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CMOS system for high throughput fluorescence lifetime sensing using time correlated single photon countingTyndall, David January 2013 (has links)
Fluorescence lifetime sensing using time correlated single photon counting (TCSPC) is a key analytical tool for molecular and cell biology research, medical diagnosis and pharmacological development. However, commercially available TCSPC equipment is bulky, expensive and power hungry, typically requiring iterative software post-processing to calculate the fluorescence lifetime. Furthermore, the technique is restrictively slow due to a low photon throughput limit which is necessary to avoid distortions caused by TCSPC pile-up. An investigation into CMOS compatible multimodule architectures to miniaturise the standard TCSPC set up, allow an increase in photon throughput by overcoming the TCSPC pile-up limit, and provide fluorescence lifetime calculations in real-time is presented. The investigation verifies the operation of the architectures and leads to the selection of optimal parameters for the number of detectors and timing channels required to overcome the TCSPC pile-up limit by at least an order of magnitude. The parameters are used to implement a low power miniaturised sensor in a 130 nm CMOS process, combining single photon detection, multiple channel timing and embedded pre-processing of the fluorescence lifetime, all within a silicon area of < 2 mm2. Single photon detection is achieved using an array of single photon avalanche diodes (SPADs) arranged in a digital silicon photomultiplier (SiPM) architecture with a 10 % fill-factor and a compressed 250 ps output pulse, which provides a photon throughput of > 700 MHz. An array of time-interleaved time-to-digital converters (TI-TDCs) with 50 ps resolution and no processing dead-time records up to eight photon events during each excitation period, significantly reducing the effect of TCSPC pile-up. The TCSPC data is then processed using an embedded centre-of-mass method (CMM) pre-calculation to produce single exponential fluorescence lifetime estimations in real-time. The combination of high photon throughput and real-time calculation enables advances in applications such as fluorescence lifetime imaging microscopy (FLIM) and time domain fluorescence lifetime activated cell sorting. To demonstrate this, the device is validated in practical bulk sample fluorescence lifetime, FLIM and simulated flow based experiments. Photon throughputs in excess of the excitation frequency are demonstrated for a range of organic and inorganic fluorophores for minimal error in lifetime calculation by CMM (< 5 %).
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Implementing Fluorescence Lifetime Imaging on a Confocal MicroscopeChiu, Yi-Chun 06 July 2005 (has links)
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.
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Self-assembled quantum dots in advanced structuresCreasey, Megan Elizabeth 09 July 2013 (has links)
Advances in nanofabrication have bolstered the development of new optical devices with potential uses ranging from conventional optoelectronics, such as lasers and solar cells, to novel devices, like single photon or entangled photon sources. Quantum encryption of optical communications, in particular, requires devices that couple efficiently to an optical fiber and emit, on demand, indistinguishable photons. With these goals in mind, ultrafast spectroscopy is used to study the electron dynamics in epitaxially grown InAs/GaAs quantum dots (QDs). Quantifying the behavior of these systems is critical to the development of more efficient devices. Studies of two newly developed InGaAs QD structures, quantum dot clusters (QDCs) and QDs embedded in photonic wires, are presented herein.
GaAs photonic wires with diameters in the range of 200 to 250 nm support only the fundamental HE11 guided mode. To fully quantify these new systems, the emission dynamics of QDs contained within wires in a large range of diameters are studied. Time correlated single photon counting measurements of the ground state exciton lifetimes are in very good agreement with predicted theoretical values for the spontaneous emission rates. For diameters smaller than 200 nm, QD emission into the HE11 mode is strongly inhibited and non-radiative processes dominate the decay rate. The best small diameter wires exhibit inhibition factors as high as 16, on par with the current state of the art for photonic crystals.
