This thesis investigates the use of Raman tweezers microspectroscopy to interrogate the biochemistry of single biological cells. Raman tweezers microspectroscopy is a powerful technique, which combines traditional Raman microspectroscopy and optical trapping, allowing the manipulation and environmental isolation of a biological cell of interest whilst simultaneously probing its biochemistry gleaning a wealth of pertinent information. The studies carried out in this thesis can be split into two broad categories: firstly, the exploitation of Raman tweezers microspectroscopy to study biological cells and secondly developments to the Raman tweezers microspectroscopy technique that extend its capabilities and the range of samples that can be studied. In the application of Raman tweezers, the stacking and interrogation of multiple cells is reported allowing a rapid representative Raman signal to be recorded from a small cell population with improved signal to noise. Also demonstrated is the ability of Raman spectroscopy to identify and grade the development of Human Papillomavirus induced cervical neoplasia with sensitivities of up to 96 %. These studies demonstrate the potential of Raman spectroscopy to study biological cells but it was noted that the traditional Raman tweezers system struggled to manipulate large cells thus a decoupled Raman tweezers microspectroscopy system is presented where a dual beam fibre optical trap is used to perform the trapping function and a separate Raman probe is introduced to probe the biochemical nature of the trapped cell. This development allowed the trapping and examination of very large cells whilst opening up the possibility of creating Raman maps of trapped objects. Raman tweezers microspectroscopy could potentially become an important clinical diagnostic and biological monitoring tool but is held back by the long signal integration times required due to the weak nature of Raman scattering. The final study presented in this thesis examines the potential of wavelength modulated Raman spectroscopy to improve signal to noise ratios and reduce integration times. All these studies aim to demonstrate the potential and extend the performance of Raman tweezers microspectroscopy.

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.

Trata-se de um estudo experimental aleatório: os tratamentos e suas repetições foram arranjados no delineamento experimental Inteiramente Casualizados. Foi realizado no Instituto Agronômico IAC de Campinas, no período de 17 a 23 de março (Fase 1) e de 1 a 7 de agosto (Fase 2) de 2006. Buscou, dentro da área da Biofísica, novos aparatos e tecnologias que pudessem avaliar o efeito das Essências Florais em seres vivos. Utilizaram-se sementes de feijoeiro que receberam três tipos de tratamentos (regas), sendo eles: Tratamento 1(água), Tratamento 2 (água com veículo alcoólico, - aqui utilizado conhaque) e Tratamento 3 (água com veículo alcoólico e Essência Floral - Walnut). Foram analisadas a emissão biofotônica só na Fase 1 e os parâmetros de crescimento biológico durante a germinação nas Fase 1 e 2. No resultado da medição biofotônica, não foi possível se obter gráficos que caracterizassem com precisão a emissão de fótons, pois os fatores externos, dentre eles a luminosidade, os climatológicos e a escolha de areia como substrato, impossibilitaram a leitura dos dados. Na medição biológica da Fase 1, nos primeiros três dias de germinação, elas permaneceram em Germinador de Sementes só com água. A Análise de Variância para a altura das plântulas obteve o valor F de 0,1200 , com o coeficiente de variação de 10,51%, e para o comprimento das raízes o valor F de 0,4800 com o Coeficiente de Variação de 12,49% e a posteriori realizou-se o Teste de Tukey , não sendo significativos estatisticamente nenhum deles. Na Fase 2 utilizou-se as regas diferenciadas desde o primeiro momento da germinação, com isso na Análise de Variância para a altura das plântulas o valor F foi de 0,0584 e o Coeficiente de Variação de 13,76% e para o comprimento das raízes o valor F de 0,0538 e o Coeficiente de Variação de 12,40% e a posteriori realizou-se o Teste de Tukey , não sendo significativos estaticamente nenhum deles. Nessa fase acrescentou-se mais uma medida, o peso das raízes, no qual a Análise de Variância o valor F foi de 0,005, mostrando que houve diferença estatisticamente significativa na média dos pesos das raízes, segundo o tratamento utilizado e o valor do coeficiente de variação obtido foi de 6,25%. Para o Teste de Tukey obteve-se significância estatística, sendo esse resultado