Orientadores: Evandro Conforti, Cristiano de Mello Gallep / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Elétrica e de Computação / Made available in DSpace on 2018-08-22T19:51:13Z (GMT). No. of bitstreams: 1 Bertogna_EduardoGiometti_D.pdf: 5503419 bytes, checksum: 6db94039ae848127cc6ba86810a7cd7d (MD5) Previous issue date: 2013 / Resumo: O estudo das emissões de fótons ultra-fracas de amostras biológicas tem se mostrado promissor em áreas como biomedicina, agronomia, ciências dos alimentos e meio ambiente, onde estas emissões se apresentam como um eficiente indicador biológico das condições fisiológicas das amostras e, portanto, uma ferramenta alternativa de análise e diagnóstico a ser explorada. Esta tese de doutorado visou apresentar uma solução em instrumentação para a eficiente medição destas emissões de fótons ultra-fracas e desenvolver aplicações desta em controle ambiental. O sistema de medição é dotado de controle de temperatura da amostra no seu interior, sistema de iluminação para medidas de emissões estimuladas utilizando modernas técnicas de detecção fotônica, integrando-se a um programa de computador na forma de um instrumento virtual que executa funções de controle e pós-tratamento dos dados adquiridos via placa de aquisição microcontrolada. Utilizando-se o sistema de medição projetado, foram realizadas medidas das emissões de fótons de amostras de sementes de trigo em germinação sob condições controladas de temperatura, umidade e iluminação no interior da câmara escura do sistema, tendo sido possível se desenvolver uma metodologia de aplicação das técnicas exploradas, tendo como base a análise comparativa dos padrões temporais das emissões simultâneas de duas amostras biológicas, em duas câmaras idênticas de medição, uma contendo a amostra de controle, e a outra a amostra sob estresse químico. A técnica possibilita correlacionar à presença do contaminante químico com desvios nas emissões de fótons em apenas algumas horas. Os experimentos de emissão estimulada por fonte externa de luz, também aplicados em análises em controle ambiental, se mostraram outra possibilidade, porém estas não foram extensivamente exploradas nesta tese, tendo apenas apresentado resultados preliminares / Abstract: Studies of ultra-weak photon emissions from biological samples has been shown promising in areas such as biomedicine, agronomy, food science and environmental studies, where those emissions could be used as an efficient biological indicator of the physiological conditions of the samples, and therefore an alternative tool for analysis and diagnostics to be explored. This thesis aimed to present an efficient solution for measuring ultra-weak photon emissions from small biological samples and also develop applications in environmental control. The measuring system, equipped with temperature control of the sample inside it and a lighting system for measuring stimulated emissions, was developed using modern photonic detection techniques, integrated to a computer program as a virtual instrument that performs control and post-processing of the data acquired via a microcontrolled board. Using the measurement system designed, several measurements of ultra-weak photon emissions from wheat seed samples germinating under controlled temperature, humidity and lighting inside the dark space of the system were performed, and it was possible to develop a methodology using the techniques exploited, based on a comparative analysis of simultaneous temporal patterns of the emissions from two biological samples, using two identical measuring chambers, one containing the control sample, and the other the sample under chemical stress. The technique makes it possible to correlate the presence of chemical contaminants with deviations in those photon emissions in a few hours period. Experiments of stimulated emissions by an external source of light, also applied to analysis in environmental control, proved to be another possibility, but these were not extensively explored in this thesis, only submitted as preliminary results / Doutorado / Telecomunicações e Telemática / Doutor em Engenharia Elétrica