The QDCs are the product of a hybrid growth technique that combines droplet heteroepitaxy with standard Stranski-Krastanov growth to create many different geometries of QDs. The work presented in this dissertation concentrates specifically on hexa-QDCs consisting of six InAs QDs around a GaAs nanomound. The first ever spectral and temporal properties of QDs within individual hexa-QDCs are presented. The QDs exhibit narrow exciton resonances with good temperature stability, indicating that excitons are well confined within individual QDs. A distinct biexponential decay is observed even at the single QD level. This behavior suggests that non-radiative decay mechanisms and exciton occupation of dark states play a significant role in the recombination dynamics in the QDCs. / text
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Compressive Transient ImagingSun, Qilin 04 1900 (has links)
High resolution transient/3D imaging technology is of high interest in both scientific research and commercial application. Nowadays, all of the transient imaging methods suffer from low resolution or time consuming mechanical scanning. We proposed a new method based on TCSPC and Compressive Sensing to achieve a high resolution transient imaging with a several seconds capturing process. Picosecond laser sends a serious of equal interval pulse while synchronized SPAD camera's detecting gate window has a precise phase delay at each cycle. After capturing enough points, we are able to make up a whole signal. By inserting a DMD device into the system, we are able to modulate all the frames of data using binary random patterns
to reconstruct a super resolution transient/3D image later. Because the low fill factor of SPAD sensor will make a compressive sensing scenario ill-conditioned, We designed and fabricated a diffractive microlens array. We proposed a new CS reconstruction
algorithm which is able to denoise at the same time for the measurements suffering from Poisson noise. Instead of a single SPAD senor, we chose a SPAD array because it can drastically reduce the requirement for the number of measurements and its
reconstruction time. Further more, it not easy to reconstruct a high resolution image with only one single sensor while for an array, it just needs to reconstruct small patches and a few measurements.
In this thesis, we evaluated the reconstruction methods using both clean measurements and the version corrupted by Poisson noise. The results show how the integration over the layers influence the image quality and our algorithm works well while the measurements suffer from non-trival Poisson noise. It's a breakthrough in the areas of both transient imaging and compressive sensing.
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Measuring the Radiative Lifetimes of the Vibrational Levels in the 6 sSg State of Sodium Dimers Using Time-Resolved SpectroscopySaaranen, Michael W. 03 May 2019 (has links)
No description available.
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The signal transduction of synapse formation and it's failure in Rett syndromeEbrecht, René 12 May 2016 (has links)
No description available.
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Investigating the structure and dynamics of DNA with fluorescence and computational techniquesSmith, Darren Andrew January 2015 (has links)
Nucleic acids, such as DNA, play an essential role in all known forms of life; however, despite their fundamental importance, there is still a significant lack of understanding surrounding their functional behaviour. This thesis explores the structure and dynamics of DNA by employing methods based on fluorescence and through the use of computational calculations. Time-resolved fluorescence experiments have been performed on dinucleotides containing 2-aminopurine (2AP) in various alcohol-water mixtures. 2AP, a fluorescent analogue of the nucleobase adenine, has been used extensively to investigate nucleic acids because of its ability to be incorporated into their structures with minimal perturbation and its high sensitivity to its local environment. Direct solvent effects on 2AP were established through measurements on the free fluorophore. Analysis of the complex fluorescence decays associated with the dinucleotides was challenging but has provided insight into their conformational dynamics. Solvent polarity was found to play a significant role in determining both photophysical and conformational properties in these systems. The complicated fluorescence decay of 2AP in nucleic acids highlights the need for accurate and unbiased analysis methods. Various time-resolved fluorescence analysis methods, including iterative reconvolution and the exponential series method, have been investigated with real and simulated data to obtain an overview of their benefits and limitations. The main outcome of the evaluation is that no single method is preferred in all situations and there is likely to be value in using a combination when there is ambiguity in the interpretation of the results. Regardless of the analysis technique used, the parameterised description of the observed fluorescence decay is meaningless if the underlying physical model is unrealistic. The advance of computational methods has provided a new means to rigorously test the viability of proposed models. Calculations have been performed at the M06-2X/6-31+G(d) level of theory to investigate the stability of 2AP-containing dinucleotides in conformations similar to those observed in the double-helical structure of DNA. The results help to explain the similarity of the time-resolved fluorescence behaviour of 2AP in dinucleotide and DNA systems but also bring to light subtle differences that could perhaps account for experimental discrepancies. The recent emergence of advanced optical microscopy techniques has offered the prospect of being able to directly visualise nucleic acid structure at the nanoscale but, unfortunately, limitations of existing labelling methods have hindered delivery of this potential. To address this issue, a novel strategy has been used to introduce reversible fluorescence photoswitching into DNA at high label density. Photophysical studies have implicated aggregation and energy-transfer as possible quenching mechanisms in this system, which could be detrimental to its future application. The reliability of fluorescence photoswitching was investigated at ensemble and single-molecule level and by performing optical lock-in detection imaging. These developments lay the foundations for improved and sequence-specific super-resolution microscopy of DNA, which could offer new insights into the 3D nanoscale structure of this remarkable biopolymer. In summary, the work presented in this thesis outlines important observations and developments that have been made in the study of the structure and dynamics of nucleic acids.