indicativo que as Essências Florais, neste experimento estimularam maior brotamento das raízes (fasciculamento), sendo que esse fenômeno auxilia a plântula a superar os estresses hídricos e também estabelecer maior resistência ao ataque de patógenos radiculares. Para a realização desse experimento foram necessárias as interações de três grandes áreas de conhecimento: Enfermagem, Agronomia e Engenharia Elétrica. Demonstrando assim a necessidade e importância na promoção de pesquisas multidisciplinares, pois só assim se promoverá a busca de aparatos que evidenciem a ação das Terapias Alternativas/Complementares quando utilizadas / A random experimental study is presented, where the treatments and their repetitions were arranged in an Entirely Casualized experimental delineation. The experiments were conducted in the Agronomic Institute IAC in Campinas, from the 17th to 23rd of March, 2006 (Phase 1) and from the 1st to 7th of August, 2006 (Phase 2). One of the study´s goals was to search within the Biophysics area, new devices and technology, which could help to identify the effect of Floral Essences in living systems. Bean seeds were adopted and received three types of treatment (wetting): Treatment 1 (water), Treatment 2 (water using alcohol as vehicle - in this case brandy) and Treatment 3 (water using alcohol as a vehicle plus Flower Essence Walnut). The biophotonic emission and the biological growth parameters were analyzed during the germination in Phases 1 and 2. For the biophotonic measurement results, it was not possible to obtain graphics that characterized with precision the photons emission, because external factors such as luminosity, climatologic ones and the choice of sand as substrate made the reading of the data impossible. In the biological measurement of Phase 1, in the first three days of germination the seeds remained in the germination chamber only with water. The Variance Analysis for the seedlings height produced an F value of 0.1200, with a variation Coefficient of 10.51%, and for the roots length an F value of 0.4800 with a Variation Coefficient of 12.49%. The Tukey Test was realized a posteriori, producing results with none statistical significance. In Phase 2, the differentiated rules were used from the very beginning of the germination, as a consequence the variance analysis for the seedlings height produced an F value of 0.0584 with a Variation Coefficient of 13,76%, and for the roots length the F value was 0,0538 with a Variance Coefficient of 12.40%. The Tukey Test was realized a posteriori, producing results with none statistical significance. In this phase another measurement was included: the roots weight, for which F was 0.005, showing that there occurred a statistically significant difference in the roots weight average according to the treatment used and the Variance Coefficient obtained was 6.25%. For the Tukey Test statistic significance was obtained, and this result indicates that the Flower Essence in this experiment stimulated a better blossom of the roots (fasciculation), and this phenomenon helped the seedlings to overcome the water stress as well as to establish a bigger resistance to the attack of radiculars patogenous. For this experiment the interaction of three big areas of knowledge was necessary: Nursing, Agronomy and Electric Engineering. This demonstrated the importance of promoting interdisciplinary research, because only in this way one could promote the search for devices that showed the action of Alternative/Complementary Therapies when utilized

O presente trabalho compreende a execução de 16 experimentos, realizados em duplicata, visando avaliar a influência de fatores, tais como iluminância, concentração de NaH2PO4.H2O, concentração de NaNO3, fotoperíodo e comprimento de onda, além da interação entre a iluminância e os demais fatores, na produção de biomassa e lipídios pela microalga marinha Chlorella minutíssima. Os experimentos estiveram dispostos em 2 arranjos ortogonais L8 de Taguchi, cada um dos quais recebeu dados obtidos em experimentos com diferentes comprimentos de onda (470 nm, além da luz branca), nos quais eram variados, em dois níveis, a iluminância (26 klx e 13 klx), as concentrações de NaH2PO4.H2O (5 mg/L e 15 mg/L) e NaNO3 (45 mg/L e 75 mg/L) e o fotoperíodo (12: 12 horas claro: escuro e 8 ciclos diários de 2: 1 horas claro: escuro). Os experimentos se processaram em reatores de vidro com formato de prisma de base quadrada com volume útil de 1,1 L, isolados da influência da luminosidade externa. Para a luz branca, o melhor resultado para a produção de