O desenvolvimento de sistemas laser com pulsos ultracurtos trouxe a possibilidade de usinagem de estruturas muito pequenas em praticamente qualquer tipo de material. Neste trabalho foi dada continuidade a estudos já iniciados no Centro de Lasers e Aplicações (CLA) com os materiais dielétricos, introduzindo a largura temporal dos pulsos laser como mais uma variável e utilizando os conhecimentos adquiridos para a determinação de limiares de ablação e parâmetros de incubação em alguns metais como: aço AISI 1045, aço inoxidável VI138, cobre eletrolítico e molibdênio. A ausência de calor no processo de ablação dos metais torna-se muito difícil, pois a criação de uma camada de íons é muito prejudicada pela mobilidade eletrônica ao seu redor. Assim a ablação de metais com pulsos ultracurtos, tem como principal mecanismo a explosão de fase associada a outros processos que também contribuem na ablação, porém em menor escala, como a explosão coulombiana e a fusão ultrarrápida. Além disso, propriedades como a constante de acoplamento elétron-fônon e a condutividade térmica assumem um papel importante e devem ser levadas em conta na investigação do processo de ablação dos metais. Este trabalho possibilitou a obtenção de parâmetros de operação nos quais o calor transferido para a rede é minimizado, possibilitando a microusinagem de precisão e alterações controladas na morfologia da superfície de diversos metais. Os resultados propiciaram assim condições para novos desenvolvimentos e aplicações práticas de usinagem com pulsos ultracurtos. / The development of laser systems with ultrashort pulses brought the possibility of machining very small structures in virtually any type of material. In this work was continued the studies already started in Lasers and Applications Center (CLA), with dielectric materials, introducing temporal width of the laser pulses as another variable, and using the knowledge acquired to determine ablation threshold and incubation parameters of some metals such as AISI 1045 steel, VI 138 stainless steel, electrolytic copper and molybdenum. The absence of heat in the ablation process of metals is much more difficult since the creation of a layer of ions is greatly impaired by electronic mobility in its vicinity. Hence, the ablation process for metals with ultrashort pulses, has, as main mechanism, the phase explosion associated with other processes that also contribute in the process, but on a smaller scale, such as Coulomb explosion and ultrafast fusion. Moreover, properties such as electron-phonon coupling constant and thermal conductivity play an important role and should be taken into account in investigating the process of ablation of metals. This study made it possible to obtain operation parameter where the heat transferred to the lattice is minimized, enabling precision micromachining and controlled changes in the morphology of the surface of metals. The results provided conditions for new developments and real machining applications with ultrashort pulses.

>Magister Scientiae - MSc / Recent research shows that the study of optical properties of organic material natural dyes has gained much consideration. The specific functional groups in several natural dyes remain essential for the large nonlinear absorption expressed in terms of nonlinear optical susceptibilities or other mechanism of absorption such as two photon absorption (TPA), reverse saturable absorption (RSA) or intensitydependent refractive index characteristic. In this thesis we highlight the optical limiting responses of selected natural dyes as nonlinear response in the femtosecond regime. This technique refers to the decrease of the transmittance of the material with the increased incident light intensity.Three dyes derived from beetroot, flame flower and mimosa flower dyes were investigated. The results showed a limiting behaviour around 795 mW for the beetroot and the flame dye while there is total transmission in the flame dye sample. The performance of the nonlinearity i.e. the optical limiting is related to the existence of alternating single and double bonds(i.e. C-C and C=C bonds) in the molecules that provides the material with the electron delocalization, but also it is related to the light intensity.Beside nonlinearity study, crystallographic investigation was carried out for more possible applicability of the selected dyes and this concerned only the mimosa and flame flower dye thin film samples since the beetroot thin film was very sensitive to strong irradiation (i.e. immediately destroyed when exposed to light with high intensity). For more stability,dye solutions were encapsulated in gels for further measurements.

