Fátima Böttcher-Luiz

Second harmonic generation microscopy as a powerful diagnostic imaging modality for human ovarian cancer

In this study we showed that second-harmonic generation (SHG) microscopy combined with precise methods for images evaluation can be used to detect structural changes in the human ovarian stroma. Using a set of scoring methods (alignment of collagen fibers, anisotropy, and correlation), we found significant differences in the distribution and organization of collagen fibers in the stroma component of serous, mucinous, endometrioid and mixed ovarian tumors as compared with normal ovary tissue. This methodology was capable to differentiate between cancerous and healthy tissue, with clear cut distinction between normal, benign, borderline, and malignant tumors of serous type. Our results indicated that the combination of different image-analysis approaches presented here represent a powerful tool to investigate collagen organization and extracellular matrix remodeling in ovarian tumors.

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.

Optical Biomarkers of Serous and Mucinous Human Ovarian Tumor Assessed with Nonlinear Optics Microscopies

Background Nonlinear optical (NLO) microscopy techniques have potential to improve the early detection of epithelial ovarian cancer. In this study we showed that multimodal NLO microscopies, including two-photon excitation fluorescence (TPEF), second-harmonic generation (SHG), third-harmonic generation (THG) and fluorescence lifetime imaging microscopy (FLIM) can detect morphological and metabolic changes associated with ovarian cancer progression. Methodology/Principal Findings We obtained strong TPEF + SHG + THG signals from fixed samples stained with Hematoxylin & Eosin (H&E) and robust FLIM signal from fixed unstained samples. Particularly, we imaged 34 ovarian biopsies from different patients (median age, 49 years) including 5 normal ovarian tissue, 18 serous tumors and 11 mucinous tumors with the multimodal NLO platform developed in our laboratory. We have been able to distinguish adenomas, borderline, and adenocarcinomas specimens. Using a complete set of scoring methods we found significant differences in the content, distribution and organization of collagen fibrils in the stroma as well as in the morphology and fluorescence lifetime from epithelial ovarian cells. Conclusions/Significance NLO microscopes provide complementary information about tissue microstructure, showing distinctive patterns for serous and mucinous ovarian tumors. The results provide a basis to interpret future NLO images of ovarian tissue and lay the foundation for future in vivo optical evaluation of premature ovarian lesions.

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.

Harmonic Optical Microscopy and Fluorescence Lifetime Imaging Platform for Multimodal Imaging

In this work, we proposed and built a multimodal optical setup that extends a commercially available confocal microscope (Olympus VF300) to include nonlinear second harmonic generation (SHG) and third harmonic generation (THG) optical (NLO) microscopy and fluorescence lifetime imaging microscopy (FLIM). We explored all the flexibility offered by this commercial confocal microscope to include the nonlinear microscopy capabilities. The setup allows image acquisition with confocal, brightfield, NLO/multiphoton and FLIM imaging. Simultaneously, two‐photon excited fluorescence (TPEF) and SHG are well established in the biomedical imaging area, because one can use the same ultrafast laser and detectors set to acquire both signals simultaneously. Because the integration with FLIM requires a separated modulus, there are fewer reports of TPEF+SHG+FLIM in the literature. The lack of reports of a TPEF+SHG+THG+FLIM system is mainly due to difficulties with THG because the present NLO laser sources generate THG in an UV wavelength range incompatible with microscope optics. In this article, we report the development of an easy‐to‐operate platform capable to perform two‐photon fluorescence (TPFE), SHG, THG, and FLIM using a single 80 MHz femtosecond Ti:sapphire laser source. We described the modifications over the confocal system necessary to implement this integration and verified the presence of SHG and THG signals by several physical evidences. Finally, we demonstrated the use of this integrated system by acquiring images of vegetables and epithelial cancer biological samples. Microsc. Res. Tech. 2012.

