Colon adenocarcinoma diagnosis in human samples by multicontrast nonlinear optical microscopy of hematoxylin and eosin stained histological sections

Combined multimodal nonlinear optical (NLO) microscopies were used to detect and quantify morphological changes associated with stroma and epithelial transformation in colon cancer. Our findings provide complementary information about tissue microstructure, displaying distinctive patterns between normal and malignant human colon. Additionally, we have demonstrated the usefulness of using fixed tissues for the disease diagnostic and prognostic. The present work provides a framework for using NLO techniques as a clinical diagnostic tool for human colon cancer. NLO metrics could be applied to other disorders, which are characterized by abnormal cell proliferation and collagen assembly.Fil: Adur, Javier Fernando. Universidad Nacional de Entre Ríos. Facultad de Ingeniería; Argentina. National Institute of Science and Technology on Photonics Applied to Cell Biology; Brasil. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia de Entre Ríos. Universidad Nacional de Entre Ríos. Centro de Investigaciones y Transferencia de Entre Ríos; ArgentinaFil: Bianchi, Mariana. Universidad Nacional de Entre Ríos. Facultad de Ingeniería; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Pelegati, Vitor B.. National Institute of Science and Technology on Photonics Applied to Cell Biology; BrasilFil: Viale, Silvia. Universidad Nacional de Entre Ríos. Facultad de Ingeniería; ArgentinaFil: Izaguirre, María Fernanda. Universidad Nacional de Entre Ríos. Facultad de Ingeniería; ArgentinaFil: Carvalho, Hernandes Faustino. Universidade Estadual de Campinas; BrasilFil: Cesar, Carlos L.. Universidade Estadual de Campinas; BrasilFil: Casco, Victor Hugo. Universidad Nacional de Entre Ríos. Facultad de Ingeniería; Argentin

Quantitative Changes In Human Epithelial Cancers And Osteogenesis Imperfecta Disease Detected Using Nonlinear Multicontrast Microscopy.

We show that combined multimodal nonlinear optical (NLO) microscopies, including two-photon excitation fluorescence, second-harmonic generation (SHG), third harmonic generation, and fluorescence lifetime imaging microscopy (FLIM) can be used to detect morphological and metabolic changes associated with stroma and epithelial transformation during the progression of cancer and osteogenesis imperfecta (OI) disease. NLO microscopes provide complementary information about tissue microstructure, showing distinctive patterns for different types of human breast cancer, mucinous ovarian tumors, and skin dermis of patients with OI. Using a set of scoring methods (anisotropy, correlation, uniformity, entropy, and lifetime components), we found significant differences in the content, distribution and organization of collagen fibrils in the stroma of breast and ovary as well as in the dermis of skin. We suggest that our results provide a framework for using NLO techniques as a clinical diagnostic tool for human cancer and OI. We further suggest that the SHG and FLIM metrics described could be applied to other connective or epithelial tissue disorders that are characterized by abnormal cells proliferation and collagen assembly.17081407-

Recognition Of Serous Ovarian Tumors In Human Samples By Multimodal Nonlinear Optical Microscopy.

We used a multimodal nonlinear optics microscopy, specifically two-photon excited fluorescence (TPEF), second and third harmonic generation (SHG∕THG) microscopies, to observe pathological conditions of ovarian tissues obtained from human samples. We show that strong TPEF + SHG + THG signals can be obtained in fixed samples stained with hematoxylin and eosin (H&E) stored for a very long time, and that H&E staining enhanced the THG signal. We then used the multimodal TPEF-SHG-THG microscopies in a stored file of H&E stained samples of human ovarian cancer to obtain complementary information about the epithelium∕stromal interface, such as the transformation of epithelium surface (THG) and the overall fibrillary tissue architecture (SHG). This multicontrast nonlinear optics microscopy is able to not only differentiate between cancerous and healthy tissue, but can also distinguish between normal, benign, borderline, and malignant specimens according to their collagen disposition and compression levels within the extracellular matrix. The dimensions of the layers of epithelia can also be measured precisely and automatically. Our data demonstrate that optical techniques can detect pathological changes associated with ovarian cancer.1609601

Second-harmonic generation imaging analysis can help distinguish sarcoidosis from tuberculoid leprosy