Neste trabalho investigamos as modificações na morfologia superficial e estrutura de filmes finos de silício amorfo hidrogenado, resultantes da irradiação com pulsos ultracurtos de femtossegundos (150 fs, 775 nm e 1 kHz). Os processos de microfabricação foram conduzidos varrendo, a velocidade constante, um feixe laser com diferentes fluências (1,8 a 6,2 MJ/m2) sobre a amostra. Os espectros de transmissão apresentaram queda para amostras irradiadas, cujas imagens de microscopia eletrônica de varredura mostraram estruturas superficiais condizentes com o fenômeno de LIPSS (Laser Induced Periodic Surface Structures). Uma análise estatística das imagens de microscopia de força atômica foi realizada com um programa que identifica e caracteriza os domínios (picos) produzidos pela microfabricação. O histograma de altura da amostra irradiada com uma fluência de 3,1 MJ/m2 mostrou que a altura média dos picos produzidos é de 15 nm, menor que o centro da distribuição de alturas para uma amostra não irradiada. Porém, para fluências acima de 3,7 MJ/m2 a morfologia é dominada pela formação de agregados. Medidas de espectroscopia Raman revelaram a formação de uma fração de silício cristalino, após a irradiação com pulsos de femtossegundos, de até 77% para 6,2 MJ/m2. Determinamos ainda uma diminuição da dimensão dos nanocristais produzidos com o aumento da fluência do laser de excitação. Portanto, nossos resultados mostram que há um compromisso entre as propriedades obtidas pela microfabricação (transmissão, distribuição de picos, fração de cristalização e tamanho dos nanocristais produzidos) que deve ser levado em conta ao aplicar a técnica de microestruturação com laser de femtossegundos. / In this work we investigated surface morphology and structural modification on hydrogenated amorphous silicon (a-Si:H) thin films, resulting from femtosecond laser irradiation (150 fs, 775 nm and 1 kHz). Microfabrication processes were carried out scanning sample´s surface, at constant speed, with distinct laser fluencies (from 1.8 to 6.2 MJ/m2). A decrease was observed in the transmission spectra of irradiated samples, whose scanning electron microscopy images revealed surface structures compatible with the Laser Induced Periodic Surface Structure (LIPSS) phenomenon. A statistical analyzes of Atomic Force Microcopy images was performed using a specially developed software, that identifies and characterizes the domains (spikes) produced by the laser irradiation. The height histogram for a sample irradiated with 3.1 MJ/m2 reveals that the average height of the produced spikes is at 15 nm, which is smaller than the center of height distribution for non-irradiated sample. For fluencies higher than 3.7 MJ/m2, however, aggregation of the produced spikes dominates the sample morphology. Raman spectroscopy revealed the formation of a crystalline fraction of 77% for laser fluence irradiation of 6.2 MJ/m2, as well as a decrease in size of the produced crystals as a function of fluence. Therefore, our results indicate that there is a compromise of the sample transmission, spikes distribution, crystallization fraction and size of nanocrystals obtained by fs-laser irradiation, which has to be taken into consideration when using this material processing method.

This thesis explores a new technique for investigating the photochemical mechanisms of femtosecond laser inscription of permanent photonic structures in Poly(methylmethacrylate) (PMMA). The refractive index (RI) structures were fabricated with a direct writing method without ablation, and analysed using a non-invasive method - namely: Raman mapping spectrometry. The writing conditions for the photonic structures under investigation are mainly represented by 800nm and 400nm wavelength with 44fs and 100fs pulse length and a low repetition rate in the kHz domain. The mass percentage of the induced monomer and end groups modification (MMA) as a measure of the modification of the ratio of C=C and C=O Raman transition varies linearly with the total fluence (total). The mass percentage of the induced monomer and end groups change is defined by the modification of normalised ratio of the Raman intensity of C=C bond (I(C=C)) and the Raman intensity of C=O bond (I(C=O)) which is denoted by I(C=C/C=O)n. The modification of this ratio is denoted by I (C=C/C=O)n and also by MMA. MMA varies linearly with total with a positive slope for both writing conditions due to the induced main chain scission and unzipping. If total increases by 1J/cm2, it is predicted an increase in MMA, by (1.550±0.11)x10-2 (cntsxcm2)/J, for the near infrared (NIR) irradiated samples that is higher than the increase of MMA for the ultraviolet (UV) irradiated sample that show a value of (1.9200.274)x10-3 ( (cntsxcm2)/J). The same trend was found for the variation of MMA with diffraction efficiency () for NIR irradiated structures and also for UV irradiated structures. If  increases by 1cnt, it is predicted that there will be an increase in MMA, by (4.233±0.383) cnts for NIR irradiated samples that is lower than the increase of MMA for the UV irradiated sample that shows a value of (14.3922.477) cnts. The variation of MMA with  is higher for UV irradiated samples than for NIR irradiated samples, and this indicates that the nonlinear absorption of two photons produces a larger percentage of the monomer and end groups than the nonlinear absorption of three photons. Gel Permeation Chromatography (GPC), which is a destructive analytical method, was applied only for the investigation of the time dependent behaviour of the molecular weight of the photonics structures which were written with the parallel writing technique using 775nm wavelength and 160fs pulse length that shows an increase of 66 in  after seven days from the laser irradiation. Twenty-four hours after laser irradiation, the GPC results show that the weighted average molecular weight (Mw) of the exposed sample of 28,610,000 Daltons is about thirty times higher than the MW