Respostas ao adoecimento: mecanismos de defesa utilizados por mulheres com síndrome de Turner e variantes

OBJETIVO:Conhecer as defesas utilizadas por mulheres com sindrome de Turner (ST) ou formas variantes para lidar com a doenca. METODO: Pesquisa qualitativa com desenho exploratorio, nao experimental. O instrumento consistiu em entrevista psicologica semidirigida, aplicada em 13 mulheres, cuja amostragem deu-se por saturacao, as quais fazem acompanhamento semestral no Centro de Atendimento Integral a Saude da Mulher. Os dados foram interpretados utilizando-se da abordagem psicodinâmica, aliada a um quadro ecletico de referenciais teoricos para discussao no espirito da interdisciplinaridade. RESULTADOS: Essas mulheres apresentaram conflitos psicossociais como dificuldades de relacionamento interpessoal; sentimentos de resignacao, raiva, impotencia, desvalia e quadros de depressao. As defesas utilizadas foram: repressao, negacao, anulacao, fantasia, adaptacao e sublimacao. CONCLUSOES:As mulheres com ST ou formas variantes tem de lidar com as intercorrencias orgânicas e psiquicas da enfermidade provocando grande sofrimento que, frequentemente, dificultam uma insercao social mais sadia. Neste caso, os achados deste estudo poderao nortear acompanhamento psicologico ambulatorial concomitantemente ao protocolo clinico de rotina.

Multimodal nonlinear optical microscopy used to discriminate epithelial ovarian cancer

We used human specimens of epithelial ovarian cancer (serous type) to test the feasibility of nonlinear imaging as complementary tools for ovarian cancer diagnosis. Classical hematoxylin-and-eosin stained sections were applied to combining two-photon excitation fluorescence (TPEF), second (SHG), and third (THG) harmonic microscopy within the same imaging platform. We show that strong TPEF + SHG + THG signals can be obtained in fixed samples stained with Hematoxylin & Eosin (H&E) stored for a very long time and that H&E staining enhanced the THG signal. We demonstrate using anisotropy and morphological measurements, that SHG and THG of stained optical sections allow reproducible identification of neoplastic features such as architectural alterations of collagen fibrils at different stages of the neoplastic transformation and cellular atypia. Taken together, these results suggest that, with our viable imaging system, we can qualitatively and quantitatively assess endogenous optical biomarkers of the ovarian tissue with SHG and THG microscopy. This imaging capability may prove to be highly valuable in aiding to determine structural changes at the cellular and tissue levels, which may contribute to the development of new diagnostic techniques.

Combined nonlinear laser imaging (two-photon excitation fluorescence, second and third-harmonic generation, and fluorescence lifetime imaging microscopies) in ovarian tumors

We applied Two-photon Excited Fluorescence (TPEF), Second/Third Harmonic Generation (SHG and THG) and Fluorescence Lifetime Imaging (FLIM) Non Linear Optics (NLO) Laser-Scanning Microscopy within the same imaging platform to evaluate their use as a diagnostic tool in ovarian tumors. We assess of applicability of this multimodal approach to perform a pathological evaluation of serous and mucinous tumors in human samples. The combination of TPEF-SHG-THG imaging provided complementary information about the interface epithelium/stromal, such as the transformation of epithelium surface (THG) and the overall fibrillar tissue architecture (SHG). The fact that H&E staining is the standard method used in clinical pathology and that the stored samples are usually fixed makes it important a re-evaluation of these samples with NLO microscopy to compare new results with a library of already existing samples. FLIM, however, depends on the chemical environment around the fluorophors that was completely changed after fixation; therefore it only makes sense in unstained samples. Our FLIM results in unstained samples demonstrate that it is possible to discriminate healthy epithelia from serous or mucinous epithelia. Qualitative and quantitative analysis of the different imaging modalities used showed that multimodal nonlinear microscopy has the potential to differentiate between cancerous and healthy ovarian tissue.

Second harmonic generation in human ovarian neoplasias

Metastasis is the main cause of death in cancer patients; it requires a complex process of tumor cell dissemination, extra cellular matrix (ECM) remodeling, cell invasion and tumor-host interactions. Collagen is the major component of ECM; its fiber polymerization or degradation evolves in parallel with the evolution of the cancerous lesions. This study aimed to identify the collagen content, spatial distribution and fiber organization in biopsies of benign and malignant human ovarian tissues. Biopsies were prepared in slides without dyes and were exposed to 800nm Ti:Sapphire laser (Spectra Physics, 100 fs pulse duration, 800mW average power, 80MHz repetition rate). The obtained images were recorded at triplets, corresponding to clear field, multiphoton and second harmonic generation (SHG) mycroscopy. Data showed considerable anisotropy in malignant tissues, with regions of dense collagen arranged as individual fibers or in combination with immature segmental filaments. Radial fiber alignment or regions with minimal signal were observed in the high clinical grade tumors, suggesting degradation of original fibers or altered polymerization state of them. These findings allow us to assume that the collagen signature will be a reliable and a promising marker for diagnosis and prognosis in human ovarian cancers.