biomassa foi de 1300 mg/L, ao passo que o melhor resultado para o teor lipídico foi de 22,75% em relação à biomassa seca. Para a luz de comprimento de onda equivalente a 470 nm, o melhor resultado para a produção de biomassa foi de 1270 mg/L, ao passo que o melhor resultado para o teor lipídico foi de 19,03% em relação à biomassa seca. / This work comprises the execution of 16 experiments, performed in duplicate, in order to evaluate the influence of factors such as illuminance, NaH2PO4.H2O concentration, NaNO3 concentration, photoperiod and wavelenght, in addition to the interaction between the illuminance and the others factors, in the production of biomass and lipids by the m arine microalgae Chlorella minutíssima. The experiments were arranged in 2 Taguchi\'s L8 orthogonal arrays, each of which received data obtained in experiments with different wavelengths (470 nm, in addition to the white light), in which the illuminance (26 klx and 13 klx), the NaH2PO4.H2O (5 mg/L and 15 mg/L) and NaNO3 (45 mg/L and 75 mg/L) concentrations and the photoperiod (12: 12 hours light: dark and 8 daily cycles of 2: 1 hours light: dark) were varied, in two levels. The experiments were performed in quadrangular prism glass reactors with a useful volume of 1.1 L, isolated from the influence of external luminosity. For white light, the best result for biomass production was 1300 mg/L, while the best resulto f lipid contente was 22,75% in relation on the dry biomass. For light with a wavelength of 470 nm, the best result for biomass production was 1270 mg/L, while the best result of lipid contente was 19,03% in relation to the dry biomass

We aim to develop an implantable, optical neural imaging device by fabricating lasers and photodiodes onto a gallium arsenide substrate. Some studies suggest that lasers exhibit higher noise than light emitting diodes (LEDs) due to coherence effects – my studies aim to quantify this noise and to guide device development. To this end, I developed a model of a fluorescent imaging device which agreed with experiment. Noise analysis performed in phantom showed that laser sources exhibit temporal and spatial noise up to 10x higher than LED sources, and in vivo noise analysis also demonstrated this trend. I studied a neural injury model called cortical spreading depression in vitro in mouse brain slices and in vivo in the rat brain using laser and LED sources. Signal magnitudes in vitro are on the order of 10% and in vivo results are inconclusive. Future work will aim to reduce coherence related noise.

We aim to develop an implantable, optical neural imaging device by fabricating lasers and photodiodes onto a gallium arsenide substrate. Some studies suggest that lasers exhibit higher noise than light emitting diodes (LEDs) due to coherence effects – my studies aim to quantify this noise and to guide device development. To this end, I developed a model of a fluorescent imaging device which agreed with experiment. Noise analysis performed in phantom showed that laser sources exhibit temporal and spatial noise up to 10x higher than LED sources, and in vivo noise analysis also demonstrated this trend. I studied a neural injury model called cortical spreading depression in vitro in mouse brain slices and in vivo in the rat brain using laser and LED sources. Signal magnitudes in vitro are on the order of 10% and in vivo results are inconclusive. Future work will aim to reduce coherence related noise.

<p>In 2011, an estimated 230,480 new cases of invasive breast cancer were diagnosed among women, as well as an estimated 57,650 additional cases of in situ breast cancer [1]. Breast conserving surgery (BCS) is a recommended surgical choice for women with early stage breast cancer (stages 0, I, II) and for those with Stage II-III disease who undergo successful neo-adjuvant treatment to reduce their tumor burden [2, 3]. Cancer within 2mm of a margin following BCS increases the risk of local recurrence and mortality [4-6]. Margin assessment presents an unmet clinical need. Breast tissue is markedly heterogeneous which makes identifying cancer foci within benign tissue challenging. Optical spectroscopy can provide surgeons with intra-operative diagnostic tools. Here, ex-vivo breast tissue is evaluated to determine which sources of optical contrast have the potential to detect malignancy at the margins in women of differing breast composition. Then, H&E images of ex-vivo breast tissue sites are quantified to further deconstruct the relationship between optical scattering and the underlying tissue morphology. </p><p>Diffuse