of the unexposed sample of 963,425 Daltons. This is an indication of the photo-cross-linking reaction. As a result of this reaction, the polymer chains link together through intermolecular forces to form a 3D network which produces an increase of molecular weight. It was also observed that there was a further decrease of molecular weight after three days to 437,441 Daltons due to main chain scission and unzipping. The main chain scission is actually the breaking of C-C bonds between structural units and the formation of radicals which further produce the monomer and end groups (MMA) through the unzipping reaction which leads to a decrease of the molecular weight. The main chain scission occurred with the greatest efficiency after three days following the end of irradiation, when the number of the main chain scissions (Ns) reached the maximum value of 1.193. An increase of molecular weight signifies an increase of the refractive index since the optical density has increased. The mechanical properties of PMMA optical fibres (e.g., Young's modulus) and of bulk PMMA (e.g., glass transition temperature) were investigated using Dynamical Mechanical Analysis (DMA) tests (e.g., stress-strain test and temperature ramp/frequency sweep test). These measurements were performed to study the effect of the manufacturing process that involves stretching and heating or cooling on the mechanical properties of PMMA optical fibres and unmodified PMMA material. T he ultimate aim of this section was to see the effect of the laser irradiation on the strain properties of an optical fibre sensor with gratings. The stress strain results show an increase of Young's modulus of the PMMA optical fibre of 5%, and this is an indication of decreased elasticity which is induced during the fabrication process. For a femtosecond laser irradiated region with UV wavelength, it is expected that there will be an increase of Young's modulus to 65%. This variation was obtained inthe research group from The Photon Science Institute by measuring Young's modulus for a diffraction grating which was written in PMMA with 180fs pulse length and 387nm wavelength and which was subjected to a strain. The elasticity was measured using the displacement of the first order diffracted beams as a result of a modification due to the applied strain [ ]. The temperature ramp/frequency sweep test shows an increase of glass transition temperature of the bulk PMMA of 54.12% which is also an indication of decreased elasticity induced during the fabrication process. A further increase in this temperature is expected for UV irradiated samples.

1. Transfection is a key technique in cell and molecular biology with many important biochemical applications. We selected a fiber fs laser at 1554 nm, an instrument widely used in optical communication research, as the excitation source. Our results demonstrated that the fs laser could perforate the cell membrane and the hole would close in sub-second interval after the laser exposure. We determined the safe exposure duration by detecting if there was any sign of mitochondrial depolarization at 1.5 hours after photoporation. Furthermore, we had successfully transfected HepG2 cells with a plasmid DNA containing the OFP gene, whose fluorescence could still be detected 24 hours after exposure. The transfection efficiency was as high as 77.3%. We also observed the proliferation of the transfected cells after 48 hours. / 2. Cell-cell fusion is a powerful tool for the analysis of gene expression, chromosomal mapping, monoclonal antibody production, and cancer immunotherapy. One of the challenges of in vitro cell fusion is to improve the fusion efficiency without adding extra chemicals while maintaining the cells alive and healthy. We show here that targeted human cancer cells could be selected by an optical tweezer and fused by a finely focused fs laser beam at 1554 nm with a high fusion eftlciency. The result confirmed that human cells could be fused exclusively by fs laser pulses, and this is the first time human cells are fused together all-optically. Mixing of cytoplasm in the fused cells was subsequently observed, and cells from different cell lines were also fused. Based on these, we firstly developed the method of optical cell-cell fusion. / 3. Failure in the induction of apoptosis or programmed cell death is one of the major contributions to the development of cancer and autoimmune diseases. Here we used a fs laser as a novel method to provide a direct apoptosis trigger to observe dynamic changes at subcellular level during apoptosis. First, we examined the effect of fs laser irradiation on the creation of reactive oxygen species (ROS) in exposed cells, which could trigger programmed cell death. By controlling the mitochondria electron transport chain (ETC), we investigated the mechanism of ROS generation by the fs pulses, including thermal effect and direct free electron liberation. Second, we induced apoptosis to targeted cells by the fs laser and found that the nuclear envelope (NE) formed tubular or tunnel-like structures (nuclear tubules - NT) inside the nucleus. The average number of NTs in each cell with laser treatment was significantly larger than in the control. Besides, the development of a NT was observed since its inception and it eventually merged with another one to form a larger NT. Meanwhile, mitochondria and tubulin were found inside the NT, and the NT formation always occurred after an upsurge of cellular Ca2+ concentration. More DNA fragmentation were also found in the region around the NTs. Based on this, we propose that NTs are developed during apoptosis and mitochondria migrate into the nucleus through the NTs to release death signals to trigger DNA fragmentation. Third, we used the fs laser to induce Ca2+ in cells in the form of a slow release, and firstly discovered that most Ca2+ was stored in the cytoplasm, and could diffuse into the nucleus after the optical trigger. Using fast confocal scanning, we