The research presented in this thesis set out to contribute to knowledge seeking to advance the state-of-the-art in laser based, multi-functional, non-invasive diagnostic systems. The results of the work have contributed to the development of methodological generic approaches to the development of devices using light to detect and analyse biomarkers relevant to human health and disease conditions. Following the development of methodologies the research then progresses to examine in-vivo the potential of the integrated spectroscopic technologies to detect changes and rhythms in the micro-vasculature of the skin relevant to the response of the subjects to emotional and physical stress. The results uncover myogenic rhythm synchronisation as a potential marker of adaptive response. The complexity of monitoring and interpreting key biomarkers of metabolism, NADH and FAD and the derived redox ratio is addressed in detail. Analysis of results of around 3,000 functional scans, including tissue oxygen saturation and laser Doppler flowmetry, provides new insights into bio-technological issues. The challenges identified with biological characteristics include sampling zones and physiological features of the skin. Technological, photonics, electronics and computing challenges emerge from the results. These should be considered in advancing the integration of bio-photonic technologies toward realising meaningful diagnostic poly-bio-markers relevant to developing algorithms capable of delivering consistent, reliable and meaningful diagnostic information with utility in clinical practice for early diagnosis of disease conditions such as cancers and cardio-vascular diseases in individuals from the global population. The results have been published in peer-reviewed international journals and presented at major international conferences in the field.

BiophotonicE. coli O157:H7(BEC) was evaluated for growth and stability to evaluate the effectiveness of BEC as a tool to validate pathogen reduction interventions. Escherichia coliO157:H7 containing Xen14 and pAK1 lux plasmids were monitored for growth and stability over 10 d at 2.2˚C and 37˚C. Concentration of BECat 2.2˚C remained constant (P> 0.05) but photonic emissions (RLU/s) changed (P<0.05), resulting in a positive correlation between RLU/s and CFU/mL (P< 0.01; r=0.5718). BEC grown at 37˚C yielded no change after a decrease on day 2 (P>0.05). In phase I, purge was monitored for photonic emission after trim was inoculated with BEC. BECwas found at 4 h in Combo C and D. Phase II purge collection used inoculated trim indicative of trim used currently in the beef industry stored in simulated combos. Neither photonic emission nor CFU’s were enumerated in phase II.

