Dissertations / Theses on the topic 'Fluorescence Microscopy, Image Correlation Spectroscopy'

This thesis explores the benefits of intensity modulation for the purpose of extending the range of applications of fluorescence spectroscopy and imaging in cellular and molecular biology and medicine. Long-lived transient states of fluorescent molecules can, because of their long lifetimes, be used to detect subtle changes in the microenvironment of the molecule. A method for determining the kinetic rates for transitions to and from such states by registration of changes in the average fluorescence intensity related to different modulation of the excitation source is introduced. It combines the detection sensitivity of fluorescence with the environmental sensitivity of the long-lived transient states and allows the use of slow detectors such as CCD cameras, making parallelization and wide-field imaging possible developments. An extension of this method, generating image contrast based on triplet state population using a standard laser scanning microscope, is also shown. A strategy to combine fluorescence correlation spectroscopy (FCS) with modulated excitation, in a way that allows extraction of correlation data for all correlation times, is presented. This enables the use of modulation to optimize measurement conditions with respect to photophysical properties of the dyes used. FCS with modulated excitation will probably prove useful in future studies involving multiple kinetic processes occurring in overlapping time ranges. One of the ideas from this project also constitutes a powerful method for generating artifact free correlation curves from data sets where sections have been removed. This is potentially very useful in biological studies where spikes in the measurements often cause problems. In the final project, cross-correlation and alternating excitation are combined in measurements on a pH-sensitive ratiometric dye to clearly distinguish the protonation–deprotonation dynamics from other processes. The presented approach makes the protonation related fluctuations manifest themselves as a very distinct anti-correlating component in the correlation curve. This enables robust data analysis using a simple model.<br>QC 20100805

Blood flow through microcirculation in both simple and complex geometry has been difficult to predict due to the composition and complex behavior of blood at the microscale. Blood is a dense suspension of deformable red blood cells that is comparable in dimensions to the microchannels that it flows through. As a result, rheological properties at the microscale can vastly differ from bulk rheological properties due to non-continuum effects. To further develop our understanding of blood microflow; experimental techniques should be explored. In this work, we explore micro-particle image velocimetry (μPIV) and two-beam fluorescence cross-correlation spectroscopy (2bFCCS) in the application of characterizing blood in microflow conditions. For the development of the μPIV analysis, a polydimethylsiloxane co-flow channel is used to observe blood flow in controlled conditions. Flow conditions (velocity profile and blood layer thickness) are selected based on an analytical model and compared to experimental measurement. The experimental results presented indicate that current flow conditions are inadequate in providing a controlled rate of shear on the blood layer in the co-flow channel and further optimization are required to improve the measurement of the velocity profile. For the development of the 2bFCCS application for blood flow analysis, a wide glass capillary microfluidic device is used to complete the verification of fluorescence fluid admissibility, the effect of laser intensity on inducing photobleaching and the velocity measurement performance. The experimental measurement of the velocity profile is validated against the theoretical profile for a rectangular channel. Results of the velocity profile of high concentration red blood cells show promise in the technique’s ability to measure blood microflows closer to physiological conditions.

The object of this thesis is to present two novel applications of Spatiotemporal Image Correlation Spectroscopy (STICS) to biological systems. STICS is a technique which uses the correlations in pixel intensity fluctuations of an image time series, captured under fluorescence microscopy, to measure the speed and direction of a flowing population of fluorescently labeled molecules. The method was first applied to measure the dynamics of transport vesicles inside growing pollen tubes of lily flowers. The measured vector maps allowed to confirm the presence of actin filaments along the periphery of the tubes, as well as the presence of a reverse-fountain pattern in the apical region. In a second set of experiments, STICS was used to measure the retrograde flow of filamentous actin in migrating chick DRG neuronal growth cones. These results serve as proof of principle that STICS can be used to probe the response of the growth cone cytoskeleton to external chemical cues.

Thesis advisor: Mary F. Roberts<br>Thesis advisor: Steven D. Bruner<br>The bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis is specifically activated by low concentrations of a non-substrate lipid, phosphatidylcholine (PC), presented as an interface. However, if the PC concentration in the interface is too high relative to substrate, the enzyme exhibits surface dilution inhibition. Understanding this bacterial enzyme, which shares many kinetic features with the larger and more complex mammalian PI-PLC enzymes, requires elucidating the mechanism for PC activation and inhibition. Various techniques were applied to study the interaction of the protein with vesicles composed of both the activator lipid PC and the substrate lipid (or a nonhydrolyzable analogue). Fluorescence correlation spectroscopy (FCS), used to monitor bulk partitioning of the enzyme on vesicles, revealed that both the PC and the substrate analogue are required for the tightest binding of the PI-PLC to vesicles. Furthermore, the tightest binding occurred at low mole fractions of substrate-like phospholipids. Field cycling 31P NMR (fc-P-NMR) spin-lattice relaxation studies provided information on how bound protein affects the lipid dynamics in mixed substrate analogue/PC vesicles. The combination of the two techniques could explain the enzyme kinetic profile for the PC activation and surface dilution inhibition: small amounts of PC in an interface enhanced PI-PLC binding to substrate-rich vesicles while high fractions of PC tended to sequester the enzyme from the bulk of its substrate leading to reduced specific activity. FCS binding profiles of mutant proteins were particularly useful in determining if a specific mutation affected a single or both phospholipid binding modes. In addition, an allosteric PC binding site was identified by fc-P-NMR and site directed spin labeling. A proposed model for PC activation suggested surface-induced dimerization of the protein. Experiments in support of the model used cysteine mutations to create covalent dimers of this PI-PLC. Two of these disulfide linked dimers, formed from W242C or S250C, exhibited higher specific activities and tighter binding to PC surfaces. In addition, single molecule total internal reflection fluorescence microscopy was used to monitor the off-rate of PI-PLC from surface tethered vesicles, providing us with a direct measure of off-rates of the protein from different composition vesicles<br>Thesis (PhD) — Boston College, 2009<br>Submitted to: Boston College. Graduate School of Arts and Sciences<br>Discipline: Chemistry