reflectance spectra were measured from benign and malignant sites from the margins of lumpectomy specimens. Benign and malignant sites were compared and then stratified by tissue type and depth. The median and median absolute deviance (MAD) was calculated for each category. The frequencies of the benign tissue types were separated by menopausal status and compared to the corresponding optical properties. </p><p>H&E images were then taken of the malignant and benign sites and quantified to describe the % adipose, % collagen and % glands. Adipose sites, images at 10x, were predominantly fatty and quantified according to adipocyte morphology. H&E-stained adipose tissue sections were analyzed with an automated image processing algorithm to extract average cell area and cell density. Non-adipose sites were imaged with a 2.5x objective. Grids of 200µm boxes corresponding to the 3mm x 2mm area were overlaid on each non-adipose image. The non-adipose images were classified as the following: adipose and collagen (fibroadipose); collagen and glands (fibroglandular); adipose, collagen and glands (mixed); and malignant sites. Correlations between <&mus&#8242;> and % collagen in were determined in benign sites. Age, BMI, and MBD were then correlated to <&mus&#8242;> in the adipose and non-adipose sites. Variability in <&mus&#8242;> was determined to be related to collagen and not adipose content. In order to further investigate this relationship, the importance of age, BMI and MBD was analyzed after adjusting for the % collagen. Lastly, the relationship between % collagen and % glands was analyzed to determine the relative contributions of % collagen and % glands <&mus&#8242;>. Statistics were calculated using Wilcoxon rank-sum tests, Pearson correlation coefficients and linear fits in R. </p><p> The diagnostic ability of the optical parameters was linked to the distance of tumor from the margin as well as menopausal status. [THb] showed statistical differences from <&mus&#8242;> between malignant (<&mus&#8242;>: 8.96cm-1±2.24MAD, [THb]: 42.70&muM±29.31MAD) compared to benign sites (<&mus&#8242;>: 7.29cm-1±2.15MAD, [THb]: 32.09&muM±16.73MAD) (p<0.05). Fibroglandular (FG) sites exhibited increased <&mus&#8242;> while adipose sites showed increased [&beta-carotene] within benign tissues. Scattering differentiated between ductal carcinoma in situ (DCIS) (9.46cm-1±1.06MAD) and invasive ductal carcinoma (IDC) (8.00cm-1±1.81MAD), versus adipose sites (6.50cm-1±1.95MAD). [&beta-carotene] showed marginal differences between DCIS (19.00&muM±6.93MAD, and FG (15.30&muM±5.64MAD). [THb] exhibited statistical differences between positive sites (92.57&muM±18.46MAD) and FG (34.12&muM±22.77MAD), FA (28.63&muM±14.19MAD), and A (30.36&muM±14.86MAD). Due to decreased fibrous content and increased adipose content, benign sites in post-menopausal patients exhibited lower <&mus&#8242;>, but higher [&beta-carotene] than pre-menopausal patients.</p><p>Further deconstructing the relationship between optical scattering and tissue morphology resulted in a positive relationship between <&mus&#8242;> and % collagen (r=0.28, p=0.00034). Increased variability was observed in sites with a higher percentage of collagen. In adipose tissues MBD was negatively correlated with age (r=-0.19, p=0.006), BMI (r=-0.33, p=2.3e-6) and average cell area (r=-0.15, p=0.032) but positively related to the log of the average cell density (r=0.17, p=0.12). In addition, BMI was positively correlated to average cell area (r=0.31, p=1.2e-5) and negatively related to log of the cell density (r=-0.28, p=7.6e-5). In non-adipose sites, age was negatively correlated to <&mus&#8242;> in benign (r=-0.32, p=4.7e-5) and malignant (r=-0.32, p=1.4e-5) sites and this correlation varied significantly by the collagen level (r=-0.40 vs. -0.13). BMI was negatively correlated to <&mus&#8242;> in benign (r=-0.32, p=4e-5) and malignant (r=-0.31, p=2.8e-5) sites but this relationship did not vary by collagen level. MBD was positively correlated to <&mus&#8242;> in benign (r=0.22, p=0.01) and malignant (r=0.21, p=4.6e-3) sites. Optical scattering was shown to be tied to patient demographics. Lastly, the analysis of collagen vs. glands was narrowed to investigate sites with glands between 0-40% (the dynamic range of the data), the linear model reflected an equivalent relationship to scattering from % glands and the % collagen in benign sites (r=0.18 vs. r=0.17). In addition, the malignant sites showed a stronger positive relationship (r=0.64, p=0.005) to <&mus&#8242;> compared to the benign sites (r=0.52, p=0.03).