Time-correlated single photon counting (TCSPC) systems are used for range profiling. The systems offer cm precision at kilometer ranges. This opens up for long range imaging with high resolution, for example by reflective tomography. With range profiles from various aspect angles around a target reflective tomography can be used to create an image. The tomographic image is a reconstruction of the boundary of the cross-section of the target. Images can be used for various purposes, e.g. identification of satellites. The quality of the tomographic reconstruction depends on the accuracy of the TCSPC system. Range profiles with a cm precision allows studies and reconstruction of complex objects. With this work we investigated the current limitations when reconstructing complex targets with reflective tomography and present possible solutions to existing problems. The limitations were investigated by studying parameters such as the intensity of the laser beam, SNR, center of rotation, angular resolution, and the angular sector. We also present methods that can improve the tomographic image. A new pre-processing method that adjusts range profiles after estimating responses with RJMCMC was introduced. We also studied different types of filters in the reconstruction process. Lastly we introduced two new post-processing methods. One that removes artifacts by considering the convex hull and one that sharpens edges in the tomographic image. The performance study showed that reflective tomography using a TCSPC system is robust in a controlled environment. Details in the low cm-range of an object can be reconstructed with high precision. However, for some target types issues appear. Of the tested performance parameters a high angle resolution was deemed to be the most important. When considering moving targets the importance of the center of rotation and integration time will also increase. The study of improvement methods showed that choosing the generalized ramp filter in the FBP more then doubled the SNR. Adjusting the range profiles, considering the convex hull, and sharpening edges are methods that work well for specific signal types. We showed that many issues that arise when measuring on complex objects can be solved with signal processing. Therefore we believe that reflective tomography can be used in various applications in the future. / Tidskorrelerad räkning av fotoner (time-correlated single photon counting, TCSPC) är en teknik som används för att skapa avståndsprofiler med cm-precision på upp emot flera kilometers håll. Tekniken kan användas till att skapa avbildningar av föremål på långa avstånd, till exempel med reflektiv tomografi. Reflektiv tomografi kan användas när man har avståndsprofiler runt om ett föremål. Den tomografiska återskapningen beskriver de yttre kanterna av ett föremåls tvärsnitt. Bildernas kvalité är starkt beroende av noggrannheten i TCSPC-systemet. En cm-precision möjliggör studier och återskapningar av föremål med små detaljer. Detta arbete går ut på att undersöka begränsningarna med återskapandet av detaljerade föremål och framlägga metoder som förbättrar återskapningarna. Begränsningarna undersöktes genom att studera olika parametrar, såsom intensiteten i lasern, SNR, rotationscentrum, vinkelupplösning och vinkelsektorer. Vi presenterade också nya metoder som förbättrar återskapningarna. Vi tittade bland annat på en metod som korrigerar avståndsprofilerna med hjälp av anpassningar med RJMCMC. Sedan undersöktes olika filter i återskapningen. Avslutningsvis introducerades två nya metoder i efterbehandlingen. En som tar hänsyn till konvexa höljet och en annan som gör kanter i bilderna skarpare. Prestandaundersökningen visade att reflektiv tomografi baserad på ett TCSPC-system är robust i en kontrollerad miljö. Centimeterstora detaljer kan återskapas med hög upplösning. För vissa förem\aa l uppstår dock problem. Av de testade prestandaparametrarna var en hög vinkelupplösning den viktigaste. Valet av rotationscentrum och integrationstiden kommer spela en större roll med föremål i rörelse. Studien av förbättringsmetoder visade att bilders SNR mer än dubblas om det generella rampfiltret används i FBP. Att korrigera avståndsprofilerna med RJMCMC, ta hänsyn till komplexa höljet och att göra kanter skarpare är metoder som fungerar bra med vissa signaltyper. Mer arbete finns att göra men vi visade att många problem i reflektiv tomografi går att lösa med signalbehandling. Vi tror därför att reflektiv tomografi går en ljus framtid till mötes.

This thesis used TCSPC (Time-Correlated Single Photon Counting) apparatus to study the time-resolve photoluminescence (TRPL) of InGaN multi-quantum-well light emission diode and the photoluminescence of Zn1-xMgxSe properties at different Mg concentration. We obtained the activation energy form Arrhenius Plot, internal quantum efficiency (IQE), the radiative lifetime, and the radiative recombination critical at 180K of In0.25Ga0.75N multi-quantum well LED. Furthermore, the variation of PL peak location and FWHM with Mg concentration of Zn1-xMgxSe thin film with x=0.1¡B0.25¡B0.34¡B0.37¡B0.4¡B0.42 are observed.