Fluorescence based microscopy techniques have been extensively used in biological sciences. The most common approach is the steady-state fluorescence microscopy. Although the said approach is powerful, it often lacks sensitivity to detect several biochemical processes that may indicate relevant conditions of biological tissues. The fluorescence dynamics analysis not only brings intrinsic information about the tissue, but is also less sensitive to the medium scattering and absorption, and sometimes capable of distinguishing between fluorescent structures with indistinguishable spectra. The intrinsic fluorescence lifetime of biological tissues is usually affected by some clinical conditions, especially when those conditions cause or are correlated with metabolic modifications. Time-resolved spectroscopy techniques can be used to detect those modifications and may be used as a tool to improve the detection and diagnosis rate of such conditions. Fluorescence Lifetime Imaging Microscopy (FLIM) combines the temporal resolution and the microscopy concept, so fluorescence lifetime images can be generated. This technique has a great potential for clinical applications since it may be able to detected lesions and delineate its borders. However, FLIM usually demands a more sophisticated instrumentation than most techniques based on the steady-state approach, what creates a difficulty for moving such a system to a clinical setting. We report the assembly, characterization, validation, and clinical application of a multispectral FLIM system featuring a handheld probe composed of a laser scanning rigid endoscope. The assembled system uses a 355 nm short pulsed laser as excitation and has three spectral channels, targeting the emission of collagen, NADH, and FAD, which are important endogenous fluorophores. The system acquires images of 8.65 x 8.65 mm2 areas in ~ 2.4 s. MATLAB codes were written to process the images using a biexponential model and a modified phasor approach. In vivo validation measurements of tumors induced in mice were performed. The system was also validated with in vivo imaging of skin of healthy volunteers. The assembled FLIM system was moved to Hospital Amaral Carvalho, where we performed a pilot clinical study, in which different types of skin lesions were imaged in vivo in a clinical setting. A significant contrast was achieved on seborrheic keratosis, Bowen´s disease, and sclerodermiform basal cell carcinoma tumors. These results indicate the potential of this technique for clinical imaging of skin lesions. / Técnicas de microscopia baseadas em fluorescência têm sido extensamente utilizadas em ciências biológicas. A abordagem mais comum se baseia na microscopia de fluorescência de estado estacionário. Apesar de poderosa, essa abordagem frequentemente não apresenta sensibilidade suficiente para detectar diversos processos bioquímicos que podem ser indicadores de relevantes problemas em tecidos biológicos. A análise da dinâmica da fluorescência não apenas trás informações intrínsecas sobre o tecido, mas também é menos sensível a espalhamento e absorção pelo meio, além de ser capaz de distinguir entre estruturas fluorescentes com espectros indistinguíveis em alguns casos. O tempo de vida intrínseco de tecidos biológicos é normalmente afetado por condições clínicas, especialmente quando estas condições causam ou são relacionadas a modificações metabólicas. As técnicas de espectroscopia resolvidas no tempo podem detectar essas modificações e podem ser utilizadas como uma ferramenta para melhorar a detecção e o diagnóstico dessas condições. A Microscopia de Tempo de Vida de Fluorescência (FLIM) combina a resolução temporal ao conceito de microscopia, de forma que imagens de tempos de vida de fluorescência podem ser gerados. Essa técnica tem um grande potencial para aplicações clínicas uma vez que ela pode ser capaz de detectar lesões e delinear suas bordas. No entanto, FLIM requer uma instrumentação muito mais sofisticada do que a maior parte das técnicas baseadas no estado estacionário, o que cria uma dificuldade para que tais sistemas possam ser levados a ambientes clínicos. Nós reportamos a montagem, caracterização, validação e aplicação clínica de um sistema FLIM multiespectral com uma sonda manual composta de um endoscópio rígido de varredura laser. O sistema montado utiliza um laser pulsado de 355 nm como fonte de excitação e conta com três canais espectrais, visando a emissão do colágeno, do NADH e do FAD, três importantes fluoróforos endógenos. O sistema é capaz de adquirir imagens de áreas de 8,65 x 8,65 mm2 em ~ 2,4 s. Códigos em MATLAB foram escritos para processar as imagens usando um modelo biexponencial e uma abordagem modificada dos fasores. Medidas in vivo de tumores induzidos em camundongos foram realizadas para validação do sistema. O sistema também foi validado com a realização de medidas in vivo da pele de voluntários sadios. O sistema montado foi levado ao Hospital Amaral Carvalho, onde realizamos um teste clínico piloto no qual diferentes tipos de lesões de pele foram imageados in vivo em um ambiente clínico. Um contraste significante foi alcançado em tumores de queratose seborreica, doença de Bowen e carcinoma basocelular esclerodermiforme. Esses resultados indicam o potencial desta técnica para o imageamento clínico de lesões de pele.

No description available.

