Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 89-94).

Thesis--University of Oklahoma. / Bibliography: leaves 62-63.

Thesis (Ph.D)--Biomedical Engineering, Georgia Institute of Technology, 2010. / Committee Chair: Griffin, Paul; Committee Member: Butera, Robert; Committee Member: Halpern, Michael; Committee Member: Nichols, Richard; Committee Member: Vidakovic, Brani. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [140]-152).

Thesis (Ph. D.)--Duke University, 2007. / Includes bibliographical references.

Protein-DNA interactions govern the fundamental cellular processes of DNA replication, transcription, repair, and chromosome organisation. Despite their importance, the detailed molecular mechanisms of protein-DNA interactions and their organisation in the cell remain elusive. The complexity of molecular biology demands new experimental concepts that resolve the structural and functional diversity of biomolecules. In this thesis, I describe fluorescence methods that give a direct view on protein-DNA interactions at the single-molecule level. These methods employ photoswitching to control the number of active fluorophores in the sample. Forster Resonance Energy Transfer (FRET) measures the distance between a donor and an acceptor fluorophore to report on biomolecular structure and dynamics in vitro. Because a single distance gives only limited structural information, I developed "switchable FRET" that employs photoswitching to sequentially probe multiple FRET pairs per molecule. Switchable FRET resolved two distances within static and dynamic DNA constructs and protein-DNA complexes. Towards application of switchable FRET, I investigated aspects of the nucleotide selection mechanism of DNA polymerase. I further explored application of single-molecule imaging in the complex environment of the living cell. Photoswitching was used to resolve the precise localisations of individual fluorophores. I constructed a super-resolution fluorescence microscope to image fixed cellular structures and track the movement of individual fluorescent fusion proteins in live bacteria. I applied the method to directly visualise DNA repair processes by DNA polymerase I and ligase, generating a quantitative account of their repair rates, search times, copy numbers, and spatial distribution in the cell. I validated the approach by tracking diffusion of replisome components and their association with the replication fork. Finally, super-resolution microscopy showed dense clusters of SMC (Structural Maintenance of Chromosomes) protein complexes in vivo that have previously been hidden by the limited resolution of conventional microscopy.

O citoesqueleto é a estrutura celular mais importante em células eucariotas e é responsável por manter a forma da célula e as junções celulares, auxiliando nos movimentos celulares. Esta é composta de filamentos de Actina, Microtúbulos e filamentos intermediários. Recentemente, a análise de duas dessas estruturas tornaram-se importantes, pois é possível obter micrografias usando microscópios de alta resolução, que contém microscopia de fluorescência, em combinação com métodos complexos de aplicação de substâncias de contraste para rotulagem e posterior análises visuais. A combinação dessas técnicas, entretanto, limita-se a ser descritiva e subjetiva. Neste trabalho, são avaliadas cinco técnicas de análise de imagens, as quais são: Bag of Visual Words (BoVW), Local Binary Local (LBP), Textons baseados em Discrete Fourier Transform (TDFT), Textons baseados em Gabor Filter Banks (TGFB) e Textons baseados em Complex Networks (TCN) sobre o conjunto de dados 2D Hela e FDIG Olympus. Experimentos extensivos foram conduzidos em ambos os conjuntos de dados, e seus resultados podem servir de base para futuras pesquisas como análises do citoesqueleto em imagens de microscopia fluorescente. Neste trabalho, é apresentada uma comparação quantitativa e qualitativa dos métodos acima mencionados para entender o comportamento desses métodos e propriedades dos microfilamentos de actina (MA) e Microtúbulos (MT) em ambos os conjuntos de dados. Os resultados obtidos evidenciam que é possível classificar o conjunto de dados da FDIG Olympus com uma precisão de até 90:07% e 98:94% para 2D Hela, além de obter 86:05% e 96:84%, respectivamente, de precisão, usando teoria de redes complexas. / The cytoskeleton is the most important cellular structure in eukaryotic cells and is responsible for maintaining the shape of the cell and cellular junctions, aiding in cell movements. This is composed of filaments of Actin, Microtubules and intermediate filaments. Recently, the analysis of two of these structures has become important because it is possible to obtain micrographs using microscopes of high resolution and fluorescence technology, in combination with complex methods of application of substances of contrast for labeling and later visual analysis. The use of these techniques, however, is limited to being descriptive and subjective. In this work, we evaluate some of the most popular image analysis techniques such as Bag of Visual Words (BoVW), Local Binary Pattern (LBP), Textons based on Discrete Fourier Transform(TDFT) , Gabor Filter banks (TGFB), and approaches based on Complex Networks theory (TCN) over the famous dataset 2D Hela and FDIG Olympus. Extensive experiments were conducted on both datasets in which their results can serve as a baseline for future research with cytoskeleton classification in microscopy fluorescence images. In this work, we present the quantitative and qualitative comparison of above mentioned methods for better understand the behavior of these methods and the properties of Actin microfilaments (MA) and Microtubules (MT) on both datasets. The results showed that it is possible to classify the FDIG Olympus data set with accuracy of up to 90:07% and 98:94% for 2D Hela, in addition to reaching 86:05% and 96:84% respectively, using complex network theory.