obtained the path way of Ca2+ diffusion after the trigger in different cases. Our findings thus provide a new method of regulating the rate of apoptosis. / Biophotonics is an exciting and fast-expanding frontier which involves a fusion of advanced photonics and biology. It has not only developed many novel methodologies for biomedical research, but also achieved significant results as an independent field. Aided with femtosecond (fs) laser technologies, important progresses have been made on manipulating, imaging, and engineering of biological samples from single molecules to tissues in the last 10 years. The laser beam of ultra-short pulses at near-infrared band enjoys a lot of advantages: high nonlinear efficiency, low absorption by biological samples, high spatial and temporal resolution with tight confinement, low photo-toxicity, non-invasive, and ease of control. In this thesis, we report new findings from cell manipulation by fs laser, including transfection, cell-cell fusion, and induction of apoptosis in cells, which are detailed as follows: / He, Hao. / Adviser: Kam Tai Chan. / Source: Dissertation Abstracts International, Volume: 73-03, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references. / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Neste trabalho foi estudada a geração de harmônicos perturbativos por pulsos laser ultracurtos em bicos de gás no vácuo. Os harmônicos foram gerados nas regiões espectrais do UV e VUV e sua geração ocorreu em fluxo de gases nobres. Esta técnica possibilita a produção de luz coerente numa região espectral de interesse considerável para várias aplicações, tais como a espectroscopia resolvida no tempo. Para a geração dos harmônicos foi utilizado um sistema laser amplificado de Ti:Safira que produz pulsos de 25 fs, centrados em 785 nm, a 4 kHz. Estes pulsos ultracurtos são injetados em uma câmara de vácuo, onde são focalizados em um bico de gás. Os harmônicos são selecionados por um monocromador e têm a sua intensidade medida por um cintilador e uma fotomultiplicadora. Buscamos abranger uma visão geral sobre a geração de harmônicos perturbativos em gases, com o objetivo de consolidar um conhecimento teórico e experimental no laboratório. Confeccionamos e comparamos o desempenho de dois sistemas distintos de injeção de gás, o bico metálico e o bico de vidro, que são dispositivos pouco explorados na literatura. Os melhores resultados foram obtidos com o bico de vidro. Pudemos estimar a eficiência de geração dos harmônicos do ponto de vista das propriedades microscópicas e macroscópicas de geração, e foi verificada a conversão de energia do feixe fundamental para a geração dos harmônicos, plasma e outros fenômenos não-lineares em argônio. Geramos até o 9º harmônico em argônio, atingindo a região de 85 nm. Foi estudado o casamento de fase e a dependência da eficiência de geração dos harmônicos com a pressão do gás, potência média do laser, chirp e a posição do foco para o 3º, 5° e 7º harmônicos. Com a variação dos parâmetros, observou-se que é possível sintonizar os harmônicos, e que sua geração compete com outros fenômenos, como a ionização do gás. Adicionalmente, foi observada uma queda da eficiência de geração para alta intensidades, que foi associada à depleção do 1º elétron de valência dos átomos do gás, reduzindo a quantidade de centros geradores dos harmônicos. Além do argônio, foram realizadas medidas em hélio, neônio e criptônio, visando estudar como a eficiência de geração dos harmônicos depende das propriedades eletrônicas do gás. Adicionalmente, esses resultados corroboraram a hipótese da depleção dos elétrons de valência. / In this work the generation of perturbative harmonics by ultrashort laser pulses in gas nozzles in vacuum was studied. The harmonics were generated in the UV and VUV regions and the frequency conversion occurred in flowing gas. This technique ensures the generation of coherent light in a spectral region of interest for various applications, such as ultrafast time-resolved spectroscopy. For the harmonics generation an amplified Ti:Shapphire laser system was used, generating 25 fs pulses, centered at 785 nm, at 4 kHz. These ultrashort pulses are injected into a vacuum chamber, where they are focused on a gas nozzle. The harmonics are selected by a monochromator and their intensity is measured by a scintillator and photomultiplier. We have sought to comprise an overview of the generation of perturbative harmonics in gases, in order to consolidate a theoretical and experimental knowledge in the laboratory. Two distinct gas injection systems were manufactured and had their performances compared, a metallic and a gas nozzle, devices that are poorly explored in literature. The best results were obtained with the glass nozzle. We could estimate the harmonics generation efficiency in argon from the point of view of their macroscopic and microscopic generation properties, and the energy conversion from the fundamental beam to the harmonics was verified, along with plasma creation and other nonlinear phenomena. We generated up to the 9th harmonic in argon, reaching the 85 nm. We studied the phase matching and the dependence of the harmonics generation efficiency with the gas pressure, laser average power, chirp and focus position for the 3rd, 5th and 7th harmonics. By varying the parameters, we observed that it is possible to tune the harmonics wavelengths, and that their generation competes with other nonlinear phenomena, such as the gas ionization. Additionally, a drop in the generation efficiency was observed at high intensities, which was associated to the depletion of the first valence electron in the gas atoms, decreasing the number of harmonic generating centers. In addition to argon, measurements were performed in helium, neon and krypton, aiming to study how the harmonic generation efficiency depends on the electronic properties of the gas. Additionally, these results corroborated the hypothesis of the valence electron depletion.