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL E NÍVEL SUPERIORCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOSarcoidosis and tuberculoid leprosy (TL) are prototypes of granulomatous inflammation in dermatology, which embody one of the histopathology limitations in distinguishing some diseases. Recent advances in the use of nonlinear optical microscopy in skin have enabled techniques, such as second-harmonic generation (SHG), to become powerful tools to study the physical and biochemical properties of skin. We use SHG images to analyze the collagen network, to distinguish differences between sarcoidosis and TL granulomas. SHG images obtained from skin biopsies of 33 patients with TL and 24 with sarcoidosis retrospectively were analyzed using first-order statistics (FOS) and second-order statistics, such as gray-level co-occurrence matrix (GLCM). Among the four parameters evaluated (optical density, entropy, contrast, and second angular moment), only contrast demonstrated statistical significance, being higher in sarcoidosis (p = 0.02||4908.31 versus 2822.17). The results may indicate insufficient differentiating power for most tested FOS and GLCM parameters in classifying sarcoidosis and TL granulomas, when used individually. But in combination with histopathology (H&E and complementary stains, such as silver and fast acid stains), SHG analysis, like contrast, can contribute to distinguishing between these diseases. This study can provide a way to evaluate collagen distribution in granulomatous diseases.231217FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL E NÍVEL SUPERIORCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL E NÍVEL SUPERIORCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO11/51959-02014/50938-82016/22927-72017/17149-801-P-3368/2017312049/2014-5465699/2014-

Valores humanos no esporte de competição : reflexões a partir da pedagogia do treinamento e do metodo de analise de conteudo aplicado a declarações midiaticas

A relação entre o esporte de competição e a educação vem se constituindo como um tema extremamente controverso dentro da Educação Física. Na perspectiva da pedagogia do treinamento, enfatizamos a importância de tornar o esporte de competição um ambiente propício ao desenvolvimento moral e aberto à temática dos valores humanos. Em nossa revisão bibliográfica, discutimos temas como a relação esporte-educação, a competição esportiva e as dicotomias entre educação e competição e entre cooperação e competição, além de abordarmos a temática dos valores humanos, ética e moralidade dentro do esporte. A partir deste referencial, o objetivo deste trabalho configurou-se em discutir temáticas referentes aos valores humanos que, conforme sua literatura, encontram-se presentes no cotidiano das práticas esportivas de alto-rendimento. Fizemos isso através do método de análise de conteúdo, aplicado a declarações midiáticas de atletas, dirigentes, comentaristas e demais pessoas envolvidas com o esporte, buscando encontrar um diagnóstico que nos permitiu contribuir para uma melhor compreensão desta relação e do tema como um todo. As análises nos permitiram fazer algumas afirmações a respeito dos valores humanos no esporte de competição: 1.Possuem natureza imprevisível; 2. Valores aceitos em grande escala não são necessariamente bons ou imutáveis; 3. A subjetividade na expressão de valores é muito presente e deve ser levada em conta. Por fim, podemos afirmar que as análises apresentadas não deixam dúvidas quanto à grande abertura que o esporte competitivo traz à discussão sobre ética e valores humanos, pois concluímos que a presença dos mesmos é constante nas declarações apresentadas. Propomos então que a educação moral e suas finalidades sejam parte integrante do âmbito do esporte competitivo, a fim de humanizá-lo e creditar seu potencial como instrumento de formação integral do ser humano.The relation between sports competition and education has been constituted as an extremely controversial subject inside the Physical Educations studies. On the perspective of training pedagogy, we stress the importance of turning the sports competition into a propitious environment to the moral development and opened to the thematic of the human values inside the sports competition scope. In our literature review, we have discussed themes like the sporteducation relation, the sports competition and the antagonism between education and competition and between cooperation and competition, and also approached the thematic of human values, ethics and morality inside sports. Through this reference, the objective of this study has been configured as to discuss the thematics related to human values that, according to its literature, are found inside the routine of the practices of highly-performance sports. We have done that through the content analysis method, applied to mediathic declarations of athletes, directors, commentarists and other people involved with sports, expecting to find a diagnosis that allows us to contribute with a better understanding of this relation and of the subject on the whole. The analyses have allowed us to make affirmatives about the human values inside the sports competition: 1. They have unforeseeable nature; 2. Values that are accepted in large scale are not necessarily good or immutable; 3. The subjectivity in the expression of values is very often and must be considered. At last, we can affirm that the presented analysis have left no doubt about the large opening that sports competition brings to the discussion about ethics and human values, because we have concluded that its presence is constant at the presented declarations. We propose than that the moral education and its finalities become part of the scope of sports competition, to humanize it and give credit to its potential as an instrument to the human integral formation