Fluorescence based microscopy techniques have been extensively used in biological sciences. The most common approach is the steady-state fluorescence microscopy. Although the said approach is powerful, it often lacks sensitivity to detect several biochemical processes that may indicate relevant conditions of biological tissues. The fluorescence dynamics analysis not only brings intrinsic information about the tissue, but is also less sensitive to the medium scattering and absorption, and sometimes capable of distinguishing between fluorescent structures with indistinguishable spectra. The intrinsic fluorescence lifetime of biological tissues is usually affected by some clinical conditions, especially when those conditions cause or are correlated with metabolic modifications. Time-resolved spectroscopy techniques can be used to detect those modifications and may be used as a tool to improve the detection and diagnosis rate of such conditions. Fluorescence Lifetime Imaging Microscopy (FLIM) combines the temporal resolution and the microscopy concept, so fluorescence lifetime images can be generated. This technique has a great potential for clinical applications since it may be able to detected lesions and delineate its borders. However, FLIM usually demands a more sophisticated instrumentation than most techniques based on the steady-state approach, what creates a difficulty for moving such a system to a clinical setting. We report the assembly, characterization, validation, and clinical application of a multispectral FLIM system featuring a handheld probe composed of a laser scanning rigid endoscope. The assembled system uses a 355 nm short pulsed laser as excitation and has three spectral channels, targeting the emission of collagen, NADH, and FAD, which are important endogenous fluorophores. The system acquires images of 8.65 x 8.65 mm2 areas in ~ 2.4 s. MATLAB codes were written to process the images using a biexponential model and a modified phasor approach. In vivo validation measurements of tumors induced in mice were performed. The system was also validated with in vivo imaging of skin of healthy volunteers. The assembled FLIM system was moved to Hospital Amaral Carvalho, where we performed a pilot clinical study, in which different types of skin lesions were imaged in vivo in a clinical setting. A significant contrast was achieved on seborrheic keratosis, Bowen´s disease, and sclerodermiform basal cell carcinoma tumors. These results indicate the potential of this technique for clinical imaging of skin lesions. / Técnicas de microscopia baseadas em fluorescência têm sido extensamente utilizadas em ciências biológicas. A abordagem mais comum se baseia na microscopia de fluorescência de estado estacionário. Apesar de poderosa, essa abordagem frequentemente não apresenta sensibilidade suficiente para detectar diversos processos bioquímicos que podem ser indicadores de relevantes problemas em tecidos biológicos. A análise da dinâmica da fluorescência não apenas trás informações intrínsecas sobre o tecido, mas também é menos sensível a espalhamento e absorção pelo meio, além de ser capaz de distinguir entre estruturas fluorescentes com espectros indistinguíveis em alguns casos. O tempo de vida intrínseco de tecidos biológicos é normalmente afetado por condições clínicas, especialmente quando estas condições causam ou são relacionadas a modificações metabólicas. As técnicas de espectroscopia resolvidas no tempo podem detectar essas modificações e podem ser utilizadas como uma ferramenta para melhorar a detecção e o diagnóstico dessas condições. A Microscopia de Tempo de Vida de Fluorescência (FLIM) combina a resolução temporal ao conceito de microscopia, de forma que imagens de tempos de vida de fluorescência podem ser gerados. Essa técnica tem um grande potencial para aplicações clínicas uma vez que ela pode ser capaz de detectar lesões e delinear suas bordas. No entanto, FLIM requer uma instrumentação muito mais sofisticada do que a maior parte das técnicas baseadas no estado estacionário, o que cria uma dificuldade para que tais sistemas possam ser levados a ambientes clínicos. Nós reportamos a montagem, caracterização, validação e aplicação clínica de um sistema FLIM multiespectral com uma sonda manual composta de um endoscópio rígido de varredura laser. O sistema montado utiliza um laser pulsado de 355 nm como fonte de excitação e conta com três canais espectrais, visando a emissão do colágeno, do NADH e do FAD, três importantes fluoróforos endógenos. O sistema é capaz de adquirir imagens de áreas de 8,65 x 8,65 mm2 em ~ 2,4 s. Códigos em MATLAB foram escritos para processar as imagens usando um modelo biexponencial e uma abordagem modificada dos fasores. Medidas in vivo de tumores induzidos em camundongos foram realizadas para validação do sistema. O sistema também foi validado com a realização de medidas in vivo da pele de voluntários sadios. O sistema montado foi levado ao Hospital Amaral Carvalho, onde realizamos um teste clínico piloto no qual diferentes tipos de lesões de pele foram imageados in vivo em um ambiente clínico. Um contraste significante foi alcançado em tumores de queratose seborreica, doença de Bowen e carcinoma basocelular esclerodermiforme. Esses resultados indicam o potencial desta técnica para o imageamento clínico de lesões de pele.