A primeira etapa do projeto foi realizar testes para usinagem controlada e otimizada de vidro ótico de borosilicato (BK7) por laser de femtossegundos. Parâmetros como energia, pulsos sobrepostos e a variação da posição focal foram investigados para controle da taxa de remoção do material e extensão da cratera ablacionada. Especial atenção foi dada à condição física e topográfica da superfície resultante da usinagem para torná-la menos rugosa e evitar a retenção de reagentes que possam contaminar e alterar as reações pretendidas. Microcanais, microválvulas, microbombas, misturadores, microrreatores, aquecedores e outros componentes foram desenvolvidos para compor sistemas microfluídicos. Os microcanais construídos sobre a superfície de vidro BK7 vedados por uma lâmina de polidimetilsiloxano (PDMS) são a base dos sistemas microfluídicos. O controle de fluxo de reagentes é feito por miniválvulas pneumáticas controladas por um microcontrolador Arduino através de uma plataforma Labview. Este trabalho mostra os componentes desenvolvidos e dois sistemas microfluídicos criados. O primeiro contém um circuito capaz de replicar ensaios imunoenzimáticos (ELISA) com um custo muito menor de insumos. O segundo é um sistema para a produção de nanocristais fluorescentes de NaYF4 especialmente utilizados como marcadores em imagens de sistemas biológicos. / The first stage of the project was to perform tests for controlled and optimized machining of borosilicate optical glass (BK7) by femtosecond laser. Parameters such as energy, number of overlapped pulses, and the focal position variation were investigated for a better extraction of material. Microchannels, microvalves, micropumps, mixers, reactors, heaters and other components were developed to compose applied microfluidic systems. Microchannels built on the surface of BK7 glass sealed by a polydimethylsiloxane (PDMS) sheet form the basis of the microfluidic circuits. The reagents flow control is done by pneumatic mini-valves controlled by an Arduino microcontroller through a Labview platform. This work shows the components developed and two microfluidic systems created. The first contains a microfluidic circuit capable of replicating enzyme-linked immunosorbent assays (ELISA) with a much lower cost of materials. The second has a microfluidic circuit for the production of NaYF4 fluorescent nanocrystals specially used as markers in images of biologic systems.

