Theses (M.S.)--Marshall University, 2007. / Title from document title page. Includes abstract. Document formatted into pages: contains ix, 193 pages. Includes vitae. Bibliography: p. 157-165.

Microscope optics have long been used to observe biological samples, when used in conjunction with a laser light source, they can also be a powerful means to probe and manipulate cells. This dissertation describes the development of methodologies for laser-based microfabrication in biological environments. These techniques use pulsed laser light at a wavelength transparent to the experimental medium except in a region with submicron dimensions defined by the focus of a high numerical aperture objective. At the focal region, high intensity light can modify sample material. The localized nature of this energetic event allows it to be accomplished in the vicinity of living cells, enabling microfabrication strategies that are used to probe and modify extracellular environments with high resolution. The basic principles of this process are explored and its use in several applications for cell culture manipulation are described. In one methodology the focused laser induces physical and chemical events that lead to the formation of a micron-scale solid from a precursor protein solution. By translating the relative position of the beam in the solution, arbitrary three-dimensional structures can be formed. The use of protein microstructures as a platform for probing and manipulating cellular microenvironments is investigated and an advanced method of rapidly patterning elaborate structures with a spatial light modulator is demonstrated. The high intensity laser focus is used in a second strategy to create microfluidic conduits in a device consisting of two stacked flow channels, one containing adherent cells in buffer and the other a reagent solution. With laser ablation of a pore, highly defined reagent plumes are directed into the cell-containing chamber where they can dose multiple specifically targeted regions with subcellular specificity. The unique microfabrication technologies described in this dissertation could prove to be of use for researchers developing diagnostic and therapeutic devices and could lead to more advanced tools for studying the basic biology of cells.

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

The ultimate objective of this project was to develop a small, transportable X-ray microscope which would be able to view a wide range of biological specimens without the need for any type of sample preparation at a resolution greater than that obtainable by conventional light microscopy (ie. about 250nm). Of the various possible implementations of X-ray microscopy currently being investigated, contact microscopy was chosen as being the most suitable for the development of such a small-scale instrument, while at the same time minimizing the effects on image quality of radiation damage to the biological specimen. The requirement for a high brightness pulsed X-ray source of less than 50ns duration for illumination of the specimen was met by the production of laser generated plasmas. These were formed by focusing a 2.2J KrF laser beam, of wavelength 248nm and duration 20ns, onto the surface of one of a number of different target materials. In order to obtain the large intensities required for the production of a sufficiently high temperature plasma, a doubly pre-ionized, discharge-pumped amplifier KrF laser was developed. This was seeded by a smaller oscillator laser by means of a coupled unstable resonator configuration. A number of different cavity arrangements were investigated and an output beam divergence of 2.5 times the diffraction limit was achieved. The plasmas generated by focusing the laser beam to an intensity of 10¹⁴W/cm² onto carbon, titanium, molybdenum and tungsten targets were characterized as fully as was necessary for their use in the X-ray microscope. Preliminary investigations on the use of a grazing incidence ellipsoidal mirror to focus the emitted X-rays onto the specimen of the microscope were made and such an optical component was manufactured and tested. Finally, numerous images of a number of different biological specimens were made and resolutions of better than 100nm were achieved. Images were read out using a Park Scientific Instruments atomic force microscope, which enabled the entire microscopy process to be carried out in a single working day. The system is now in routine use and can produce more than ten images per session.

Thesis (M.S.)--Ohio University, August, 1998. / Title from PDF t.p.

Thesis (Ph.D.) - University of St Andrews, March 2007.

No description available.

In this thesis, the development of a femtosecond Cr⁴⁺:forsterite solid-state laser is described where the mode-locking procedure was initiated using two novel saturable absorbers. One was a GaInNAs quantum-well device and the other a quantum-dot-based saturable absorber. These devices had not previously been exploited for the generation of femtosecond pulses from a solid-state laser but in the course of this project, successful mode-locked laser operation in the femtosecond domain was demonstrated for both devices. When the GaInNAs device was incorporated in the Cr⁴⁺:forsterite laser, transform-limited pulses with durations as short as 62fs were obtained. The performance of this femtosecond laser was significantly superior to that for previous quantum-well based saturable absorbers in the 1300nm spectral region. The dynamics of the device were investigated with the aim of refining subsequent devices and to explore the potential to grow future devices for use at longer wavelengths. At the outset of my research work quantum-dot based saturable absorbers had not be used for the mode locking of solid-state lasers in the femtosecond regime. The work presented in this thesis showed that quantum-dot structures could be exploited very effectively for this purpose. This was initially achieved with the quantum-dot element being inclined at an off-normal incidence within the cavity but experimental assessment together with further development of the device allowed for implementation at normal incidence. Reliable operation of the femtosecond laser was demonstrated very convincingly where transform-limited pulses of 160fs duration were generated. Having developed practical femtosecond Cr⁴⁺:forsterite lasers, the final part of the project research was directed towards exemplar applications for a laser operating in the 1300nm spectral region. These were biophotonics experiments in which assessments of both deep tissue penetration and two-photon chromosome cutting were undertaken. This work confirmed the suitability of the 1300nm laser radiation for propagation through substantial thicknesses of biological tissue (~15cm). The demonstration of highly localised two-photon cutting of Muntjac deer chromosomes also represented a novel result because single-photon absorption could be avoided effectively and the temporal broadening of the femtosecond pulses in the delivery optics arising from group velocity dispersion around 1300nm was minimal.