The cell cycle culminates with the segregation of sister chromatids, which is a fundamental step in ensuring the transmission of unaltered genetic material. Chromosome segregation is carried out by the mitotic spindle, which captures and pulls sister chromatids towards the opposite poles. Anaphase starts when the correct bipolar attachment is achieved. Chromosomes migrate evenly to the two daughter cells, both inheriting the same genetic material. The presence of unattached kinetochore at anaphase onset is dangerous, since it may lead to unbalanced ploidy of daughter cells, with severe consequences for their survival. For this reason, improperly attached chromosomes activate the mitotic checkpoint that arrests cell division before anaphase. Cells can maintain an arrest for several hours but eventually will resume proliferation, a process we refer to as adaptation. Whether adapting cells bypass an active block or whether the block has to be removed to resume proliferation is not clear. Likewise, it is not known whether all cells of a genetically homogeneous population are equally capable to adapt. Here, we show that the mitotic checkpoint is operational when yeast cells adapt and that each cell has the same propensity to adapt. Our results are consistent with a model of the mitotic checkpoint where adaptation is driven by random fluctuations of APC/CCdc20 , the molecular species inhibited by the checkpoint. Our data provide a quantitative framework for understanding how cells overcome a constant stimulus that halts cell cycle progression.

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&reg;) 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.<br>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.

This thesis work aims to present a novel approach to reconstruct Structured Illumination Microscopy, SIM, raw data and to analyze SIM reconstructed images. These new approaches will be demonstrated in the study of chromatin organization. The dissertation will be articulated as follows: Chapter 1 provides an introduction to chromatin nanoscale organization and optical fluorescence microscopy, which is one of the main tools involved in life sciences studies. Indeed, optical microscopy allowed investigating, with high specificity and sensitivity, living samples such as cells, and even tissues. The reader will be presented with a summary on the fluorescence optical microscopy and on the super-resolution, SR, techniques available today including SIM, which is the microscope used in this thesis work. In Chapter 2 the focus is on the introduction of a new reconstruction tool for specific SR-SIM microscopy powered by the Separation of Photons by Lifetime Tuning, SPLIT, method. The introduction of the concept, applied in other works to different SR techniques, will be followed by the practical implementation of the method on the SIM microscope. Then, the applicability of the technique, which we called SPLIT-SIM, will be demonstrated on several different samples. Indeed, it will be used on Simulated data, on test experimental beads, on biological samples both in one and two-color staining. In Chapter 3 the focus will move on the coupling of SIM reconstructed data to colocalization analysis. In particular, for the first time, SIM was coupled to Image Cross-Correlation Spectroscopy, ICCS, in the study of two-color images of a model sample. DNA origami-based structures were chosen as a model sample with precise distances allowing for evaluation of the analysis results. Moreover, all the images analyzed by the pixel-based technique, SIM-ICCS, were analyzed also with an object-based technique as a comparison to evaluate which could be the best choice in SIM acquisitions. Finally, Chapter 4 will be focused on the application of the analysis, performed in chapter 3, to two-color SIM images of nuclear structure. The analysis will be performed on ‘positive control’ in which the target structures will be colocalized and on a negative control in which the structured are spatially segregated within the nucleus. Both object-based and pixel-based analysis will be able to extract coherent results thus showing how SIM-ICCS can become an interesting and useful tool to analyze SIM multicolor acquisitions.

The diffusible soluble oligomeric amyloid β-peptide (Aβ) has been identified as a toxic agent in Alzheimer’s disease that can cause synaptic dysfunction and memory loss, indicating its role as potential therapeutic targets for AD treatment. Recently an oligomer-specific sandwich biotin-avidin interaction based assay identified the Aβ oligomer dissociation potency of a series of dihydroxybenzoic acid (DHBA) isomers. Because the sandwich assay is an ensemble method providing limited size information, fluorescence correlation spectroscopy (FCS) was employed to provide single molecule resolution of the disassembly mechanism. Using FCS coupled with atomic force microscopy, we investigated the size distribution of fluorescein labeled synthetic Aβ oligomers at physiological concentrations, and monitored in real time the change of size and mole fraction of oligomers in the presence of dissociating agents or conditions. The higher-order dissociation process caused by DHBA isomers produced no transient oligomeric intermediates, a desirable feature for an anti-oligomer therapeutic. Urea and guanidine hydrochloride, in contrast, produced a linear dissociation with a progressive decrease of size and mole fraction of oligomers. FCS allows the facile distinction of small molecule Aβ oligomer dissociators that do not produce stable potentially toxic oligomeric Aβ intermediates.