Cell wounding, the loss of plasma membrane integrity, is a common event in the life of many cell types. Most cells are subjected to physiological events during normal functions that can lead to disruption of their plasma membranes, especially cells in the load bearing organs such as muscle, skin and bone. The capacity of the cell to repair day-to-day wear-and-tear injuries, as well as traumatic ones, is fundamental for maintaining tissue integrity. / In this thesis, we were trying to uncover single cell wound responses by applying the femtosecond laser (fs laser) technology. A well-characterized tunable fs laser was coupled with a laser scanning confocal microscopic system. Combining real-time observations of the fs laser-induced wound and 3D reconstruction of the cells, post-damage cell and nucleus morphological transformation and wound dynamics were reported. The major findings of this study include: (1) Fs laser could induce a small hole on the plasma membrane of the targeted cell. With the same laser irradiation time, the initial hole size were positively correlated with the laser power. (2) Four typical hole evolution scenarios were reported. Hole resealing was a fast process mostly within 100 seconds in normal condition. Whether a cell could reseal the hole is dependent on the initial hole size. Cells had difficulty to cope with the bigger holes. Three ranges of hole size were given in the thesis to predict the hole resealing result. (3) After fs laser damage, the whole cell underwent a contraction. The post-damage nucleus area, footprint, and each section layer of the cell all shrank, only the thickness remained the same. The nucleus retreated a bit from the damage site after damage. (4) Oxidative stress altered some of the cellular responses to the laser damage. The fs laser- induced holes in oxidative groups were bigger than the normal condition. The cells underwent an overall swelling after fs laser damage instead of the contraction in the normal group. Section layer areas and the thickness of the cell increased after damage. But similar to the normal condition, nucleus shrinkage and retreat from the damage site were also found in the oxidative stress groups. (5) Although both acute and chronic oxidative stresses compromised the integrity of the plasma membrane, chronic oxidative stress compromised more severely with several critical post-damage cell transformations and low resealing ratio. Acute oxidative stress on the other hand may somehow promote the resealing ability of the cells. (6) The section layers closer to the bottom of the cells transformed less than the layers further away from the bottom. This probably suggested that the cell basal attachment provided a constraint force to the plasma membrane for morphological changes. / 細胞創傷，即細胞膜的完整性受損，是多種細胞生命週期中一種常見的現象。細胞在執行正常功能時可能遭遇不同程度的生理性損傷，其中大部分會導致細胞膜的破壞。這一現象對存在於承受壓力器官中的細胞更為頻繁，例如肌肉，皮膚和骨骼。細胞對於日常磨損性傷害以及意外創傷的修復能力，是維持組織完整性的基石。 / 在本論文中，我們通過使用飛秒鐳射技術模擬單細胞創傷，觀察並試圖揭示單細胞對於創傷的反應過程。在實驗中，參數可調的飛秒鐳射器與共軛聚焦顯微鏡整合為一個系統，用於在單細胞膜上進行定點損傷。我們結合了對損傷的即時觀測，細胞的三維結構重建技術，完整記錄了損傷前後的損傷部位，細胞整體以及細胞核的形態變化。以下是本研究的主要發現：（1）飛秒鐳射能夠在目標細胞的細胞膜上進行局部穿孔。在鐳射照射時間相同的情況下，鐳射穿孔的大小與鐳射的平均功率呈正相關。（2）我們發現了鐳射穿孔後穿孔部位有四種不同的變化情況。穿孔後細胞封孔是一相當快的過程，在細胞成功封孔的情況下，大部分細胞將在100秒以內將穿孔部位重新填滿。細胞是否可以將穿孔封住取決於鐳射照射後初始穿孔的大小。細胞很難修復較大的孔。我們將細胞初始穿孔大小分為三個範圍，根據這三個範圍可以利用初始孔的尺寸大致預測穿孔後細胞的封孔情況。（3）飛秒鐳射損傷細胞後，細胞將會收縮，並且細胞核的平面面積，細胞的平面面積（或稱細胞足跡），以及細胞各分層面積都有不同程度的縮小。僅細胞厚度未發生顯著變化。同時，細胞核的位置相對於損傷部位有所後退。（4）細胞在氧化應激過後，對於飛秒鐳射造成的損傷反應有所變化。具體表現為：鐳射穿孔的尺寸比正常情況下更大；穿孔後細胞將會整體腫脹而非收縮。各分層面積和細胞厚度都有不同程度的增大。但是細胞核的反應與正常情況類似，即細胞核將會收縮，並且後退以遠離鐳射損傷部位。（5）儘管急性氧化應激和慢性氧化應激都一定程度上損傷了細胞膜的完整性，但是從細胞對於鐳射創傷的反應觀察，長期慢性氧化應激對於細胞膜的損害更為嚴重，具體表現為鐳射損傷後細胞的嚴重形變和細胞膜修復比例的降低。而另一方面，急性氧化應激在某種程度上可以增強細胞對於鐳射穿孔的修復能力。（6）細胞膜穿孔後，細胞各層的面積變化不一，位置越靠近底層的分層面積變化越小。這可能表明細胞貼壁行為形成了一個對細胞形變的約束力。 / Duan, Xinxing. / Thesis M.Phil. Chinese University of Hong Kong 2014. / Includes bibliographical references (leaves 73-85). / Abstracts also in Chinese. / Title from PDF title page (viewed on 24, October, 2016). / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

A terapia fotodinâmica (TFD) é uma modalidade de tratamento para o câncer baseado na interação da luz com um agente fotossensibilizador (FS) e o oxigênio molecular presente na célula alvo. A TFD apresenta vantagens sobre os métodos tradicionais de tratamentos como o dano seletivo às células neoplásicas, ausência de intervenção cirúrgica, possibilidade de repetição do procedimento e efeitos colaterais controlados. Uma das limitações da técnica é a profundidade de pouca penetração da luz no tecido biológico e consequentemente o volume tecidual tratado. Uma alternativa para superar esta limitação é o emprego de fonte de luz pulsada que comparativamente a irradiação com luz contínua (CW), apresenta maior potência de pico levando a uma maior profundidade de penetração e maior formação de espécies reativas de oxigênio. O objetivo deste trabalho é a avaliação da TFD utilizando fonte de luz pulsada no regime de femtossegundos através de ensaios