Optical microscopies of coherent processes

Orientador: Carlos Lenz CésarTese (doutorado) - Universidade Estadual de Campinas, Instituto de Física Gleb WataghinResumo: Técnicas de microscopias ópticas são as principais ferramentas capazes de observar células e tecidos biológicos em tempo real e com mínimo dano. Essa área foi revolucionada recentemente através das microscopias confocais de varredura a laser e as microscopias de óptica não linear, naturalmente confocais. Entre os processos não lineares temos, a fluorescência excitada por dois ou mais fótons, geração de segundo harmônico [Second Harmonic Generation - SHG] e terceiro harmônico [Third Harmonic Generation - THG]. SHG e THG são técnicas de óptica não linear coerentes, não necessitam de marcadores exógenos e permitem reconstrução de imagens em três dimensões com resolução espacial subcelular. As técnicas de fluorescência permitem visualizar estruturas específicas no espaço, mas não permitem discriminar as substâncias químicas nas estruturas celulares, e as técnicas de SHG e THG não possuem especificidade química. Espectroscopia Raman possui especificidade química através das propriedades vibracionais das moléculas e pode ser usada como mecanismo de contraste na aquisição de imagens. Comparada com a espectroscopia/microscopia infravermelho, a microscopia Raman traz a informação das vibrações moleculares do infravermelho para o visível, eliminando os problemas da baixa resolução espacial e opacidade das amostras. Entretanto a baixa sensibilidade dessa técnica implica em tempos de aquisição de imagens muito longos, da ordem de horas, inviabilizando acompanhar a dinâmica de processos celulares em tempo real. Como solução para essa baixa sensibilidade do espalhamento Raman espontâneo, surgiu a microscopia por espalhamento Raman Coerente anti-Stokes [Coherent Anti-Stokes Raman Scattering - CARS]. Comparado com Raman espontâneo, a microscopia CARS representa aumento de 4 a 5 ordens de grandeza na sensitividade da técnica, diminuindo os tempos de aquisição ao ponto de viabilizar a aquisição em taxas de vídeos (mais rápido do que 30 quadros por segundo) e estudos em tempo real. Essa tese é dedicada ao estudo experimental e teórico, assim como de algumas aplicações, das técnicas de óptica não linear, com destaque para processos de óptica não linear coerentes. Apresentamos de forma detalhada três sistemas experimentais para a aquisição de imagens de Raman coerente e um sistema integrado com várias técnicas de óptica não linear. Mostramos as primeiras imagens de CARS realizadas no Brasil. Além do CARS convencional, trabalhamos com outra técnica de CARS de ordem mais alta, o CARS cascata [cascade CARS - CCARS], e, no melhor do nosso conhecimento, apresentamos as primeiras imagens internacionais obtidas com essa metodologia. CCARS aumenta o contraste da técnica CARS, diminuindo o fundo não ressonante, um problema que aflige a comunidade científica dedicada ao uso dessa técnica. Além da diminuição do fundo não ressonante, a emissão do CCARS acontece em um comprimento de onda diferente de qualquer outro efeito não linear coerente, significando um acréscimo de complexidade mínimo para sua detecção quando comparado com o CARS. Por último mostramos algumas aplicações realizadas com o sistema experimental desenvolvido para integrar diversas modalidades ópticas em paralelo, especialmente da geração de harmônicos com a fluorescência excitada por dois fótons e suas variantes, como microscopia de tempo de vida de fluorescência (Fluorescence Lifetime Imaging ¿ FLIM)Abstract: Optical microscopies techniques are the main tools capable of observing cell and biological tissues in real time and with minimum damage. This area have recently been revolutionized by confocal laser scanning microscopies and non-linear microscopies, naturally confocal. Among the non-linear process we have, the two or more photons excited fluorescence, second harmonic generation [SHG] and third harmonic generation [THG]. SHG and THG are coherent nonlinear techniques, they do not require exogenous markers and allow three dimension imaging reconstruction with subcellular resolution. The fluorescence techniques allow visualizing specific structures in space, but do not allow discriminating the chemical substances in cellular structures, SHG and THG techniques do not have chemical specificity. Raman spectroscopy has chemical specificity through the vibrational properties of the molecules and can be used as a contrast mechanism for imaging acquisition. Compared to infrared spectroscopy/microscopy, Raman microscopy brings information about molecular vibration from infrared to visible, eliminating the low resolution and sample opacity problems. However, this technique low sensibility implies in very long imaging acquisition times, order of hours, making it not viable for following cellular process dynamics in real time. As an answer for the spontaneous Raman scattering low sensibility, the coherent anti-Stokes Raman scattering [CARS] emerged. Compared to spontaneous Raman, CARS microscopy presents an increase of 4 to 5 orders of magnitude in the sensitivity of the technique, lowering the acquisition times to the point of making video acquisition (faster than 30 frames per second) and real time studies possible. This thesis is dedicated to the experimental and theoretical study, as well as some applications, of the non-linear techniques, with emphasis on coherent non-linear optical processes. We present in detailed form three experimental systems for the acquisition of coherent Raman images, and a system with the integration of various non-linear techniques. We show the first CARS images acquired in Brazil. In addition to conventional CARS, we worked with other higher order CARS technique, the cascade CARS [CCARS], and, in the best of our knowledge, we present the first international image acquired with this methodology. CCARS increases the contrast from CARS technique, decreasing the non-resonant background, a problem that afflicts the scientific community dedicated to the use of this technique. Besides the decrease of the non-resonant background, the CCARS emission occurs in a different wavelength from any other non-linear coherent effect, meaning a minimum complexity increase for its detection when compared with CARS. Finally we show some applications performed with the experimental system developed to integrate several optical modalities in parallel, especially the generation of harmonics with two photons excitation fluorescence and their variants such as Fluorescence Lifetime Imaging [FLIM]DoutoradoFísicaDoutor em Ciências830406/2010CAPE