A cinética enzimática dos derivados de quinizarina com cadeias homólogas por lipases imobilizadas foi investigada por espectroscopia de fluorescência. Este estudo foi realizado em nível macroscópico e microscópico. Para o estudo macroscópico, foi utilizada a lipase suportada CALB (Novozyme® 435) e para o estudo microscópico a lipase Rhizopus niveus imobilizada em nanopartículas de sílica. Os derivados de quinizarina são espécies que não apresentam fluorescência, porém, quando são hidrolisados, tornam-se fluorescentes (quinizarina). Com um modelo cinético considerando um mecanismo de dois processos sequenciais do tipo Michaelis-Menten, foi possível fazer uma descrição adequada da evolução temporal da formação da quinizarina. O tempo médio de reação da hidrólise enzimática, em nível macroscópico, foi determinado para os derivados diacetato, dibutirato, dihexanoato e dioctanoato de quinizarina nos solventes hexano, ciclo-hexano e decalina saturados com água. No estudo microscópico, a lipase de Rhizopus niveus foi incorporada em nanopartículas de sílica de 200nm. A hidrólise enzimática foi monitorada por imagens e pela flutuação da intensidade de fluorescência com o tempo, por meio da microscopia de fluorescência confocal. Os resultados mostraram que, após a adição do substrato (derivados da quinizarina), começam a aparecer regiões fluorescentes devido ao trabalho enzimático (formação da quinizarina). As imagens de microscopia de fluorescência confocal não mostraram uma nítida diferença entre os substratos avaliados. Entretanto, o estudo da flutuação da intensidade de fluorescência mostrou que há uma diferença entre os substratos e que é possível estimar constantes de tempo de relaxação da atividade enzimática. Além disso, a atividade da lipase depende da forma em que a mesma está distribuída nas nanopartículas (ligada ou adsorvida) e também do tamanho da cadeia de alquílica que compões os derivados. O decaimento de fluorescência da quinizarina produzida pela hidrólise dos derivados pela lipase foi adquirido por microscopia de fluorescência confocal usando excitação de 2-fótons. / The kinetics of enzymatic hydrolysis of quinizarin diester by supported lipase dispersed beads in organic solvents was investigated by fluorescence spectroscopy. This study was performed on macroscopic and microscopic levels. For the macroscopic study was used CALB immobilized lipase (Novozyme ® 435) on acrylate beads, and for microscopic study Rhizopus niveus lipase immobilized on silica nanoparticles. The quinizarin derivatives (substrates) are non-fluorescent species, and only the end product quinizarin has fluorescence. A kinetic model considering two sequential Michaelis-Menten mechanisms provides a suitable description of the time evolution of the quinizarin formation monitored by emission spectroscopy and photon counting measurements. The average reaction time of the enzymatic hydrolysis was determined for quinizarin diacetate, dibutirate, dihexanoate and dioctanoate in hexane, cyclohexane and decaline water saturated solvents. In the microscopic study, the Rhizopus niveus lipase was dispersed into and bound silica mesoporous 200nm particles. In both systems, dispersed silica nanoparticles and a small fraction of aggregates are found in thin film. The enzyme activity was monitored by images and fluctuations of fluorescence intensity over time using confocal fluorescence microscopy. The results showed that after addition of substrate fluorescent spots due to enzyme activity start to appear. Confocal fluorescence images showed no clear difference among substrates. However, the study of fluorescence intensity fluctuations showed that enzyme activity depends on the type of substrate and enzyme support. In addition, the lipase activity depends on the form in which it is distributed in the nanoparticles (bound or entrapped) and the size of the alkyl diester derivatives. The fluorescence decay of quinizarin produced by lipase hydrolysis of diester was measured by confocal fluorescence microscopy using 2-photon pulse excitation.