Orientador: Maria Alice da Cruz Hofling / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-07T07:40:42Z (GMT). No. of bitstreams: 1 Dourado_DorotyMesquita_D.pdf: 6138629 bytes, checksum: 583afdc02d280fcf5b42546f6eeb5981 (MD5) Previous issue date: 2006 / Resumo: O estudo de substâncias naturais visa não só a caracterização dos seus princípios ativos e efeitos biológicos, mas também a compreensão de fenômenos patofisiológicos que muitas vezes são reproduzidos experimentalmente por essas substâncias. Dentre estas, nos países de clima tropical, os venenos ofídicos merecem importância particular pela riqueza de constituintes farmacologicamente ativos e pela freqüência dos acidentes causados por picada de serpentes venenosas, particularmente abundantes em países tropicais. No Brasil, esses acidentes adquirem relevância em termos de saúde pública porque podem causar graves alterações sistêmicas e no local da picada, sendo o gênero Bothrops responsável por 90% deles. Neste trabalho, as alterações patológicas locais e sistêmicas (mionecrose, edema e hemorragia) induzidas pela injeção intramuscular (i.m.) do veneno de Bothrops moojeni (40 µg/mL, 0,1 ml/100 g do peso do animal) em camundogos foram estudadas histologicamente, através da contagem de capilares e pela imunomarcação do fator de crescimento endotelial de vasos (VEGF) no músculo gastrocnêmio e pelas alterações nos níveis séricos das enzimas creatino kinase (CK), fosfatase ácida (AcPase), fosfatase alcalina (AlkPase), desidrogenase lática (LDH), transaminase oxalo-acética (AST) e da proteína mioglobina ao longo de 3, 12, 24 horas, 3, 7 e 21 dias (n = 5/período) após o envenenamento. Os efeitos de protocolos de irradiação por lasers de baixa potência, HeNe (632,8 nm) e o diodo AsGa (904 nm), ambos com densidade de energia de 4 J/cm², sobre esses parâmetros foram avaliados. A primeira sessão de irradiação foi administrada logo após a injeção i.m. do veneno e depois a cada 24 h, de forma que o número de irradiações feito foi uma, uma, duas, 4, 8 e 22, respectivamente, incididas perpendicularmente no local da injeção por 120 s (HeNe) e 18,3 s (GaAs) de duração de cada sessão. Para as dosagens de LDH, AST e mioglobina o tempo zero, sem irradiação foi acrescentado. Os resultados foram comparados com animais controles, injetados com solução salina estéril (0,9%). Morfologicamente, o veneno produziu imediata série de alterações, a qualidade das quais mostrou-se semelhante das 3 às 24 h (pós-injeção) p.i., porém com aumento do número de células mionecróticas e de infiltrado inflamatório. A fase regenerativa iniciada 3 d p.i., mostrou crescente número de fibras em regeneração, decrescente número de fibras degeneradas e substituição de polimorfos nuclerares por população de macrófagos. Histologicamente, a irradiação pelo HeNe atenuou as alterações iniciais (3 h), porém a do AsGa foi mais efetiva em longo prazo (21 d), induzindo ao maior número de miotubos e à melhor citoarquitetura do tecido. A contagem do número de vasos sangüíneos no local e vizinhanças da lesão, mostrou que o grupo tratado com veneno e não irradiado com laser de baixa energia teve diminuição do número médio de capilares na área afetada, e que a irradiação laser acelerou a neovascularização, sendo o HeNe mais eficaz do que o AsGa. Os resultados também mostraram que o VEGF e seus receptores foram expressos em parte dos neutrófilos, em esparsas células satélites nos períodos iniciais do envenenamento (3 ¿ 24 h), nos capilares das áreas periféricas à lesão, e nos interstícios de fibras em degeneração. A irradiação com HeNe induziu o aparecimento desse marcador nos polimorfonucleares, mas o AsGa aboliu a positividade a eles. O HeNe promoveu a expressão de VEGF nos capilares dos tendões e nas células musculares intactas. Aos 7 d p.i., na região das fibras regenerativas a irradiação com HeNe levou ao aparecimento de positividade em células do interstício semelhantes a fibroblastos, além de marcação em células ¿satélites¿ de miotubos em formação. A irradiação por ambos os lasers induziu também o aparecimento de VEGF positivo nos fibroblastos do tendão que atravessava o músculo, nas camadas média e adventícia de pequenas artérias e veias e nos fusos neuromusculares, além do interstício dos ramos nervosos intramusculares, principalmente nos períodos de 7 e 21 dias. Os resultados da análise das enzimas mostraram que a irradiação pelo laser HeNe reduziu marcadamente a atividade de CK de 3 h até 7 dias, enquanto o laser AsGa de 12 h até 21 dias p.i. A atividade da AcPase aumentou dramaticamente às 12 h p.i., após o que houve um declínio que foi mais acentuado com o laser AsGa. A irradiação com o laser HeNe induziu aumento gradual da atividade de AlkPase até 24 h e então diminuiu-a, ao passo que o laser AsGa causou um extraordinário aumento entre 12 h e 24 h, mantendo diferença signifitiva em relação ao grupo veneno de camundongos não irradiados. A irradiação com HeNe não foi eficaz em diminuir os níveis de LDH, ao invés foi prejudicial elevando os seus níveis em diferentes momentos. Já a irradiação com AsGa foi eficaz em diminuir significativamente os níveis da enzima ao longo de todos os períodos observados. Com relação a AST, a irradiação com HeNe foi eficaz em diminuir os níveis da enzima apenas às 3 h p.i., após o que teve efeitos deletérios. Por outro lado, a irradiação com AsGa não foi eficaz em diminuir os níveis de AST, exceto aos 7 dias, quando houve redução significativa de16,6%. Os níveis elevados de mioglobina sérica causados pelo veneno de B. moojeni, foram diminuídos significativamente com o laser AsGa das 12 h p.i. até os 7 d, ao passo que o HeNe só mostrou eficácia dos 3 aos 7 d p.i. O grupo de camundongos injetados com solução salina não mostrou nenhuma diferença significante em todos os tempos analisados para esses parâmetros. Concluímos que a terapia laser de baixa energia, arsenieto de Gálio aplicada localmente é capaz de alterar o conteúdo sérico de biomarcadores de dano da fibra muscular (CK e mioglobina), como também de marcadores de alterações sistêmicas, como o laser HeNe foi eficaz para aumentar a