Submitted by Geandra Rodrigues (geandrar@gmail.com) on 2018-05-15T15:14:29Z No. of bitstreams: 1 geissydeazevedomendes.pdf: 36976754 bytes, checksum: 80ab46b7f0b567167c8b9d332ae25d24 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2018-05-22T15:06:38Z (GMT) No. of bitstreams: 1 geissydeazevedomendes.pdf: 36976754 bytes, checksum: 80ab46b7f0b567167c8b9d332ae25d24 (MD5) / Made available in DSpace on 2018-05-22T15:06:38Z (GMT). No. of bitstreams: 1 geissydeazevedomendes.pdf: 36976754 bytes, checksum: 80ab46b7f0b567167c8b9d332ae25d24 (MD5) Previous issue date: 2018-02-22 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O café é um dos mais importantes produtos alimentares na economia mundial. O Brasil é o maior produtor e exportador do grão e o segundo maior consumidor do produto no mundo. A maior parte do café disponível comercialmente são as espécies do tipo café arábica (Coffea arabica L.) e café robusta/conilon (Coffea canephora L.), além de suas misturas. O café arábica tem maior valor comercial e suas características sensoriais gerais são mais apreciadas quando comparadas ao robusta. O objetivo deste trabalho é diferenciar as espécies de café após a torrefação por espectroscopia óptica. Assim, misturas com diferentes proporções de cafés arábica/robusta torrado e moído, em três graus de torrefação (claro, médio e escuro) foram estudados utilizando as técnicas de espectroscopia no infravermelho por transformada de Fourier (FTIR), fluorescência e fluorescência resolvida no tempo (TCSPC). Notória a similaridade existente na composição química das amostras, dois métodos multivariados foram aplicados para avaliação dos espectros de infravermelho: análise de componentes principais (PCA) e regressão por mínimos quadrados parciais (PLS). A partir dos espectros avaliados com o PCA, foi possível diferenciar as espécies de café e identificar compostos que são bons discriminantes entre elas, como a cafeína e os lipídeos, presentes em quantidades maiores no café robusta e arábica, respectivamente. Além disso, ao associar os espectros do FTIR ao PLS, foi desenvolvido um modelo capaz de predizer quantitativamente a proporção de cada espécie nas amostras estudadas, com coeficiente de determinação superior a 0,99. As análises de fluorescência e fluorescência resolvida no tempo foram realizadas utilizando-se comprimentos de onda de excitação/emissão em 280/480 nm, 310/470 nm e 400/605 nm. Os espectros de emissão obtidos pela técnica discriminam as espécies e indicam a presença de fluoróforos em apenas um dos cafés analisados. Já pela contagem de fóton único correlacionado no tempo (TCSPC), obteve-se intensidade média do tempo de vida de emissão, no qual o conjunto 310/470 nm apresentou diferença entre as espécies superior a 20%. Os resultados obtidos neste trabalho, evidenciam o fato de que as técnicas empregadas são promissoras, uma vez que são capazes de distinguir com rapidez as espécies ou até mesmo quantificar a porcentagem das misturas. / Coffee is one of the most important food products in the world economy. Brazil is the largest producer and exporter of the grain and the second largest consumer of coffee in the world. Most commercially available coffee is arabica coffee (Coffea arabica L.) and robusta/conilon coffee (Coffea canephora L.) type species as well their blends. The arabica coffee has higher commercial value and its general sensorial characteristics are more favorable when compared to robusta coffee one. This work aimed to differentiate coffee species after roasting by optical spectroscopy. Thus, blends with different ratios of roasted and ground arabica/robusta coffees in three grades of roasting (light, medium and dark) were studied using the Fourier-Transform Infrared Spectroscopy (FTIR), fluorescence and time resolved fluorescence (TCSPC) spectroscopy techniques. Notably similar in their chemical composition, two multivariate methods were used to analyze the sample spectra: principal component analysis (PCA) and partial least squares regression (PLS). From the spectra analyzed with PCA, it was possible to differentiate the coffee species and to identify compounds that are good discriminants among them, such as caffeine and lipids, present in larger quantities in the robusta and arabica coffee, respectively. In addition, by associating the FTIR spectra to the PLS, a model capable of quantitatively predict the proportion of each specie in the studied samples was developed, with a determination coefficient higher than 0.99. Time resolved fluorescence and fluorescence analyzes were performed using excitation/emission wavelengths at 280/480 nm, 310/470 nm and 400/605 nm. The emission spectra obtained by the technique discriminate the species and indicate the presence of fluorophores in only one of the coffees analyzed. As for the time-correlated single photon count (TCSPC), the mean intensity of the emission lifetime was calculated, in which the set 310/470 nm showed a difference between species of more than 20%. The results obtained in this work demonstrate the fact that the techniques employed are promising since they are able to quickly distinguish the species or even quantify the percentage of the blends.