Over the past few decades, fluorescence microscopy has proven to become the most widely used imaging technique in the field of life sciences. Unfortunately, all classical optical microscopy techniques have one thing in common: their resolution is limited by the diffraction. Thankfully, due to the very strong interest, development of fluorescent microscopy techniques is very intense, with novel solutions surfacing repeatedly. The major breakthrough came with the appearance of super-resolution microscopy techniques, enabling imaging well below the diffraction barrier and opening the new era of nanoscopy. Among the fluorescent super-resolution techniques, Stimulated Emission Depletion (STED) microscopy has been particularly interesting, as it is a purely optical technique which does not require post image processing. STED microscopy has proven to resolve structures down to the molecular resolution. However, super-resolution microscopy is not a cure to all the problems and it also has its limits. What has shown to be particularly challenging, was the super-resolution imaging of thick samples. With increased thickness of biological structures, the aberrations increase and signal-to-noise (SNR) decreases. This becomes even more evident in the super-resolution imaging, as the nanoscopic techniques are especially sensitive to aberrations and low SNR. The aim of this work is to propose and develop a 3D STED microscope that can successfully image thick biological samples with nanoscopic resolution. In order to achieve that, adaptive optics (AO) has been employed for correcting the aberrations, using the indirect wavefront sensing approach. This thesis presents a custom built 3D STED microscope with the AO correction and the resulting images of thick samples with resolution beyond diffraction barrier. The developed STED microscope achieved the resolution of 60nm in lateral and 160nm in axial direction. What is more, it enabled super-resolution imaging of thick, aberrating samples. HeLa, RPE-1 cells and dopaminergic neuron differentiated from human IPS cells were imaged using the microscope. The results shown in this thesis present 3D STED imaging of thick biological samples and, what is particularly worth to highlight, 3D STED imaging at the 80μm depth, where the excitation and depletion beams have to propagate through the thick layer of tissue. 3D STED images at such depth has not been reported up to date.

FLIM is branch of microscopy mainly used in biology which is quickly improving thanks to a rapid enhancement of instrumentation and techniques enabled by new sensors. In FLIM, the most precise method of measuring fluorescent decays is called TCSPC. High voltage PMT detection devices together with costly and bulky optical setups which scan the sample are usually required in TCSPC instrumentation. SPADs have enabled a big improvement in TCSPC measurement setup, providing a CMOS compatible device which can be designed in wide arrays format. However, sensors providing in-pixel TCSPC do not scale in size and in large array like the time-gated SPAD pixel sensors do. Time-gated pixels offer a less precise lifetime estimation, discarding any photon information outside a given time window, but this loss in photon-efficiency is offset by gains in pixel size. This work is aimed at the development of a wide field TCSPC sensor with a pixel size and fill factor able to reduce the cost of such devices and to obtain a high resolution time-resolved fluorescence image in the shortest time possible. The study focuses on SPAD and pixel design required to maximise the fill factor in sub 10 μm pixel pitch. Multiple pixel designs are proposed in order to reduce pixel area and so enable affordable wide array TCSPC systems. The first proposed pixel performs the CMM lifetime estimation in order to reduce the frame rate needed to stream the data out of the SPAD array. This pixel is designed in a 10 μm pitch and attains with the most aggressive design a fill factor of 10:17 %. A second design proposes an analogue TCSPC which consists in a S/H TAC circuitry. This simpler pixel can achieve a higher fill factor of 19:63% as well as smaller pitch of 8 μm thanks to the adoption of SPAD n-well and electronics area sharing. This last design is implemented in a 320 x 256 SPAD array in which is included part of a novel ADC aimed at reduction of the processing time required to build a TCSPC histogram. A more conventional analogue readout is used to evaluate the pixel performance as well as a more fine TCSPC histogram. The device was used to measure the fluorescence lifetime of green micro-spheres while the 2b flash ADC is used to demonstrate rapid resolution and separation of two different fluorescence decays.