quantidade de vasos sanguíneos no tecido lesado. A fotoestimulação promovida pela irradiação com laser de baixa energia pode alterar favoravelmente indicadores de alterações locais e sistêmicas causadas por envenenamento por Bothrops moojeni, apontando esse recurso como promissor na recuperação do músculo afetado e nos distúrbios sistêmicos advindos / Abstract: The study of natural substances aims not only characterization of its active principles and biological effects, but also the comprehension of pathophysiologic phenomena that frequently are reproduced experimentally by these substances. Among these substances, snake venoms deserve special importance for the richness of pharmacologically active components and the frequency of accidents caused by venomous snake bites, particularly abundant in tropical countries. In Brazil these accidents acquires relevance in terms of public health because they may cause severe systemic and local alterations, being the Bothrops genus responsible for 90% of them. In this work, the pathologic local and systemic alterations induced by the intramuscular (i.m.) injection of Bothrops moojeni venom (40 µg/mL, 0.1 ml/100 g of animal weight) in mice were studied histologically, by capillary counting, immunolabeling of the vascular endothelial growth factor (VEGF) in the gatrocnemius and by the changes of creatinekinase (CK), acid phosphatase (AcPase), alkaline phosphatase (AlkPase), lactic acid desidrogenase (LDH), transaminase oxaloacética (TGO) and mioglobin serum levels at 3, 12, 24 hours, 3, 7 and 21 days (n = 5/period) after envenoming. The effects of protocols of irradiation using low potency lasers, HeNe (632.8 nm) and GaAs diode (904 nm), both with a energy density of 4 J/cm-2, on these parameters were evaluated. The first section of irradiation was administered soon after the i.m. injection of venom and then at each 24 h interval, in such a way that the number of irradiations done were one, one, two, 4, 8 and 22, respectively, applied perpendicularly right at the injection site during 120 s (HeNe) and 18.3 s (GaAs) duration for each session. For determination of LDH, TGO and mioglobin, a zero time without irradiation was added. The results were compared with control animals, injected with sterile saline (0.9%). Morphologically, the venom produced a immediate series of changes, the quality of which were similar from 3 to 24 h p.i., but with a progressive number of myonecrotic fibers and inflammatory infiltrate. The regenerative phase starting at day 3, showed a crescent number of regenerating and decreasing number of degenerated fibers with polymorphonuclear cells being replaced by macrophages. Histolgically, HeNe irradiation attenuated the first stages of degeneration (3 h), but GaAs¿s was more effective considering the whole period of observations (21 d), leading to the presence of higher number of miotubes and better cytoarchitecture of the tissue. The counting of capillaries at the site and periphery of lesion showed that the venom-injected unirradiated mice had a decrease in the average number of capillaries, and that laser irradiation accelerated the neovascularization, being HeNe more efficient than GaAs. The results also showed that VEGF and its receptors were expressed in a parcel of neutrophils, in sparce satellite cells in the initial periods of the envenoming (3 ¿ 24 h), in capillaries of the periphery of lesion and in the middle of the degenerating fibers. The irradiation with HeNe induced enhancement of VEGF expression in the neutrophils, but the GaAs abolished it. HeNe also promoted VEGF expression in the tendon capillaries and intact skeletal muscles. At 7 d p.i., VEGFlabeled fibroblast-like cells were seen in the regenerative region, as well as ¿satellite¿-like cells around developing miotubes after irradiation with HeNe. The irradiation by both lasers also induced VEGF positivity in tenocytes, in smooth muscle cells of blood vessels, in adventitia layer, in neuromuscular spindle and in the interstices of intramuscular nerves in the periods from 7 to 21 days p.i.. The results from serum enzymes showed that HeNe irradiation reduced markedly CK activity from 3 h to 7 days, whereas GaAs reduced it from 12 h to 21 days p.i.. AcPase activity increased dramatically at 12 h p.i. after which there was decline, which was more accentuated with laser GaAs. HeNe irradiation induced a gradual increase of AlkPase activity until 24 h p.i., followed by decrease, whereas GaAs caused an extraordinary increase between 12-24 h, maintaining a significant difference in comparison to the venom unirradiated mice. The irradiation with HeNe was not efficient in decrease the LDH levels; on the contrary, it was deleterious, elevating their levels. Conversely, the irradiation with GaAs was efficient in decrease significantly the enzyme levels during all observed periods. In regard to TGO, the irradiation with HeNe was efficient in diminishing the enzyme levels in the initial 3 h p.i., after which it was deleterious. On the other hand, irradiation with GaAs was not effective in minimizing AST serum levels, except at day 7 when there was a significant 16.6% reduction. The high mioglobin serum levels induced by B. moojen venom, significantly declined with GaAs from 12 h to 7 d p.i., while HeNe irradiation showed efficacy between 3-7 days p.i. The group of mice injected (I.m.) with sterile saline did not show any significant difference in all periods analyzed for these parameters. We conclude that therapy with low potency laser applied locally is capable of alter the serum content of muscle damage biomarkers (CK and myoglobin), as well markers of systemic alterations. The photostimulation promoted by low energy laser irradiation can alter favorably markers of local and systemic alterations caused by Bothrops moojeni snake envenoming, suggesting this approach as a promising resource for recovery of affected muscle as well as in the resulting systemic disturbances. Key-words: B. moojeni, CK, seric enzymes, morphometry, VEGF, laser irradiation / Doutorado / Histologia / Doutor em Biologia Celular e Estrutural