Understanding and identifying critical protein-protein interactions is just one of the key outcomes in biological research. It can help to confirm key cellular interactions, which in some fields, such as cancer research, can result in a greater understanding of disease pathogenesis, elucidate mechanisms of therapeutic resistance and aid in the development of new specific targets, leading to new methods of prevention and treatment. Time-correlated single photon counting fluorescence lifetime imaging microscopy is just one of the tools used to carry out this line of research. Here we demonstrate a direct interaction between two proteins involved in gene regulation and expression; p21 and FMN2. Furthermore, we also show the capability of this system to measure chromatin compaction in three dimensions. However, fluorescence lifetime imaging has some drawbacks, acquisition times on such a system can range from the tens of seconds to minutes, which is often too long to comprehensively measure many biological events. But microscopy is always developing, aided by new techniques and, perhaps even more so, new technological developments. This thesis also demonstrates two new methods of light sheet microscopy, that use both new equipment made available because of technological developments to allow time resolved imaging and traditional microscopic aspects to form a light sheet system based on polarisation. It outlines the design and how to build these systems and presents their function to show their great promise. Both techniques presented in this thesis utilise aspects of light not conventionally used in light sheet microscopy. Further development of these systems and application of emerging technologies will yield a system capable of outperforming current light sheet fluorescence microscopy-based fluorescence lifetime imaging techniques. The implementation of polarisation control into such a system would enable three-dimensional anisotropy based SPIM-FLIM measurements, an indispensable tool in researching molecular orientation and mobility at a macroscopic level in developing organisms.

High resolution 3D images are of high interest in military operations, where data can be used to classify and identify targets. The Swedish defence research agency (FOI) is interested in the latest research and technologies in this area. A draw- back with normal 3D-laser systems are the lack of high resolution for long range measurements. One technique for high long range resolution laser radar is based on time correlated single photon counting (TCSPC). By repetitively sending out short laser pulses and measure the time of flight (TOF) of single reflected pho- tons, extremely accurate range measurements can be done. A drawback with this method is that it is hard to create single photon detectors with many pixels and high temporal resolution, hence a single detector is used. Scanning an entire scene with one detector is very time consuming and instead, as this thesis is all about, the entire scene can be measured with less measurements than the number of pixels. To do this a technique called compressed sensing (CS) is introduced. CS utilizes that signals normally are compressible and can be represented sparse in some basis representation. CS sets other requirements on the sampling compared to the normal Shannon-Nyquist sampling theorem. With a digital micromirror device (DMD) linear combinations of the scene can be reflected onto the single photon detector, creating scalar intensity values as measurements. This means that fewer DMD-patterns than the number of pixels can reconstruct the entire 3D-scene. In this thesis a computer model of the laser system helps to evaluate different CS reconstruction methods with different scenarios of the laser system and the scene. The results show how many measurements that are required to reconstruct scenes properly and how the DMD-patterns effect the results. CS proves to enable a great reduction, 85 − 95 %, of the required measurements com- pared to pixel-by-pixel scanning system. Total variation minimization proves to be the best choice of reconstruction method. / Högupplösta 3D-bilder är väldigt intressanta i militära operationer där data kan utnyttjas för klassificering och identifiering av mål. Det är av stort intresse hos Totalförsvarets forskningsinstitut (FOI) att undersöka de senaste teknikerna in- om detta område. Ett stort problem med vanliga 3D-lasersystem är att de saknar hög upplösning för långa mätavstånd. En teknik som har hög avståndsupplös- ning är tidskorrelerande enfotonräknare, som kan räkna enstaka fotoner med extremt bra noggrannhet. Ett sådant system belyser en scen med laserljus och mäter sedan reflektionstiden för enstaka fotoner och kan på så sätt mäta avstånd. Problemet med denna metod är att göra detektion av många pixlar när man bara kan använda en detektor. Att skanna en hel scen med en detektor tar väldigt lång tid och istället handlar det här exjobbet om att göra färre mätningar än antalet pixlar, men ändå återskapa hela 3D-scenen. För att åstadkomma detta används en ny teknik kallad Compressed Sensing (CS). CS utnyttjar att mätdata normalt är komprimerbar och skiljer sig från det traditionella Shannon-Nyquists krav på sampling. Med hjälp av ett Digital Micromirror Device (DMD) kan linjärkombi- nationer av scenen speglas ner på enfotondetektorn och med färre DMD-mönster än antalet pixlar kan hela 3D-scenen återskapas. Med hjälp av en egenutvecklad lasermodell evalueras olika CS rekonstruktionsmetoder och olika scenarier av la- sersystemet. Arbetet visar att basrepresentationen avgör hur många mätningar som behövs och hur olika uppbyggnader av DMD-mönstren påverkar resultatet. CS visar sig möjliggöra att 85 − 95 % färre mätningar än antalet pixlar behövs för att avbilda hela 3D-scener. Total variation minimization visar sig var det bästa valet av rekonstruktionsmetod.