</p><p>The data indicate that the ability of an optical parameter to differentiate benign from malignant breast tissues is dictated by patient demographics. Scattering differentiated between malignant and adipose sites and would be most effective in post-menopausal women. [&beta-carotene] or [THb] may be more applicable in pre-menopausal women to differentiate malignant from fibrous sites. Patient demographics are therefore an important component to incorporate into optical characterization of breast specimens. Through the subsequent stepwise analysis of tissue morphology, <&mus&#8242;> was positively correlated to collagen and negatively correlated to age and BMI. Increased variability of <&mus&#8242;> with collagen level was not dependent on the adipose contribution. A stronger correlation between age and <&mus&#8242;> was seen in high collagen sites compared to low collagen sites. Contributions from collagen and glands to <&mus&#8242;> were independent and equivalent in benign sites; glands showed a stronger correlation to <&mus&#8242;> in malignant sites than collagen. This information will help develop improved scattering models and additional technologies from separating fibroglandular sites from malignant sites and ultimately improve margin assessment.</p> / Dissertation

This two-part thesis focuses on biointerfaces of two different biological systems. It specifically examines the interplay of structure and functionality in these biointerfaces. Part one studies photo-bio-electrochemically active bacteria and the strong dependence of their electrical current generation on electrode structure and pigment organisation. Part two uncovers surprising design principles of photonic structures on flower petals and presents research tools to study disordered optical systems. Biophotovoltaics (BPV) is a newly described biophysical effect in which a biofilm of photosynthetic microorganisms associated with an anode produces electrical current that can be harvested and passed through an external circuit. In this thesis-part, an experimental set-up is presented to quantitatively measure photo-electric activity of cyanobacteria in BPVs. Using this set-up, a systematic study of anode morphologies reveals that large electrode surface areas enhance photocurrents by two orders of magnitude, identifying structuration as key design criterion for bioelectrochemical interfaces. Electrodes with micrometer-sized pores allow enhanced direct contact area with bacteria, but with tested cyanobacteria this did not result in a photocurrent increase, disproving recent speculations in the literature. Furthermore, a theoretic-mathematical framework is presented to estimate light-energy utilisation in biofilms. It is detailed how pigment concentration and distribution affects the light-level dependent saturation of electron harvesting biofilms. This study brings the theory together with experiments, such as genetic modification and photo-current measurements. Part two of this thesis approaches the interaction of light and biointerface structuration from a different angle. In a significant extension of the candidate’s MPhil project, it was discovered that the disorder in natural photonic structures can be an advantage rather than a limitation in biology. With biological image analysis, optics simulations and nano-manufacturing a new photonic effect is uncovered which is iridescent but surprisingly constant in chroma. In collaboration with plant scientists, it is shown that many flowers have co-evolved disordered surface structuration that generates this bee visible colouration.

Photodynamic therapy (PDT) is an alternative technique for treatment of tumors and other lesions. It results from the combination of three factors: a photosensitizer (PS), molecular oxygen and light. This combination triggers photochemical reactions that result in the destruction of cells and their neighborhoods. Because the FS presents preferential retention in tumors, its effect becomes localized, selectively destroying the tumor parenchyma and stroma only. However, the penetration of light during PDT is limited due to attenuation of the possible excitation wavelengths by the biological tissue itself. Thus, the depth of treatment is not sufficient to ensure the treatment of extensive lesions. To overcome this difficulty, one strategy is to surgically remove the bulk of the lesion, and then applying the PDT on the surgical bed. However, it is unknown how the alterations caused by surgical techniques (bleeding, coagulation, inflammatory processes, etc.) influence the