In recent years, the study of stimulated emission from and by biological systems has gained wide spread attention as a promising technology platform for novel biointegrated laser. However, the photonic properties and the associated physics of many biological laser systems are not yet fully understood and many promising resonator architectures and laser classes have not yet transitioned into the biological world. In this thesis, we investigate the fundamental photonic properties of lasers based on single biological cells and explore the potential of distributed feedback (DFB) gratings as novel biointegrated laser resonators. We show how the easy and flexible fabrication of DFB resonators helps to realize optofluidic and solid-state biological lasers. Lasing characteristics, such as tunable and single mode emission, are investigated and different applications are explored. Fourier-space emission studies on different biological lasers give insight in to the photonic dispersion relation of the system and the fundamental creation of lasing modes and their confinement in living systems. The first purely water based optofluidic DFB laser is demonstrated and novel sensing applications are suggested. This device shows low threshold lasing due to an optimized mode shape, which is achieved by a low refractive index substrate and the use of a mixed-order grating. Next, by integrating a high refractive index interlayer on a DFB resonator, a laser device incorporating the novel solid-state biological gain material green fluorescent protein (GFP) is realized. Lastly, we show how the thickness of organic polymer lasers can be reduced to its fundamental limit (< 500 nm) and the resulting membrane like laser devices can be applied to and operated on various body parts to potentially complement biometric identification.