This project investigates an image registration process, involving a method known as K-4PCS. This registration process was applied to a set of 16 long range lidar scans, acquired at different positions by a single pixel TCSPC (Time Correlated Single-Photon Counting) lidar system. By merging these lidar scans, after having been transformed by proper scan alignments, one could obtain clear information regarding obscured surfaces. Using all available data, the investigated method was able to provide adequate alignments for all lidar scans.The data in each lidar scan was subsampled and a subsampling ratio of 50% proved to be sufficient in order to construct sparse, representative point clouds that, when subjected to the image registration process, result in adequate alignments. This was approximately equivalent to 9 million collected photon detections per scan position. Lower subsampling ratios failed to generate representative point clouds that could be used in the imageregistration process in order to obtain adequate alignments. Large errors followed, especially in the horisontal and elevation angles, of each alignment. The computation time for one scan pair matching at a subsampling ratio = 100%. was, on average, approximately 120 s, and 95s for a subsampling = 50%.To summarise, the investigated method can be used to register lidar scans acquired by a lidar system using TCSPC principles, and with proper equipment and code implementation, one could potentially acquire 3D images of a measurement area every second, however, at a delay depending on the efficiency of the lidar data processing.

No description available.

Nous présentons une caméra à balayage de fente intégrée basée sur un système de comptage de photon unique résolu en temps (TCSPC-SC) employant l'architecture linéaire « streak » pour surmonter la limitation de l'espace inhérent aux systèmes TCSPC bidimensionnels. Cette solution permet l'intégration de fonctionnalités électroniques complexes dans les pixels sans l'inconvénient d'un faible facteur de remplissage conduisant à une faible efficacité de détection. Le TCSPC-SC se compose de deux blocs principaux: une photodiode à avalanche (SPAD) et un bloc de mesure de temps, les deux blocs sont intégrés en technologie 180 nm CMOS standard. La structure de la SPAD utilisée a été sélectionnée parmi 6 structures différentes après un processus de caractérisation précise et approfondie. Le bloc de mesure du temps se compose d'un TOC hybride capable d'atteindre des résolutions de temps élevées et ajustables avec une large dynamique de mesure grâce à un système de conversion de temps (TOC) hybride qui combine l'approche analogique basée sur un convertisseur de temps vers amplitude(TAC), et les approches numériques utilisant une boucle à verrouillage de retard (DLL) et un compteur numérique. Le TOC hybride a été spécialement conçu pour être utilisé dans un système TCSPC qui intègre une ligne de TOC nécessitant ainsi une conception appropriée pour limiter la consommation d'énergie et la surface d'occupation et parvenir à une architecture flexible et facilement extensible. Suite à la conception et la réalisation de ces deux blocs dans une technologie180 nm CMOS standard, une structure de test de la caméra à balayage de fente (TCSPC-SC) qui englobe 8 unités a été réalisée dans le but final de mettre en œuvre un modèle TCSPC-SC complet et plus large. / In this work we present a TCSPC Streak Camera (TCSPC-SC) that takes advantage of the streak mode imaging ta overcome the space limitation inherent ta 20 TCSPC sensor arrays. This cost-effective solution allows the integration of complex functionalities in the pixel without the inconvenience of low fill factor that leads ta low detection efficiency. The TCSPC~SC consists of two main building blacks: a SPAD and a time measurement black bath integrated in 180 nm Standard CMOS technology. The SPAD was selected among 6 different SPAD structures following a thorough characterization