La membrane plasmique constitue la première entité séparant la cellule de son environnement. A ce rôle de barrière s'ajoute celui de réguler la. Par conséquent, la membrane plasmique est une zone privilégiée pour le passage d'information. Cependant, son étude reste difficile, ne serait-ce que par l'extraordinaire complexité d'organisation de cet assemblage supramoléculaire.Mon projet de thèse vise à développer de nouvelles approches expérimentales pour explorer plus spécifiquement l'organisation et le rôle de la membrane plasmique d'une cellule dans les mécanismes de transduction de l'information. Deux axes ont été privilégiés : le premier, concerne la description de la dynamique d'organisation de la membrane ; le deuxième concerne l'inter-connectivité et la transmission du signal d'une cellule avec d'autres partenaires.Ce manuscrit se compose de plusieurs parties. Le premier chapitre introduira succinctement les questions biologiques. Dans le second chapitre, je présenterai des méthodes utilisées pour l'étude de la membrane. J'y présenterai aussi une série d'observation que j'ai réalisée sur la diffusion de l'EGFR. Le troisième chapitre sera consacré à la technique de corrélation croisée de fluorescence depuis le montage jusqu'à l'étude du modèle EGFR. Dans la quatrième partie, nous verrons comment les collaborations à l'interface biophysique ont permis des développements innovants sur un système de pinces optiques holographiques. J'y présenterai les applications de ce système à différent modèles d'intérêt biologique. Enfin, je conclurai ce document par une brève discussion autour des résultats obtenus aussi bien d'un point de vue méthodologique que biologique<br>The plasma membrane separates the cell from its environment. But it is more than a barrier any cell has to communicate with the outside world. Therefore the plasma membrane plays a prime role in transferring and exchanging information. However, the biological study of the plasma membrane remains difficult due to the extraordinary complexity of it organization.My thesis is a part of an effort to develop new experimental approaches to explore more specifically the organization and the role of the plasma membrane in the signal transduction mechanisms. Two major aspects were followed: the first one concerns the description of the dynamics of membrane organization and of molecular interactions, the second concerns the inter-connectivity and signal transduction between a cell and other biological partners.This manuscript is composed of several parts. The first chapter briefly introduces the biological questions that I tried to answer. In the second chapter, I present the methods commonly used to study the membrane with a dynamic perspective. Additionally, I include a series of observations that I made on the EGF receptor diffusion. The third chapter is devoted to the fluorescence cross-correlation technique to study the assembly of the EGFR. In the fourth part, I demonstrate how scientific collaborations at the interface between biology and physics have led to the development of innovative solutions on a holographic optical tweezers system. I present applications of this system in different biological models. Finally, I conclude this thesis with a brief discussion about my technological and biological results

In the frame of the research in biology and in particular in regenerative medicine, it is widely requested the ability to perform measurements that have a low impact on the observed biological systems. Many measurements imply sample modifications and also sample fixation avoiding living samples measurements. In this doctoral thesis it is presented the realization of an advanced optical multimodal microscope that integrates coherent anti-Stokes Raman scattering, second harmonic generation and two-photon excitation fluorescence techniques in a single powerful tool. The combination of all these microscopy techniques in a single microscope allows gathering more information during samples imaging, implementing fluorescence technique with label free techniques. A description of the experimental setup of the realized multimodal microscope is presented together with the metrological characterization of the instrument, evaluating the main uncertainty sources that influence the measurement processes. Label free microscopy techniques allow performing measurements on biological samples with low invasiveness, since to image the specimens it is not demanded any specific sample preparation. This characteristic leads to measurements on living samples with a true low impact, opening new avenues on the research in biology and in particular way in regenerative medicine. Novel applications of these microscopy techniques are presented to study the extracellular matrix production in both fixed and living samples, as well as to characterize the scaffolds topology and the scaffold-cells interactions in a time-lapse experiment using living samples. This doctorate thesis is composed by a state of the art chapter in which are discussed the advanced nonlinear optical microscopy techniques from a theoretical point of view, the main experimental implementations of CARS microscopy and the main parameters and properties to be measured relevant in regenerative medicine applications and products. A chapter is dedicated to the experimental setup for the realization of the multimodal CARS-SHG-TPEF microscope at the I.N.Ri.M. laboratory. A specific chapter is dedicated to the study of the main sources of uncertainty of the measurements using CARS, TPEF and SHG techniques. A chapter in which are discussed the biological experiments realized using the multimodal CARS-SHG-TPEF microscope and at the end a final chapter with the conclusion of this doctorate thesis. This study has been conducted at the Italian institute of metrology (Istituto Nazionale di Ricerca Metrologica, I.N.Ri.M.) as part of the projects REGENMED, METREGEN and ACTIVE with funding respectively from the UE (ERA-NET plus Grant Agreement No 217257) and from Piedmont Region on UE under the programs CIPE 2007- converging technologies, grant 0126000010-METREGEN and POR-FESR I-I.1.3-I1.1 - ACTIVE. The main accomplished results are: • The development of the whole experimental multimodal CARS-SHG-TPEF microscopy system • The theoretical study of the main sources of uncertainty in the measurements with CARS, TPEF and SHG techniques. • The realization of biological experiments using these microscopy techniques: o To study the collagen production from fixed histological sections of human dermal fibroblasts cultured in fibrin gel scaffold using CARS and SHG techniques o To study the collagen production by live human fibroblasts and mesenchymal stem cells cultured in fibrin gel scaffold using CARS and SHG techniques o To characterize polymeric scaffolds in culture media with a label-free method using CARS and SHG techniques o To study the colonization in a two days time-lapse experiment of a polymeric scaffold by human mesenchymal stem cells stained with calcein using CARS and TPEF techniques

The diploma thesis deals with the registration of images taken with the multimodal holographic microscope (MHM). The summary covers the fluorescent and holographic microscopy, and the multimodal holographic microscope combining both these microscopy types. Every pair of the images needs to be aligned in order to gain new information by combining both image types. The thesis contains an algorithm that registers images by phase correlation as well as a process created in MATLAB in accordance with the algorithm. The most important procedure parameters’ influence on the registration success is described and the results are annotated.