Simple Gaas And Inp Colloidal Quantum Dots Synthesis Using Laser Ablation

In this work we will present a simple synthesis route for obtaining both GaAs and InP colloidal quantum dots using the same laser ablation assembly. © 2010 OSA/FiO/LS 2010

Elastic Fibers And Collagen Distribution In Human Aorta

Elastic and collagen fibers are essential components of the aorta, the remodeling of these structures is accompanied with aging in various diseases and life-threatening events. While the elastic fibers confer resilience to major blood vessels collagen confers resistance to the same. Elastic fibers are easily visualized in the fluorescent light when stained with hematoxylin eosin. Second Harmonic Generation (SHG) is a non linear signal that occurs only in molecules without inversion symmetry and is particularly strong in the collagen fibers arranged in triple helices. The aim of this paper is to describe the distribution of collagen in the thickness of the thoracic aorta, and to demonstrate the distribution of between elastic fibers. The images were acquired in a multifoton microscopy and both signals, Two-phtoton excitaded fluorescence (TPEF) and SHG, were excited by a Ti:Sapphire laser. We used a band pass filter to filter the SHG signal from the TPEF signal. The thickness of the aorta varies 2-3 mm, and the image was composed of the juxtaposition of images of 220 × 220 microns. We acquired images of a histological slide of the thoracic aorta stained with picrosirius red (specific for collagen) at a wavelength of 670nm SHG subsequently acquired images with the same region and observed that the images are overlapping. Therefore, the following images were acquired by confocal microscopy (fluorescence of eosin for visualization of elastic fibers) and for collagen SHG.After reconstruction of the images, we observed the distribution of collagen along the aorta. © 2011 SPIE.7903The Society of Photo-Optical Instrumentation Engineers (SPIE),Becker and Hickl GmbH,Boston Electronics,Chroma Technology,Coherent, Inc.Christopher, M.M., Hotz, C.S., Cytologic diagnosis: Expression of probability by clinical pathologists (2004) Veterinary Clinical Pathology, 33 (2), pp. 84-95Metze, K., Adam, R.L., Quantification in histopathology - Some pitfalls (2005) Brazilian Journal of Medical and Biological Research, 38, pp. 141-143Adam, R.L., (2002) Análise Espectral Usando a Transformada de Fourier Discreta Para O Estudo de Núcleos Celulares: Elaboração de Programa e Aplicação No Desenvolvimento do Coração, , (Dissertação - Mestrado) Campinas, SP - Universidade Estadual de CampinasVieira, G., Confocal microscopy for automatic measurement of the density and distance between Elastin fibers of histologic preparations of normotensive and hypertensive patients (2010) SPIE, 7568, pp. 75680K-75680K8De Carvalho, H.F., Taboga, S.R., Fluorescence and confocal laser scanning microscopy imaging of elastic fibers in hematoxylin-eosin stained sections (1996) Histochemistry and Cell Biology, 106 (6), pp. 587-592. , DOI 10.1007/s004180050080Georgiou, E., (2000) D. Opt. Commun., 176, pp. 253-260Campagnola, P.J., (2001) J. Biomed. Opt., 6, pp. 277-286Roth, S., Freund, I., (1981) Biopolymers, 20, pp. 1271-129