process ta fully determine its performance figures. The time measurement black consists of a hybrid TOC capable of achieving high adjustable time resolutions with large dynamic range owing ta a time conversion scheme that combines traditional Analog Time to Amplitude Converter (TAC), Digital DLL-based and counter-based TOC. Furthermore, thehybrid TOC was especially designed ta be used in a TCSPC system that incorporates an array of TDCs which required a careful design ta limit power consumption and occupation area in order to achieve a flexible and easily scalable architecture. These two building blacks were bath fabricated in a 180 nm standard CMOS technology and employed ta demonstrate a TCSPC Streak Camera(TCSPC-SC) test structure that englobes 8 units in order ta demonstrate the system's operation principle with the final aim of implementing a complete and bigger TCSPC-SC model in the near future

Molecular dynamics of biomolecules, like proteins and nucleic acids dictate essential biological processes allowing life to function. They are involved in a vast number of cellular tasks including DNA replication, genetic recombination, transcription and translation, as well as signalling, translational motion, structure formation, biochemical synthesis, immune response, and many more. Developed over billions of years by evolution they constitute fine-tuned networks modulated by temperature and regulatory mechanisms. A better understanding of the thermodynamic fundamentals of inter- and intramolecular conformational changes can shed light on the underlying processes of diseases and enables the transfer of biological architectures, properties and compositions to nanotechnological applications. Dynamics of biomolecules occur on a wide range of timescales covering more than twelve orders of magnitude. Fluorescence spectroscopy techniques like time-correlated single photon counting (TCSPC), fluorescence correlation spectroscopy (FCS), and immobilized and freely diffusing single-molecule Förster resonance energy transfer (FRET) spectroscopy represent powerful tools monitoring the dynamics at different ranges within this large span of timescales. However, a unified approach covering all biological relevant timescales remains a goal in the field of fluorescence spectroscopy. This would comprise a methodological workflow for qualitative and quantitative analysis of biomolecular dynamics ranging from nanoseconds to hours. In this work, a custom built single-molecule fluorescence spectroscopy set-up was constructed combining confocal single-molecule FRET spectroscopy with TCSPC, FCS and fluorescence anisotropy techniques for multiparameter fluorescence detection (MFD). The set-up allows the complementary observation of single-molecules over an extensive timescale ranging from fast reconfiguration dynamics of polymers (nanoseconds) to slow membrane protein folding (hours) without the need of molecular synchronization. Freely diffusing molecules enable high throughput measurements in heterogeneous membrane-mimetic and denaturing environments. Additionally, routines for data acquisition and processing were developed followed by the elaboration of a methodological workflow for the qualitative and quantitative analysis of biomolecular dynamics. Finally, the applicability was demonstrated on a big diversity of biological systems (DNA hairpin, Holliday junction, soluble and membrane proteins) in aqueous, membrane-mimetic and denaturing environments covering conformational dynamics from nanoseconds to hours.:Chapter 1: Introduction Chapter 2: Dynamics of Biomolecules 2.1 Dynamics of Nucleic Acids 2.1.1 DNA Hairpin Dynamics 2.1.2 Dynamics of Holliday Junctions 2.2 Dynamics of Proteins 2.2.1 Model Systems of Protein Folding Chapter 3: Fundamentals of Fluorescence Spectroscopy 3.1 Basics of Fluorescence 3.2 Förster Resonance Energy Transfer (FRET) Chapter 4: Multiparameter Fluorescence Detection 4.1 Single-Molecule FRET Spectroscopy 4.1.1 