Endocytosis is one of the most fundamental mechanisms by which the cell communicates with its surrounding. Specific signals are transduced through the cell membrane by a complex interplay between proteins and lipids. Clathrin depended endocytosis is one of important signalling pathways which leads to budding of the plasmalemma and a formation of endosomes. The FCHo2 is an essential protein at the initial stage of the this process. In is a membrane binding protein containing BAR (BIN, Amphiphysin, Rvs) domain which is responsible for a membrane binding. Although numerous valuable work on BAR proteins was published recently, the mechanistic description of a BAR domain functionality is missing. In present work we applied in vitro systems in order to gain knowledge about molecular basis of the activity of the FCHo2 BAR domain. In our studies we used supported lipid bilayers (SLBs) and lipid monolayers as s model membrane system. The experiments were carried out with a minimal number of components including the purified FCHo2 BAR domain. Using SLBs we showed that the BAR domain can bind to entirely flat bilayers. We also demonstrated that these interactions depend on the negatively charged lipid species incorporated in the membrane. We designed an assay which allows to quantify the membrane tubulation. We found out that the interaction of the FCHo2 BAR domain with the lipid membrane is concentration dependent. We showed that an area of the bilayer deformed by the protein depends on the amount of the used BAR domain. In order to study the relation between the mobility of lipids and the activity of FCHo2 BAR domain we designed a small-volume monolayer trough. The design of this micro-chamber allows for the implementation of the light microscopy. We demonstrated that the measured lipid diffusion in the monolayer by our new approach is in agreement with literature data. We carried out fluorescence correlation spectroscopy (FCS) experiments at different density of lipids at the water-air interface.We showed that the FCHo2 BAR domain binding affinity is proportional to the mean molecular area (MMA). We additionally demonstrated that the increased protein binding is correlated with the higher lipid mobility in the monolayer. Additionally, by curing out high-speed atomic force microscopy (hsAFM) we acquired the structural information about FCHo2 BAR domains orientation at the membrane with a high spatio-temporal resolution. Obtained data indicate the BAR domains interact witheach other by many different contact sites what results in a variety of protein orientations in a protein assemble.

The goal of this project is to study novel nanoscale excitation volumes, sensitive enoughto study individual chromophores and go on to study new and exciting self assemblyapproaches to this problem. Small excitation volumes may be engineered using light con-finement inside apertures in metal films. These apertures enhance fluorescence emissionrates, quantum yields, decrease fluorescence quenching, enable higher signal-to-noiseratios and allow higher concentration single chromophore fluorescence, to be studied byrestricting this excitation volume. Excitation volumes are reported on using the chro-mophore's fluorescence by utilising fluorescence correlation spectroscopy, which monitorsfluctuations in fluorescence intensity. From the correlation in time, we can find the res-idence time, the number of chromophores, the volume in which they are diffusing andtherefore the fluorescence emission efficiency. Fluorescence properties are a probe ofthe local environment, a particularly powerful tool due to the high brightness (quantumyield) fluorescent dyes and sensitive photo-detection equipment both of which are readilyavailable, (such as avalanche photodiodes and photomultiplier tubes). Novel materialscombining the properties of conducting and non-conducting materials at scales muchsmaller than the incident wavelength are known as meta-materials. These allow combi-nations of properties not usually possible in natural materials at optical frequencies. Theproperties reported so far include; negative refraction, negative phase velocity, fluorescenceemission enhancement, lensing and therefore light confinement has also been proposed tobe possible. Instead of expensive and slow lithography methods many of these materialsmay be fabricated with self assembly techniques, which are truly nanoscopic and otherwiseinaccessible with even the most sophisticated equipment. It was found that nanoscaled volumes from ZMW and HMMs based on NW arrays wereall inefficient at enhancing fluorescence. The primary cause was the reduced fluorescencelifetime reducing the fluorescence efficiency, which runs contrary to some commentatorsin the literature. NW based lensing was found to possible in the blue region of the opticalspectrum in a HMM, without the background fluorescence normally associated with a PAAtemplate. This was achieved using a pseudo-ordered array of relatively large nanowireswith a period just smaller than lambda / 2 which minimised losses. Nanowires in the traditionalregime lambda / 10 produced significant scattering and lead to diffraction, such that they werewholly unsuitable for an optical lensing application.