optical properties of biological tissues as to compromise a subsequent PDT protocol. This work aims to study the influence of using surgical techniques prior to a PDT protocol in an animal model. It was applied four surgical tools (electrocautery, ultrasonic scalpel, long-pulsed Er:YAG laser and ultrashort-pulsed Ti: Sapphire laser) in order to verify the most recommended for a surgery + PDT combined protocol, using Photogem ® as the photosensitizer. Surface and subsurface changes were performed to simulate the remaining tissue properties treated with such techniques. The order of application between photosensitization and surgery was one of the study variables. In vivo fluorescence spectroscopy, confocal fluorescence microscopy of the necrotic tissue (ex vivo) and histopathology were used as analysis techniques of the tissue response. The analysis of ex vivo necrosis fluorescence made possible to assess the damage to biological tissues, supporting the histopathological analysis. Analyzing the depth of necrosis was important to confirm the generation of photodynamic effect in tissues previously modified by the use of surgery. Testing a surgical excision protocol, the same success was obtained. Through in vivo fluorescence it was possible to detect the concentration of the PS in all steps of the protocols. A hypothesis of increased electrical conductivity of photosensitized tissue is proposed to explain the decrease in thermal damage observed during excision. Finally, the full feasibility of the combination between surgery and PDT was verified, with space to suggest guidelines for the implementation of future protocols involving combined use of the studied techniques. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Conselho Nacional de Desenvolvimento Científico e Tecnológico / A Terapia Fotodinâmica (TFD) é uma técnica alternativa para o tratamento de tumores e outras lesões. Ela é fruto da combinação de três fatores: um fotossensibilizador (FS), o oxigênio molecular e luz. Esta junção desencadeia reações fotoquímicas que resultam na destruição de células e suas vizinhanças. Pelo fato de o FS apresentar retenção preferencial em tumores, seu efeito torna-se localizado, destruindo seletivamente apenas o parênquima e o estroma tumorais. Contudo, a penetração de luz durante a TFD é limitada, devido à atenuação dos possíveis comprimentos de onda de excitação pelo próprio tecido biológico. Assim, a profundidade de tratamento não é suficiente para garantir o tratamento de lesões extensas. Para superar esta dificuldade, uma estratégia é remover cirurgicamente a maior parte da lesão, aplicando a TFD em seguida no leito cirúrgico. No entanto, desconhecem-se como as alterações provocadas por técnicas cirúrgicas (hemorragia, coagulação, processos inflamatórios, etc.) influenciam nas propriedades ópticas dos tecidos biológicos a ponto de comprometer um subsequente protocolo de TFD. O presente trabalho se propõe a estudar a influência do uso de cirurgia anterior ao protocolo de TFD em um modelo animal. Foram aplicadas quatro ferramentas cirúrgicas (bisturi elétrico, bisturi ultrassônico, laser de Er: YAG de pulsos longos e laser de Ti: Safira de pulsos ultracurtos) com o fim de verificar a mais recomendável para o protocolo combinado de cirurgia + TFD, utilizando o Photogem® como fotossensibilizador. Simularam-se alterações de superfície e sub-superfície no tecido remanescente tratado com tais técnicas. A ordem de aplicação cirurgia-fotossensibilização foi uma das variáveis do estudo. Como técnicas de análise da resposta tecidual foram utilizadas: espectroscopia de fluorescência in vivo, microscopia confocal de fluorescência do tecido necrosado (ex vivo) e histopatologia. A análise da fluorescência da necrose ex vivo tornou possível avaliar os danos provocados nos tecidos biológicos, corroborando com a análise histopatológica. A análise da profundidade de necrose foi determinante para confirmar a geração de efeito fotodinâmico em tecidos anteriormente alterados pelo uso de cirurgia. Testando um protocolo de excisão cirúrgica propriamente dita, verificou-se o mesmo sucesso. Através da fluorescência in vivo foi possível detectar a concentração do FS em todas as etapas dos protocolos. Uma hipótese de aumento da condutividade elétrica do tecido fotossensibilizado é proposta para explicar a diminuição do dano térmico observado na excisão. Por fim, foi verificada a viabilidade total da combinação entre cirurgia e TFD, com espaço para sugerir diretrizes para a execução de futuros protocolos que envolvam uso combinado das técnicas estudadas.