Confocal Microscopy 4.1.2 Freely Diffusing Molecules 4.1.3 Fluorescence Spectroscopy 4.2 Time-Correlated Single-Photon Counting (TCSPC) 4.3 Pulsed Interleaved Excitation (PIE) 4.4 Fluorescence Anisotropy 4.5 Fluorescence Correlation Spectroscopy (FCS) 4.6 MFD Setup 4.7 Analysis Software Chapter 5: Analysis of Molecular Dynamics 5.1 Sub-Microseconds – Peptide Chain Dynamics 5.1.1 Identification of Peptide Chain Dynamics 5.1.2 Quantification of Peptide Chain Dynamics 5.1.3 Discussion 5.2 Microseconds – Dynamics of Barrier Crossing 5.2.1 Maximum Likelihood Estimation of the Transition-Path Time 5.2.2 Quantification of the Upper Bound of the Transition-Path Time 5.2.3 Discussion 5.3 Milliseconds – Fast Protein Folding Dynamics 5.3.1 Correlation of the Relative Donor Lifetime (τD(A) / τD(0)) with FRET Efficiency (E) 5.3.2 Burst-Variance Analysis (BVA) 5.3.3 FRET-Two-Channel Kernel-Based Density Distribution Estimator (FRET-2CDE) 5.3.4 Estimation of the Conformational Relaxation Rate using Bin-Time Analysis 5.3.5 Extracting Folding Kinetics using the Three-Gaussian (3G) Approximation 5.3.6 Dynamic Probability Distribution Analysis (dPDA) 5.3.7 Folding and Unfolding Rate Estimation using a Maximum-Likelihood Estimator 5.3.8 Discussion 5.4 Milliseconds to Seconds – Stacking Dynamics of DNA 5.4.1 Identification of Dynamics on the Recurrence Timescale 5.4.2 Quantification of Dynamics on the Recurrence Timescale 5.4.3 Discussion 5.5 Minutes to Hours – Slow Protein Folding Dynamics 5.5.1 Identification of Slow Protein Folding Dynamics 5.5.2 Quantification of Slow Protein Folding Dynamics 5.5.3 Discussion Chapter 6: Conclusion and Outlook Chapter 7: Appendices 7.1 Derivation of Equation 4.6 (inspired by Daniel Nettels) 7.2 Protein sequences 7.3 Identification of dynamics on the recurrence timescale 7.4 Dependency of psame on the sample concentration 7.5 Effect of fluorescence quenching on MFD parameters Chapter 8: References / Biomoleküle, wie Proteine und Nukleinsäuren, sind essentielle Bausteine des Lebens und permanent an biologischen Prozessen beteiligt. Innerhalb der Zelle nehmen sie eine Vielzahl von Aufgaben wahr, darunter DNA-Replikation, genetische Rekombination, Transkription und Translation, sowie Signalübertragung, Transport, Strukturbildung, biochemische Synthese und Immunreaktion. In Milliarden von Jahren evolutionärer Entwicklung wurden biomolekulare Prozesse immer feiner aufeinander Abgestimmt. Um den zugrundeliegenden Mechanismus von Krankheiten besser zu Verstehen und um die einzigartigen Eigenschaften und Kompositionen biologischer Systeme auf nanotechnologische Anwendungen übertragen zu können, ist es unbedingt notwendig ein besseres Verständnis thermodynamischer Grundlagen inter- und intramolekularer Konformationsänderungen zu erlangen. Dabei finden sich Dynamiken von Biomolekülen über eine Zeitskale von mehr als zwölf Größenordnungen verteilt. Fluoreszenzspektroskopietechniken, wie zeitkorrelierte Einzel-photonenzählung (TCSPC), Fluoreszenzkorrelationsspektroskopie (FCS), und Förster-Resonanzenergietransfer (FRET)–Spektroskopie von immobilisierten und frei diffundierenden Molekülen, stellen leistungsfähige Werkzeuge dar, welche es ermöglichen Dynamiken in der den Techniken entsprechenden Zeitskala aufzulösen. Dennoch, besteht der dringende Bedarf nach einer einheitlichen Methode, der in der Fluoreszenzspektroskopie alle biologisch relevanten Zeitskalen abdeckt. Dies würde einen methodischen Workflow für die qualitative und quantitative Analyse der biomolekularen Dynamik von Nanosekunden bis Stunden bedeuten. In dieser Arbeit wurde ein speziell angefertigter Multiparamter-Fluoreszenzspektroskopie-Aufbau konstruiert, welcher die konfokale Einzelmolekül-FRET-Spektroskopie mit den TCSPC-, FCS- und Fluoreszenz-Anisotropie-Techniken kombiniert. Der Aufbau ermöglicht die Beobachtung komplementärer Eigenschaften von Einzelmolekülen über eine umfangreiche Zeitskala hinweg. Dynamiken von schnell rekonfigurierenden