A new method, probability distribution of diffusivities (time scaled square displacements between succeeding video frames), was developed to analyze single molecule tracking (SMT) experiments. This method was then applied to SMT experiments on ultrathin liquid tetrakis(2-ethylhexoxy)silane (TEHOS) films on Si wafer with 100 nm thermally grown oxide, and on thin semectic liquid crystal films. Spatial maps of diffusivities from SMT experiments on 220 nm thick semectic liquid crystal films reveal structure related dynamics. The SMT experiments on ultrathin TEHOS films were complemented by fluorescence correlation spectroscopy (FCS). The observed strongly heterogeneous single molecule dynamics within those films can be explained by a three-layer model consisting of (i) dye molecules adsorbed to the substrate, (ii) slowly diffusing molecules in the laterally heterogeneous near-surface region of 1 - 2 molecular diameters, and (iii) freely diffusing dye molecules in the upper region of the film. FCS and SMT experiments reveal a strong influence of substrate heterogeneity on SM dynamics. Thereby chemisorption to substrate surface silanols plays an important role. Vertical mean first passage times (mfpt) in those films are below 1 µs. This appears as fast component in FCS autocorrelation curves, which further contain a contribution from lateral diffusion and from adsorption events. Therefore, the FCS curves are approximated by a tri-component function, which contains an exponential term related to the mfpt, the correlation function for translational diffusion and a stretched exponential term for the broad distribution of adsorption events. Lateral diffusion coefficients obtained by FCS on 10 nm thick TEHOS films, thereby, are effective diffusion coefficients from dye transients in the focal area. They strongly depend on the substrate heterogeneity. Variation of the frame times for the acquisition of SMT experiments in steps of 20 ms from 20 ms to 200 ms revealed a strong dependence of the corresponding probability distributions of diffusivities on time, in particular in the range between 20 ms and 100 ms. This points to average dwell times of the dye molecules in at least one type of the heterogeneous regions (e.g. on and above silanol clusters) in the range of few tens of milliseconds. Furthermore, time series of SM spectra from Nile Red in 25 nm thick poly-n-alkyl-methacrylate (PnAMA) films were studied. In analogy to translational diffusion, spectral diffusion (shifts in energetic positions of SM spectra) can be studied by probability distributions of spectral diffusivities, i.e. time scaled square energetic displacements. Simulations were run and analyzed to study contributions from noise and fitting uncertainty to spectral diffusion. Furthermore the effect of spectral jumps during acquisition of a SM spectrum was investigated. Probability distributions of spectral diffusivites of Nile Red probing vitreous PnAMA films reveal a two-level system. In contrast, such probability distributions obtained from Nile Red within a 25 nm thick poly-n-butylmethacrylate film around glass transition and in the melt state, display larger spectral jumps. Moreover, for longer alkyl side chains a solvent shift to higher energies is observed, which supports the idea of nanophase separation within those polymers.