in vitro da fotodegradação de dois tipos de FSs e da necrose induzida em fígado sadio de ratos (estudos in vivo). Nos estudos in vitro foram avaliadas a fotodegradação do Photogem (PG - 8μg/mL) e do Photodithazine (PDZ - 6μg/mL), para as irradiâncias de 280, 340 e 400 mW/cm2 com PG, e 15, 56 e 112 mW/cm2 para o PDZ. Nos estudos in vivo foram avaliados o perfil de necrose induzida com as fontes de luz CW e pulsado em modelo animal, que receberam PG e PDZ nas concentrações de 1,5 mg/kg e 1,0 mg/kg, respectivamente. Foram irradiados com 74 mW/cm2 (PG) e 102 mW/cm2(PDZ) e dose total de energia de 150 J/cm2. Posteriormente foi avaliada a dependência de profundidade de necrose com a irradiância (60, 80, 107, 127, 138, 188 e 229 mW/cm2) utilizando PG e com dose total entregue de 150 J/cm2. As fontes de luz empregadas foram um laser de diodo 630 nm (PG), um laser de diodo emitindo em 660 nm (PDZ) e um laser de Ti:Safira, taxa de repetição de 1 kHz, comprimento de onda 800 nm e largura de pulso de 75 fs em associação com amplificador paramétrico óptico (APO) para conversão de comprimentos de onda na região de 400 - 1150 nm. Os experimentos in vitro mostraram que taxa de fotodegradação para o PG foi maior utilizando o laser pulsado do que para o laser CW. Quando utilizado o PDZ, o laser CW promoveu uma taxa de fotodegradação maior do que o pulsado. Nos estudos in vivo, foi observada a necrose induzida com laser pulsado cerca de duas vezes mais profunda do que a induzida pelo laser CW, enquanto necrose induzida pelo laser pulsado com PDZ foi maior do que a do laser CW. No estudo da dependência da profundidade de necrose em função da irradiância, o laser pulsado induziu profundidade de necrose maior do que o laser CW para irradiâncias abaixo de 80 mW/cm2, acima desta irradiância, o laser CW e o pulsado não mostraram diferença significativa. Estes resultados mostram que a combinação da fonte de luz pulsada e PG podem ser consideradas como alternativa para aumentar o volume tecidual tratado, porém, devem-se observar os parâmetros empregados para se obter o maior volume tecidual tratado. O mesmo resultado não foi observado para o PDZ como fotossensibilizador, o que indica uma forte dependência dos mecanismos da TFD com o FS e o regime de iluminação empregado. / Photodynamic therapy (PDT) is a therapeutic modality for cancer based on light, a photosensitizer agent (PS) and molecular oxygen into the target cells. PDT has advantages over traditional treatments, such as, selective damage to tumor cells, absence of surgical intervention, possibility of repeated procedures and controlled side effects. One of the limitations of PDT is the low light penetration in biological tissues and hence the treated tissue volume. An alternative to overcome this limitation is the use of pulsed light sources that compared to continuous (CW) irradiation, present higher peak power leading to a greater penetration depth and enhancing the reactive oxygen species production. The aim of this study is to evaluate PDT using pulsed light source at femtosecond regime through in vitro photodegradation of two PSs types and in vivo induced necrosis in healthy rat liver. In the in vitro study we evaluated the photodegradation of Photogem (PG - 8μg/mL) and Photodithazine (PDZ - 6μg/mL), at different irradiances (280, 340, and 400 mW/cm2 for PG and 15, 56 and 112 mW/cm2 for PDZ). In the in vivo studies the induced necrosis profile with CW laser and Pulsed Laser were evaluated in animal model which received PG (1,5 mg/kg) and PDZ (1,0 mg/kg) and were irradiated with 74 mW/cm2 and 102 mW/cm2, respectively, and the fluence was 150 J/cm2. After that, the dependence of depth of necrosis with irradiance (60, 80, 107, 127, 138, 188 and 229 mW/cm2) with PG and 150 J/cm2 of fluence was evaluated. The light source used in those studies were a 630 and 660 nm diode laser (PG and PDZ excitation light, respectively) and a Ti:Sapphire Regenerative Amplifier laser, 1 kHz repetition rate, 800 nm wavelength and 75 fs pulse width in association with an optical parametric amplifier (OPA) to convert 400-1150 wavelengths. The in vitro results showed that the PG photodegratation rates were greater to pulsed laser when compared with CW laser. When PDZ was used, the photodegradarion rates with pulsed laser were lower than CW laser. In the in vivo studies, the induced necrosis with pulsed laser was twice the induced by CW laser with PG as photosensitizer. When PDZ was used, the induced necrosis was greater for CW laser than for pulsed laser. For different irradiances, the depth of necrosis induced by CW laser was greater than for pulsed laser below 80 mW/cm2, above this, the pulsed and CW laser did not show difference. These results demonstrated that pulsed light and PG can be considered an alternative to enhance the treated tissue volume. The same was not observed to PDZ, which indicate the strong dependence of PDT mechanisms with PS and the light regime applied.