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Zeitaufgelöste Mikroskopie an einzelnen Molekülen zur Untersuchung der Polymerdynamik in dünnen FilmenSchmidt, Ruben 30 March 2006 (has links) (PDF)
Gegenstand dieser Diplomarbeit ist die Untersuchung der Dynamik in dünnen Polymerfilmen anhand von einzelnen Molekülen. Zu diesem Zweck wurden dünne Filme (kleiner 100nm) hergestellt und mittels Einzelmoleküldetektion und zeitaufgelöster Einzelphotonenzählung analysiert, was eine orts- und zeitaufgelöste Untersuchung einzelner Farbstoffmoleküle ermöglicht.
Ziel war es, festzustellen ob, und auf welchem Weg, die Dynamik der Umgebung in Fluktuationen der Fluoreszenzlebensdauer einzelner Moleküle sichtbar wird.
Neben der Evaluierung der Untersuchungsmethoden wurden in dieser Arbeit zwei Arten von Sensormolekülen - DiD und Malachit Grün - näher untersucht. / The subject of this diploma thesis is the analysis of dynamics in thin polymer films using single molecules. Thin polymer films (less than 100nm) were produced and analysed by Single Molecule Detection (SMD) and Time Correlated Single Photon Counting (TCSPC). This allows a spatial and time resolved investigation of the single dye molecule.
The aim was to ascertain if, and in which way, the dynamics of the environment are reflected by fluctuations of the fluorescence lifetime of the single molecule.
In addition to evaluating the investigation methods two kinds of molecules - DiD and Malachite Green - were also analysed.
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Extended Förster Theory of Electronic Energy Transport within Pairs of Reorienting Chromophoric MoleculesNorlin, Nils January 2009 (has links)
An extended Förster theory (EFT), previously derived (L. B.-Å. Johansson et al. J. Chem. Phys., 1996,105) has theoretically been adapted and used in simulations of donor-acceptor energy transfer (DAET), which is a process often referred to as FRET. It was shown that the classical Förster theory is only valid in the initial part of the fluorescence decay. In this thesis an EFT is derived and outlined for electronic energy transport between two fluorescent molecules which are chemically identical, but photophysically non-identical. The energy migration within such asymmetric pairs is partially reversible and therefore referred to as partial donor-donor energy migration (PDDEM). The previously derived model of PDDEM (S. V. Kalinin et al. Spectrochim Acta Part A, 2002,58) is an approximation of the EFT. In particular, the EFT accounts for the time-dependent reorientations as well as the distance that influence the rate of electronic energy migration. The reorientation of the fluorophores transition dipole moments has been simulated using Brownian dynamics. As a result, the related “k2-problem” has been solved. The EFT of PDDEM has also been studied regarding the effect of PDDEM on experimental observables e.g. quantum yield of fluorescence and steady-state anisotropies
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Zeitaufgelöste Mikroskopie an einzelnen Molekülen zur Untersuchung der Polymerdynamik in dünnen FilmenSchmidt, Ruben 31 August 2005 (has links)
Gegenstand dieser Diplomarbeit ist die Untersuchung der Dynamik in dünnen Polymerfilmen anhand von einzelnen Molekülen. Zu diesem Zweck wurden dünne Filme (kleiner 100nm) hergestellt und mittels Einzelmoleküldetektion und zeitaufgelöster Einzelphotonenzählung analysiert, was eine orts- und zeitaufgelöste Untersuchung einzelner Farbstoffmoleküle ermöglicht.
Ziel war es, festzustellen ob, und auf welchem Weg, die Dynamik der Umgebung in Fluktuationen der Fluoreszenzlebensdauer einzelner Moleküle sichtbar wird.
Neben der Evaluierung der Untersuchungsmethoden wurden in dieser Arbeit zwei Arten von Sensormolekülen - DiD und Malachit Grün - näher untersucht. / The subject of this diploma thesis is the analysis of dynamics in thin polymer films using single molecules. Thin polymer films (less than 100nm) were produced and analysed by Single Molecule Detection (SMD) and Time Correlated Single Photon Counting (TCSPC). This allows a spatial and time resolved investigation of the single dye molecule.
The aim was to ascertain if, and in which way, the dynamics of the environment are reflected by fluctuations of the fluorescence lifetime of the single molecule.
In addition to evaluating the investigation methods two kinds of molecules - DiD and Malachite Green - were also analysed.
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