Tese de mestrado em Bioquímica, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2013<br>A membrana celular é composta por uma ampla variedade de lípidos e proteínas. Através do estabelecimento de interacções entre lípidos e proteínas, quer membranares quer solúveis, podem ser originadas heterogeneidades espaciais, levando à formação de ‘jangadas lipídicas’. Estes domínios transitórios desempenham um papel fundamental em diferentes cadeias de sinalização celular. Apesar da investigação desenvolvida na área, a membrana celular é ainda hoje um dos componentes celulares menos bem compreendidos. Nas últimas décadas têm vindo a surgir um conjunto de diferentes metodologias in vitro que permitem o estudo de variados processos biológicos sob condições definidas e controladas. Neste sentido, foram desenvolvidos variados modelos membranares minimalistas, como GUVs (giant unillamelar vesicles), GPMVs (giant plasma membrane vesicles) and SLBs (supported lipid bilayers), permitindo a análise das interacções lípido-proteína até a um nível unimolecular. Quando correctamente aplicados estes modelos permitem simplificar o sistema isolando virtualmente o fenómeno ou a partícula de interesse retendo, porém, as suas características fundamentais. Apesar da vantagem evidente do uso destes modelos, estas abordagens experimentais são tediosas, envolvendo geralmente o uso de amostras de elevado volume e em que a variação de certas características membranares (como a difusão lipídica) é apenas possível por alteração da composição lipídica ou da temperatura. As monocamadas lipídicas são historicamente descritas como o primeiro sistema lipídico observado, formando-se por distribuição espontânea de moléculas lipídicas sobre a interface água - ar e formação de filmes finos sobre superfícies. Dado o seu design único, a mobilidade e compactação lipídica podem ser facilmente ajustadas neste sistema modelo por compressão da monocamada depositada na interface. Apesar de o seu uso em ensaios de ligação proteica, este sistema tem sido negligenciado na investigação membranar recente devido à sua restrita aplicação. Mais concretamente, as tinas de Langmuir comercialmente disponíveis não são compatíveis com microscopia confocal, estando assim limitadas à monitorização da pressão superficial (π). Além disso, dado o volume de cada amostra relativamente elevado (cerca de 100 mL), são necessárias elevadas quantidades de proteína purificada, comprometendo assim a sua aplicação em estudos biológicos. Neste estudo, foi utilizada uma nova tina miniaturizada de área fixa, desenvolvida no grupo por Chwastek (Chwastek & Schwille, 2013). As suas dimensões reduzidas permitem não só o uso de amostras de pequeno volume (cerca de 200 μL) como também conferem a versatilidade necessária para o seu acoplamento a um microscópio confocal. Deste modo, é possível aplicar técnicas de imagiologia de alta resolução (permitindo a eliminação de ruído de fundo, o controlo de profundidade de campo e a compilação de secções ópticas de amostras espessas oferecido pelo microscópio confocal), assim como espectroscopia de correlação de fluorescência (FCS – técnica espectroscópica cuja sensibilidade advém do uso de baixas concentrações de sonda e na consequente medição de flutuações espontâneas da intensidade de fluorescência que resultam de desvios estocásticos do sistema relativamente ao seu equilíbrio térmico). O objectivo global deste projecto prende-se com a melhor compreensão da interacção estabelecida entre proteínas e monocamadas lipídicas. Mais concretamente foi medida a ligação a monocamadas lipídicas de duas proteínas estruturalmente bem caracterizadas: a estreptavidina – proteína tetramérica solúvel com elevada afinidade de ligação para a biotina (KD ≈ 10-15 M); e a toxina da cólera – enterotoxina, secretada pela bactéria Vibrio cholerae, constituída por um anel homo-pentamérico de subunidades B (CtxB5), responsável pela sua ligação ao receptor membranar gangliósido GM1 (KD ≈ 10-9 ou 10-10 M), e uma subunidade A com actividade de ribosilação que é parcialmente internalizada pelas células do hospedeiro, perturbando cascatas celulares de sinalização e desencadeando a infecção colérica. Foram realizadosensaios de ligação proteica a monocamadas lipídicas homogéneas e com separação de fases, na ausência ou presença dos respectivos ligandos específicos, de modo a distinguir e caracterizar os diferentes comportamentos proteicos. Em última análise, procurou-se desenvolver e optimizar um ensaio universal e quantitativo de ligação proteica a membranas lipídicas. Previamente aos estudos das interacções proteína-monocamanda, procedeu-se à caracterização morfológica das monocamadas lipídicas, assim como da mobilidade lipídica, recorrendo a microscopia confocal e a FCS, respectivamente. Observou-se pois que a fluidez e homogeneidade global das monocamadas é acompanhada pelo aumento linear do coeficiente de difusão em função da densidade lipídica no intervalo estudado (50 a 90 MMA – área molecular média em Å2), tal como se prevê do modelo de área livre (free area model). Mais, os valores obtidos para o ponto crítico do DMPC (36.0 ± 4.7 e 37.2 ± 13.5 Å2 em misturas marcadas com sondas diferentes) encontram-se dentro do erro dos valores descritos na literatura. Apesar da robustez demonstrada pelo sistema, é porém necessário ter em conta a possibilidade de ocorrerem desvios em extremos de densidade lipídica (50 e 100 MMA), por formação de fases S (sólida) e G (gás), respectivamente. Após validação da metodologia proposta, procedeu-se ao estudo da influência da compactação lipídica na ligação proteica à monocamada – quer com a estreptavidina, quer com a CtxB5 observou-se uma preferência para a ligação a monocamadas de baixa densidade lipídica e, correspondentemente baixa π e elevada difusão lipídica. Estes resultados são ainda suportados pelas experiências realizadas em misturas lipídicas com separação de fase – ambas as proteínas apresentam uma elevada afinidade para a fase fluida LE (liquid expanded) em comparação com os domínios compactos LC (liquid condensed). Adicionalmente, resultados de titulações de monocamadas com CtxB5 apontam para a mesma tendência visto que a 90 MMA se obtêm intensidades de fluorescência proteica ao nível da monocamada superiores que a 50 MMA. Este comportamento já tinha sido proposto no início da década de 80 tendo por base a análise de variações de π por adição proteica a monocamadas lipídicas (Phillips et al, 1975; Fidelio et al, 1981; Cumar et al, 1982). Porém, no caso da estreptavidina observa-se ainda um comportamento extraordinário de acumulação nas bordas de domínios quando a diferença na organização lipídica entre diferentes fases é muito elevada. A especificidade de ligação foi apenas possível para a CtxB5 por incorporação de GM1 na monocamada. Titulações de monocamadas demonstraram que a presença de uma baixa concentração do gangliósido (0.1 mol %) é suficiente para aumentar fortemente a ligação da proteína à monocamada. Além disso, em sistemas lipídicos homogéneos a proteína retém apreferência por baixas densidades lipídicas. Porém, na presença de separação de fases, a presença de GM1 promove a ligação de CtxB5 a domínios LC, dado que este se incorpora preferencialmente em domínios rígidos. Apesar da elevada reprodutibilidade associada às medições em lípidos, a aplicação do sistema a proteínas tem um erro associado bastante elevado que impede ainda uma quantificação exacta da sua ligação aos lípidos. É de salientar que para além da elevada sensibilidade do sistema de detecção usado, as monocamadas lipídicas formadas sobre a interface água-ar são altamente sensíveis a diferentes factores externos (p.e. oxidação lipídica, formação de menisco e não homogeneidades da compactação lipídica aquando da deposição lipídica, evaporação da subfase, deposição proteica, etc.) que ainda não são totalmente controladas. Deste modo, é ainda necessário proceder à optimização do método de modo a poder determinar-se KD da ligação de proteínas à superfície lipídica. Este projecto resultou pois na obtenção de novos dados semi-quantitativos de que a compactação lipídica desempenha um papel importante na interacção proteína-membrana. A separação de fases surge pois como um mecanismo de regulação da ligação proteica, não só por controlo da mobilidade lipídica mas também por segregação de ligandos. Em suma, a abordagem aqui proposta surge como uma alternativa poderosa para estudos de interacções membrana-proteina, permitindo o fácil ajuste de parâmetros membranares como a compactação lipídica, difícil de regular em sistemas de bicamadas. A sua combinação potente com a microscopia confocal e espectroscopia de correlação de fluorescência resulta não só numa elevada resolução temporal da mobilidade de partículas lipídicas, como também na alta resolução espacial da ligação proteica. Deste modo, o trabalho apresentado contribuiu para a aproximação ao desenvolvimento de um novo ensaio quantitativo global de ligação proteica a membranas lipídicas.<br>Cellular membranes are composed of a wide variety of lipids and proteins which can lead to spatial heterogeneity and formation of so-called ‘lipid rafts’, which play an important role in signaling processes. To investigate these features of lipid bilayers various in vitro models have been developed. Although a variety of experimental assays exist, they very often require large samples volume, are complicated and the membrane features can be varied only by changes in lipid composition or temperature. Here, a fixed-area miniaturized monolayer trough was used. Due to its unique design, lipid mobility in monolayers is easily accessible while keeping other factors constant. The system utilized in this study allows the use of small sample volumes (200 μL) and can be combined with a confocal microscope and thus providing access to high resolution imaging and sensitive fluorescence correlation spectroscopy (FCS) technique. The goal of the project was to design a quantitative assay by which protein-monolayer interactions can be studied. Thus, monolayers were investigated in terms of morphology and lipid mobility. Further, interactions of well-known ligand-protein pairs were studied: cholera toxin (Ctx)/ganglioside GM1 and streptavidin/biotin. The influences of phase separation and presence of lipid ligands were investigated. It was shown that the membrane affinities of cholera toxin B (CtxB5) and streptavidin depend on the surface density of lipid molecules. Moreover, FCS measurements indicate a correlation between higher protein binding and increased lipid mobility. When phase separated lipid monolayers were used, both proteins bound preferentially to liquid expanded phase (LE). However, in case of CtxB5, in the presence of the ganglioside, the protein mostly binds to the liquid condensed phase (LC). In summary, protein binding to lipid monolayers was studied by means of fluorescence microscopy and spectroscopy. During the studies, an appropriate methodology was developed and several experimental scenarios were tested.