Embora a ablação a laser venha sendo bastante utilizada em materiais em geral, pouco é entendido sobre o comportamento deste processo perto de uma interface separando dois materiais distintos. Neste contexto, o principal objetivo deste trabalho foi realizar um estudo macroscópico e microscópico dos processos que envolvem a ablação a laser em regime de femtossegundos em materiais homogêneos e estruturados. No caso de materiais estruturados, o estudo focou-se em uma situação de interface, na qual ocorrem mudanças nas propriedades de ablação. Baseado nos resultados, nós pretendemos obter subsídios científicos para entender as aplicações da ablação em regime de pulsos ultracurtos para estruturas estratificadas, tais como de dentes, ossos, interface resina-dente, dente-metal, e outras. Diferentes técnicas experimentais foram idealizadas para determinar a progressão da ablação dentro do material e obter dados extraídos da superfície. Utilizando luz espalhada de uma fonte externa, o processo de ablação foi temporalmente monitorado, permitindo determinar a velocidade de ablação em materiais transparentes, assim como perfis típicos de ablação nestes materiais. Em um segundo experimento, nosso estudo permitiu quantificar a variação da geometria de ablação perto de uma interface separando dois materiais distintos. Nossos dados foram suficientes para prever a ocorrência de uma descontinuidade no perfil da ablação entre dois meios: resina A e resina B, mostrando uma repentina descontinuidade do diâmetro da cavidade ablacionada. Adicionalmente, foi realizada uma análise dos aspectos morfológicos de diferentes tecidos biológicos irradiados e nosso estudo mostrou a eficiência da ablação utilizando laser de femtossegundos no processamento de tecidos duros e a possibilidade de utilizar esses sistemas sem causar danos térmicos e mecânicos nos tecidos remanescentes. Finalmente nós aplicamos a microperfuração a laser para produzir micro-poros na superfície de tecidos biológicos (fígados), melhorando a penetração do medicamento ALA e a aumentando a profundidade de tratamento. / Although laser ablation has been long used in general materials, little is known regarding the behavior of theses process near an interface separating two distinct materials. In this context, the main aim of this work was to perform a microscopic and macroscopic study of the processes that include femtosecond laser ablation in homogeneous and/or structured materials. In the case of structured materials, the study focused on an interface situation, in which sudden changes occurred in the properties. Based on the results, we aimed to obtain scientific subsidies to understand the application of ultrashort pulses to stratified structures, such as teeth, bones, resin-teeth or metal-teeth interface, and others. Distinct experimental techniques were used to determinate the ablation progression into the materials and to obtain data extracted from their surface. By using the scattered light from an external source, the ablation process was monitored temporally, allowing to determine the velocity of ablation in transparent materials, besides determining the typical profiles of ablated cavities in these materials. In a second experiment, our study allowed quantifying the overall variation in the ablation geometry that takes place on the interface of two different materials. Our data were sufficient to predict the occurrence of a discontinuity in the ablation profile on the interface between two media: resin A and resin B, showing a sudden discontinuity of the ablated cavity diameter. In addition, an analysis of the morphological aspects of different biological tissues irradiated by femtosecond laser pulses was performed and a comparative study showed the ablation efficiency of the femtosecond lasers in hard tissues processing and the possibility of using these systems with no thermal and mechanic damage. Finally, we applied a laser micromachining producing micro-pores on the tissue surface, improving the ALA penetration and increasing the treatment depth.