The spatiotemporal organization of membrane proteins is implicated in cellular trafficking, signalling and reception. It was proposed that biological membranes partition into lipid rafts that can promote and control the organization of membrane proteins to localize the mentioned processes. Lipid rafts are thought to be transient (microseconds) and small (nanometers), rendering their detection a challenging task. To circumvent this problem, multi-component artificial membrane systems are deployed to study the segregation of lipids at longer time and length scales. In this thesis, multi-component Droplet Interface Bilayers (DIBs) were imaged using fluorescence and interferometric scattering microscopy. DIBs were used to examine and manipulate microscopic lipid domains and to observe, for the first time, transient nanoscopic lipid domains. The techniques and results described here will have important implications on future research in this field.

Modeling of light propagation through turbid (highly-scattering) media such as living tissue is important for a number of medical applications including diagnostics and therapeutics. This thesis studies methods of performing such simulations quickly and accurately. It begins with a formal definition of the problem, a review of solution methods, and an overview of the current state of the art in fast simulation methods encompassing both traditional software and more specialized hardware acceleration approaches (GPU, custom logic). It introduces FullMonte, the fastest mesh-based Monte Carlo software model available and highlights its novel optimiza- tions. Additionally, it demonstrates the first fully three-dimensional hardware simulator using Field-Programmable Gate Array (FPGA) custom logic, offering large (40x) power-efficiency and performance (3x) gains. Next, a plan for significant future feature enhancements and performance scale-out is sketched out. Lastly, it proposes applying the simulators developed to a number of problems relevant to current clinical and research practice.

Modeling of light propagation through turbid (highly-scattering) media such as living tissue is important for a number of medical applications including diagnostics and therapeutics. This thesis studies methods of performing such simulations quickly and accurately. It begins with a formal definition of the problem, a review of solution methods, and an overview of the current state of the art in fast simulation methods encompassing both traditional software and more specialized hardware acceleration approaches (GPU, custom logic). It introduces FullMonte, the fastest mesh-based Monte Carlo software model available and highlights its novel optimiza- tions. Additionally, it demonstrates the first fully three-dimensional hardware simulator using Field-Programmable Gate Array (FPGA) custom logic, offering large (40x) power-efficiency and performance (3x) gains. Next, a plan for significant future feature enhancements and performance scale-out is sketched out. Lastly, it proposes applying the simulators developed to a number of problems relevant to current clinical and research practice.

Optical tweezers have allowed us to harness the momentum of light to trap, move, and manipulate microscopic particles with Angstrom-level precision. Position and force feedback systems grant us the ability to feel the microscopic world. As a tool, optical tweezers have allowed us to study a variety of biological systems, from the mechanical properties of red blood cells to the quantised motion of motor-molecules such as kinesin. They have been applied, with similar impact, to the manipulation of gases, atoms, and Bose-Einstein condensates. There are, however, limits to their applicability. Historically speaking, optical tweezers have only been used to trap relatively simple structures such as spheres or cylinders. This thesis is concerned with the development of a fabricational and optical manipulation protocol that allows holographical optical tweezers to trap photonic membranes. Photonic membranes are thin, flexible membranes, that are capable of supporting nanoplasmonic features. These features can be patterned to function as metamaterials, granting the photonic membrane the ability to function as almost any optical device. It is highly desirable to take advantage of these tools in a microfluidic environment, however, their extreme aspect ratios mean that they are not traditionally compatible with the primary technology of microfluidic manipulation: optical tweezers. In line with recent developments in optical manipulation, an holistic approach to optical trapping is used to overcome these limitations. Full six-degree-of-freedom control over a photonic membrane is demonstrated through the use of holographical optical tweezers. Furthermore, a photonic membrane (PM)-based surface-enhanced Raman spectroscopy sensor is presented which is capable of detecting rhodamine dye from a topologically undulating sample. This work moves towards marrying these technologies such that photonic membranes, designed for bespoke applications, can be readily deployed into a microfluidic environment. Extending the range of tools available in the microfluidic setting helps pave the way toward the next set of advances in the field of optical manipulation.