Trata-se de um estudo experimental aleatório: os tratamentos e suas repetições foram arranjados no delineamento experimental Inteiramente Casualizados. Foi realizado no Instituto Agronômico IAC de Campinas, no período de 17 a 23 de março (Fase 1) e de 1 a 7 de agosto (Fase 2) de 2006. Buscou, dentro da área da Biofísica, novos aparatos e tecnologias que pudessem avaliar o efeito das Essências Florais em seres vivos. Utilizaram-se sementes de feijoeiro que receberam três tipos de tratamentos (regas), sendo eles: Tratamento 1(água), Tratamento 2 (água com veículo alcoólico, - aqui utilizado conhaque) e Tratamento 3 (água com veículo alcoólico e Essência Floral - Walnut). Foram analisadas a emissão biofotônica só na Fase 1 e os parâmetros de crescimento biológico durante a germinação nas Fase 1 e 2. No resultado da medição biofotônica, não foi possível se obter gráficos que caracterizassem com precisão a emissão de fótons, pois os fatores externos, dentre eles a luminosidade, os climatológicos e a escolha de areia como substrato, impossibilitaram a leitura dos dados. Na medição biológica da Fase 1, nos primeiros três dias de germinação, elas permaneceram em Germinador de Sementes só com água. A Análise de Variância para a altura das plântulas obteve o valor F de 0,1200 , com o coeficiente de variação de 10,51%, e para o comprimento das raízes o valor F de 0,4800 com o Coeficiente de Variação de 12,49% e a posteriori realizou-se o Teste de Tukey , não sendo significativos estatisticamente nenhum deles. Na Fase 2 utilizou-se as regas diferenciadas desde o primeiro momento da germinação, com isso na Análise de Variância para a altura das plântulas o valor F foi de 0,0584 e o Coeficiente de Variação de 13,76% e para o comprimento das raízes o valor F de 0,0538 e o Coeficiente de Variação de 12,40% e a posteriori realizou-se o Teste de Tukey , não sendo significativos estaticamente nenhum deles. Nessa fase acrescentou-se mais uma medida, o peso das raízes, no qual a Análise de Variância o valor F foi de 0,005, mostrando que houve diferença estatisticamente significativa na média dos pesos das raízes, segundo o tratamento utilizado e o valor do coeficiente de variação obtido foi de 6,25%. Para o Teste de Tukey obteve-se significância estatística, sendo esse resultado indicativo que as Essências Florais, neste experimento estimularam maior brotamento das raízes (fasciculamento), sendo que esse fenômeno auxilia a plântula a superar os estresses hídricos e também estabelecer maior resistência ao ataque de patógenos radiculares. Para a realização desse experimento foram necessárias as interações de três grandes áreas de conhecimento: Enfermagem, Agronomia e Engenharia Elétrica. Demonstrando assim a necessidade e importância na promoção de pesquisas multidisciplinares, pois só assim se promoverá a busca de aparatos que evidenciem a ação das Terapias Alternativas/Complementares quando utilizadas / A random experimental study is presented, where the treatments and their repetitions were arranged in an Entirely Casualized experimental delineation. The experiments were conducted in the Agronomic Institute IAC in Campinas, from the 17th to 23rd of March, 2006 (Phase 1) and from the 1st to 7th of August, 2006 (Phase 2). One of the study´s goals was to search within the Biophysics area, new devices and technology, which could help to identify the effect of Floral Essences in living systems. Bean seeds were adopted and received three types of treatment (wetting): Treatment 1 (water), Treatment 2 (water using alcohol as vehicle - in this case brandy) and Treatment 3 (water using alcohol as a vehicle plus Flower Essence Walnut). The biophotonic emission and the biological growth parameters were analyzed during the germination in Phases 1 and 2. For the biophotonic measurement results, it was not possible to obtain graphics that characterized with precision the photons emission, because external factors such as luminosity, climatologic ones and the choice of sand as substrate made the reading of the data impossible. In the biological measurement of Phase 1, in the first three days of germination the seeds remained in the germination chamber only with water. The Variance Analysis for the seedlings height produced an F value of 0.1200, with a variation Coefficient of 10.51%, and for the roots length an F value of 0.4800 with a Variation Coefficient of 12.49%. The Tukey Test was realized a posteriori, producing results with none statistical significance. In Phase 2, the differentiated rules were used from the very beginning of the germination, as a consequence the variance analysis for the seedlings height produced an F value of 0.0584 with a Variation Coefficient of 13,76%, and for the roots length the F value was 0,0538 with a Variance Coefficient of 12.40%. The Tukey Test was realized a posteriori, producing results with none statistical significance. In this phase another measurement was included: the roots weight, for which F was 0.005, showing that there occurred a statistically significant difference in the roots weight average according to the treatment used and the Variance Coefficient obtained was 6.25%. For the Tukey Test statistic significance was obtained, and this result indicates that the Flower Essence in this experiment stimulated a better blossom of the roots (fasciculation), and this phenomenon helped the seedlings to overcome the water stress as well as to establish a bigger resistance to the attack of radiculars patogenous. For this experiment the interaction of three big areas of knowledge was necessary: Nursing, Agronomy and Electric Engineering. This demonstrated the importance of promoting interdisciplinary research, because only in this way one could promote the search for devices that showed the action of Alternative/Complementary Therapies when utilized