碩士<br>國立臺灣大學<br>應用力學研究所<br>96<br>In protein folding, early folding events occur on a microsecond to second time scale. In order to study protein kinetics, folding reactions must be triggered in a short time. In this thesis, we design four different micro-mixers based on hydrodynamic focusing. We use con-focal microscopy and fluorescence correlation spectroscopy (FCS) to synthesize qualitatively and quantitatively. Velocity is the most important parameter since it deeply affects the determination of mixing dead time and time resolution of our micro-mixer. Fluorescence correlation spectroscopy is a powerful technique to determine the velocity precisely. We made an overall comparison of four different micro-mixers by FCS and con-focal microscopy.

In this thesis, we have focused our attention on three different process, - diffusion of organic dyes, conformation dynamics of protein and solvation dynamics. Diffusion of organic dyes provides important information on the micro-viscosity and heterogeneity of the system. We have determined the diffusion coefficient and heterogeneity inside a niosome using fluorescence correlation spectroscopy. We also determined the micro-viscosity of different region of a live cell by measuring the diffusion coefficient of dyes in those regions. We studied the effect of solvent mixtures, such as binary mixtures of water and di-methyl sulfoxide (DMSO) and water and ionic liquid, on the structure and conformational dynamics of an enzyme, lysozyme. Finally, using time resolved confocal microscopy, we have studied solvation dynamics in different regions of a biological cell - nucleus, cytoplasm, lipid droplets and cell membrane.<br>Research was conducted under the supervision of Prof. Kankan Bhattcharyya of the Physical Chemistry division under SCS [School of Chemical Sciences]<br>Research was carried out under DST research grant and CSIR fellowship

In this thesis, we have applied time resolved confocal microscopy to five different processes, - intermittent oscillation of lipid droplets in live cells14, in situ imaging of cancer cell by fluorescent gold nanoclusters, selective killing of breast cancer cells, delivery of cytochrome c and hence imaging of live cell and effect of binary solvent mixture on structure and dynamics of a protein. Intermittent oscillation in live lung cell is observed using time resolved confocal microscopy and we have observed significant differences between normal and cancer cell. Imaging of cancer cell was done using in situ generated gold nanoclusters. We have shown how doxorubicin loaded gold nanoclusters (AuNC) selectively killed breast cancer cell. We have delivered cytochrome c, inside a cell by gold nanoclusters. We have studied the structure and dynamics of cytochrome c in water-ethanol binary mixture using FCS.<br>Research was conducted under the supervision of Prof. Kankan Bhattacharyya of the Physical Chemistry division under SCS [School of Chemical Sciences]<br>Research was carried out under DST grant and CSIR fellowship

Self-organized nanostructures play a crucial role in a wide range of natural phenomena ranging from molecular recognition (e.g. enzyme-substrate, antigenantibody interaction), to a more general case of unusual chemistry in confined environments. Due to the confinement and local interaction, the biological activity and dynamics in these assemblies are significantly different from those in ordinary solutions. In recent years, ultrafast time resolved spectroscopy revealed substantial amount of new information on these systems.The experimented results has stimulated a large body theoretical studies and in particular, large scale computer simulations. The main motivation of this thesis is to understand the role of confinement on the spectroscopy and dynamics with both temporal and spatial resolution. In the present thesis, we studied different organized media using femtosecond upconversion, time resolved confocal microscopy and fluorescence correlation spectroscopy (FCS). In bulk, all chemical informations are averaged over an exceedingly large number of molecules of the order of the Avogadro number. Recent single molecule studies have demonstrated variation of properties of individual molecular systems in an ensemble. This has completely revolutionized our understanding of chemistry.<br>The research was carried out under the supervision of Prof. Kankan Bhattacharyya of the Physical Chemistry division under SCS [School of Chemical Sciences]<br>The research was conducted under CSIR fellowship and DST research grant

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.

