecond timescale. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2015
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Espectroscopia de correlação de fluorescência aplicada em estudos de sistemas moleculares, biológicos e celulares / Fluorescence correlation spectroscopy applied in studies of molecular, biological and cellular systemsFernando Massayuki Tsutae 24 May 2016 (has links)
A espectroscopia de correlação de fluorescência (FCS) é uma das diferentes técnicas de análise por imagens de alta resolução espacial e temporal de biomoléculas em concentrações extremamente baixas. Ela se tornou uma técnica extremamente poderosa e sensível em áreas como bioquímica e biofísica. Como uma técnica bem estabelecida, ela é utilizada para medir concentrações locais de biomoléculas, através da marcação com moléculas fluorescentes. Coeficientes de difusão e constantes cinéticas também podem ser medidos através de FCS assim como detecção de molécula única. Ela também pode dar informação precisa sobre interações de antígeno-anticorpo, ácidos nucleicos e proteínas. Através de uma combinação de marcadores de alto rendimento quântico, fontes de luz estável (lasers), detecção ultrassensível e microscopia confocal, é possível realizar medidas de FCS em volumes de fentolitros (fL) e em concentrações de nanomolar (nM) em soluções aquosas ou em células vivas. Em contraste com outras técnicas de fluorescência, a sensibilidade da FCS aumenta com a diminuição da concentração do fluoróforo marcador, porque o parâmetro de interesse não é a intensidade de emissão de fluorescência, mas sim as flutuações espontâneas da fluorescência. Durante o tempo em que a partícula ou molécula atravessa o volume de medida pode ocorrer mudanças conformacionais e reações químicas e fotofísicas que alteram as características de emissão do fluoróforo e causam flutuações no sinal detectado. Estas flutuações são então monitoradas e transformadas em uma curva de autocorrelação, por intermédio de um software comercial que emprega um modelo físico apropriado para FCS. Em nosso estudo, utilizamos um marcador comercial (ALEXA 488®) para marcar proteínas. Primeiramente utilizamos a técnica de FCS para medir concentrações extremamente baixas de marcadores fluorescentes. Também realizamos um experimento testando a influência da viscosidade do meio na difusão livre do fluoróforo, assim como as melhores condições em que temos um melhor sinal de FCS. Por fim, estudamos a difusão de proteínas marcadas (PUC II e IV) em meio aquoso (PBS) e no interior de células. / Fluorescence correlation spectroscopy (FCS) is one of the many different modes of high-resolution spatial and temporal analysis of extremely low concentrated biomolecules. It has become a powerful and sensitive tool in fields like biochemistry and biophysics. As a well established technique, it is used to measure local concentrations of fluorescently labeled biomolecules, diffusion coefficients, kinetic constants and single molecule studies. Through a combination of high quantum yield fluorescent dyes, stable light sources (lasers), ultrasensitive detection and confocal microscopy is possible to perform FCS measurements in femtoliters volumes and nanomolar concentrations in aquous solution or in live cells. Unlike with other fluorescence technics, its sensibility increases with the decrease of dye concentrarion, because the main factor is not the emission intensity itself. Instead this, spontaneous statistical fluctuation of fluorescence becomes the main factor in FCS analisys. During the time that the conjugated-dye cross the volume detection can occur conformational changes, chemical reaction and photophysical processes that can change the emission properties of the dye and, then, change the detected sinal. This fluctuations are tracked and changed into a autocorrelation curve, by a specific software, appropriate to perform FCS analisys. In our study, we use comercial dye (Alexa 488) to label proteins. Firstly, we applied FCS to measure extremally diluted concentrations of dyes (~1 nM). We have performed experiments testing the influence of the viscosity medium in the free difusion of the dyes and the optical apparatus and conditions that result in the best FCS signal. We also have studied protein diffusion (PUC II e IV) in aquous medium (PBS) and toward the inner of the cells.
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Fluorescence correlation spectroscopy for studying intermediate filament assemblySchroeder, Viktor 04 August 2017 (has links)
No description available.
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Single-Focus Confocal Data Analysis with Bayesian NonparametricsJanuary 2020 (has links)
abstract: The cell is a dense environment composes of proteins, nucleic acids, as well as other small molecules, which are constantly bombarding each other and interacting. These interactions and the diffusive motions are driven by internal thermal fluctuations. Upon collision, molecules can interact and form complexes. It is of interest to learn kinetic parameters such as reaction rates of one molecule converting to different species or two molecules colliding and form a new species as well as to learn diffusion coefficients.
Several experimental measurements can probe diffusion coefficients at the single-molecule and bulk level. The target of this thesis is on single-molecule methods, which can assess diffusion coefficients at the individual molecular level. For instance, super resolution methods like stochastic optical reconstruction microscopy (STORM) and photo activated localization microscopy (PALM), have a high spatial resolution with the cost of lower temporal resolution. Also, there is a different group of methods, such as MINFLUX, multi-detector tracking, which can track a single molecule with high spatio-temporal resolution. The problem with these methods is that they are only applicable to very diluted samples since they need to ensure existence of a single molecule in the region of interest (ROI).
In this thesis, the goal is to have the best of both worlds by achieving high spatio-temporal resolutions without being limited to a few molecules. To do so, one needs to refocus on fluorescence correlation spectroscopy (FCS) as a method that applies to both in vivo and in vitro systems with a high temporal resolution and relies on multiple molecules traversing a confocal volume for an extended period of time. The difficulty here is that the interpretation of the signal leads to different estimates for the kinetic parameters such as diffusion coefficients based on a different number of molecules we consider in the model. It is for this reason that the focus of this thesis is now on using Bayesian nonparametrics (BNPs) as a way to solve this model selection problem and extract kinetic parameters such as diffusion coefficients at the single-molecule level from a few photons, and thus with the highest temporal resolution as possible. / Dissertation/Thesis / Source code related to chapter 3 / Source code related to chapter 4 / Doctoral Dissertation Physics 2020
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