Polymeren (Nanosekunden) bis hin zu langsam faltenden Membranproteinen (Stunden) sind ohne molekulare Synchronisation möglich. Darüber hinaus, ermöglicht der Einsatz frei diffundierender Moleküle einen hohen Messdurchsatz und die Anwendung heterogener membranmimetischer und denaturierender Lösungen. Zusätzlich wurden Routinen zur Datenerfassung und -verarbeitung entwickelt, gefolgt von der Ausarbeitung eines methodischen Workflows zur qualitativen und quantitativen Analyse von biomolekularen Dynamiken. Abschließend wurde die Anwendbarkeit an fünf biologischen Modelsystemen (DNA-Haarnadel, Holliday-Junction, lösliche und Membranproteine) in wässrigen, membranmimetischen und denaturierenden Umgebungen demonstriert und alle biologisch relevanten Zeitskalen von Nanosekunden bis Stunden abgedeckt.:Chapter 1: Introduction Chapter 2: Dynamics of Biomolecules 2.1 Dynamics of Nucleic Acids 2.1.1 DNA Hairpin Dynamics 2.1.2 Dynamics of Holliday Junctions 2.2 Dynamics of Proteins 2.2.1 Model Systems of Protein Folding Chapter 3: Fundamentals of Fluorescence Spectroscopy 3.1 Basics of Fluorescence 3.2 Förster Resonance Energy Transfer (FRET) Chapter 4: Multiparameter Fluorescence Detection 4.1 Single-Molecule FRET Spectroscopy 4.1.1 Confocal Microscopy 4.1.2 Freely Diffusing Molecules 4.1.3 Fluorescence Spectroscopy 4.2 Time-Correlated Single-Photon Counting (TCSPC) 4.3 Pulsed Interleaved Excitation (PIE) 4.4 Fluorescence Anisotropy 4.5 Fluorescence Correlation Spectroscopy (FCS) 4.6 MFD Setup 4.7 Analysis Software Chapter 5: Analysis of Molecular Dynamics 5.1 Sub-Microseconds – Peptide Chain Dynamics 5.1.1 Identification of Peptide Chain Dynamics 5.1.2 Quantification of Peptide Chain Dynamics 5.1.3 Discussion 5.2 Microseconds – Dynamics of Barrier Crossing 5.2.1 Maximum Likelihood Estimation of the Transition-Path Time 5.2.2 Quantification of the Upper Bound of the Transition-Path Time 5.2.3 Discussion 5.3 Milliseconds – Fast Protein Folding Dynamics 5.3.1 Correlation of the Relative Donor Lifetime (τD(A) / τD(0)) with FRET Efficiency (E) 5.3.2 Burst-Variance Analysis (BVA) 5.3.3 FRET-Two-Channel Kernel-Based Density Distribution Estimator (FRET-2CDE) 5.3.4 Estimation of the Conformational Relaxation Rate using Bin-Time Analysis 5.3.5 Extracting Folding Kinetics using the Three-Gaussian (3G) Approximation 5.3.6 Dynamic Probability Distribution Analysis (dPDA) 5.3.7 Folding and Unfolding Rate Estimation using a Maximum-Likelihood Estimator 5.3.8 Discussion 5.4 Milliseconds to Seconds – Stacking Dynamics of DNA 5.4.1 Identification of Dynamics on the Recurrence Timescale 5.4.2 Quantification of Dynamics on the Recurrence Timescale 5.4.3 Discussion 5.5 Minutes to Hours – Slow Protein Folding Dynamics 5.5.1 Identification of Slow Protein Folding Dynamics 5.5.2 Quantification of Slow Protein Folding Dynamics 5.5.3 Discussion Chapter 6: Conclusion and Outlook Chapter 7: Appendices 7.1 Derivation of Equation 4.6 (inspired by Daniel Nettels) 7.2 Protein sequences 7.3 Identification of dynamics on the recurrence timescale 7.4 Dependency of psame on the sample concentration 7.5 Effect of fluorescence quenching on MFD parameters Chapter 8: References

In this thesis, the development and implementation of fluorescence lifetime imaging microscopy that integrates time correlated single photon counting (TCSPC) and a confocal microscope will be described. The TCSPC method has high detection efficiency, with a time resolution limited only by the transit time spread of the detector, and directly delivers the decay functions in the time domain. TCSPC can also be used to obtain images that indicate the fluorescence resonance energy transfer (FRET) effect between critical fluorophores, an important method distinguish the difference between binding and co-localization. Estimation of distances between RET fluorophore pairs can also be established. Additionally, the effects of ion concentration, oxygen concentration, pH value, ..etc. can also be revealed.