This thesis is concerned with the understanding of photon propagation within turbid media with different characteristics (e.g. scattering anisotropy, optical thickness and geometric thickness). The turbid media (samples) used in the work presented in this thesis consist of polystyrene microspheres and semi-skimmed milk suspensions. In particular the work presented in this thesis is concerned with the development of novel microscopic imaging techniques that allow high quality (i.e. micrometer scale resolution, high contrast and have a high signal-to-noise ratio) imaging through turbid media. The work presented in this thesis differs from previously published work in that the techniques developed are primarily designed for microscopic imaging systems.

Les nanomatériaux sont une classe de contaminants qui est de plus en plus présent dans l’environnement. Leur impact sur l’environnement dépendra de leur persistance, mobilité, toxicité et bioaccumulation. Chacun de ces paramètres dépendra de leur comportement physicochimique dans les eaux naturelles (i.e. dissolution et agglomération). L’objectif de cette étude est de comprendre l’agglomération et l’hétéroagglomération des nanoparticules d’argent dans l’environnement. Deux différentes sortes de nanoparticules d’argent (nAg; avec enrobage de citrate et avec enrobage d’acide polyacrylique) de 5 nm de diamètre ont été marquées de manière covalente à l’aide d’un marqueur fluorescent et ont été mélangées avec des colloïdes d’oxyde de silice (SiO2) ou d’argile (montmorillonite). L’homo- et hétéroagglomération des nAg ont été étudiés dans des conditions représentatives d’eaux douces naturelles (pH 7,0; force ionique 10 7 à 10-1 M de Ca2+). Les tailles ont été mesurées par spectroscopie de corrélation par fluorescence (FCS) et les résultats ont été confirmés à l’aide de la microscopie en champ sombre avec imagerie hyperspectrale (HSI). Les résultats ont démontrés que les nanoparticules d’argent à enrobage d’acide polyacrylique sont extrêmement stables sous toutes les conditions imposées, incluant la présence d’autres colloïdes et à des forces ioniques très élevées tandis que les nanoparticules d’argent avec enrobage de citrate ont formées des hétéroagrégats en présence des deux particules colloïdales.<br>Nanomaterials are a class of contaminants that are increasingly found in the natural environment. Their environmental risk will depend on their persistence, mobility, toxicity and bioaccumulation. Each of these parameters will depend strongly upon their physicochemical fate (dissolution, agglomeration) in natural waters. The goal of this paper is to understand the agglomeration and heteroagglomeration of silver nanoparticles in the environment. Two different silver nanoparticles (nAg; citrate coated and polyacrylic acid coated) with a diameter of 5 nm were covalently labelled with a fluorescent dye and then mixed with colloidal silicon oxides (SiO2) and clays (montmorillonite). The homo- and heteroagglomeration of the silver nanoparticles were then studied in waters that were representative of natural freshwaters (pH 7.0; ionic strength 10-7 to 10-1 M of Ca2+). Sizes were followed by fluorescence correlation spectroscopy (FCS) and results were validated using enhanced darkfield microscopy with hyperspectral imaging (HSI). Results have demonstrated that the polyacrylic acid coated nAg was extremely stable under all conditions, including in the presence of other colloids and at high ionic strength, whereas the citrate coated nAg formed heteroagregates in the presence of both natural colloidal particles.

Endocytosis is one of the most fundamental mechanisms by which the cell communicates with its surrounding. Specific signals are transduced through the cell membrane by a complex interplay between proteins and lipids. Clathrin depended endocytosis is one of important signalling pathways which leads to budding of the plasmalemma and a formation of endosomes. The FCHo2 is an essential protein at the initial stage of the this process. In is a membrane binding protein containing BAR (BIN, Amphiphysin, Rvs) domain which is responsible for a membrane binding. Although numerous valuable work on BAR proteins was published recently, the mechanistic description of a BAR domain functionality is missing. In present work we applied in vitro systems in order to gain knowledge about molecular basis of the activity of the FCHo2 BAR domain. In our studies we used supported lipid bilayers (SLBs) and lipid monolayers as s model membrane system. The experiments were carried out with a minimal number of components including the purified FCHo2 BAR domain. Using SLBs we showed that the BAR domain can bind to entirely flat bilayers. We also demonstrated that these interactions depend on the negatively charged lipid species incorporated in the membrane. We designed an assay which allows to quantify the membrane tubulation. We found out that the interaction of the FCHo2 BAR domain with the lipid membrane is concentration dependent. We showed that an area of the bilayer deformed by the protein depends on the amount of the used BAR domain. In order to study the relation between the mobility of lipids and the activity of FCHo2 BAR domain we designed a small-volume monolayer trough. The design of this micro-chamber allows for the implementation of the light microscopy. We demonstrated that the measured lipid diffusion in the monolayer by our new approach is in agreement with literature data. We carried out fluorescence correlation spectroscopy (FCS) experiments at different density of lipids at the water-air interface.We showed that the FCHo2 BAR domain binding affinity is proportional to the mean molecular area (MMA). We additionally demonstrated that the increased protein binding is correlated with the higher lipid mobility in the monolayer. Additionally, by curing out high-speed atomic force microscopy (hsAFM) we acquired the structural information about FCHo2 BAR domains orientation at the membrane with a high spatio-temporal resolution. Obtained data indicate the BAR domains interact witheach other by many different contact sites what results in a variety of protein orientations in a protein assemble.