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-

Epithelial Ovarian Cancer Diagnosis of SecondHarmonic Generation Images: A Semiautomatic Collagen Fibers Quantification Protocol

A vast number of human pathologic conditions are directly or indirectly related to tissular collagen structure remodeling. The nonlinear optical microscopy second-harmonic generation has become a powerful tool for imaging biological tissues with anisotropic hyperpolarized structures, such as collagen. During the past years, several quantification methods to analyze and evaluate these images have been developed. However, automated or semiautomated solutions are necessary to ensure objectivity and reproducibility of such analysis. This work describes automation and improvement methods for calculating the anisotropy (using fast Fourier transform analysis and the gray-level co-occurrence matrix). These were applied to analyze biopsy samples of human ovarian epithelial cancer at different stages of malignancy (mucinous, serous, mixed, and endometrial subtypes). The semiautomation procedure enabled us to design a diagnostic protocol that recognizes between healthy and pathologic tissues, as well as between different tumor types.Fil: Zeitoune, Angel Alberto. 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: Luna, Johana S. J.. Universidad Nacional de Entre Ríos. Facultad de Ingeniería; ArgentinaFil: Sanchez Salas, Kynthia. Universidad Nacional de Entre Ríos. Facultad de Ingeniería; ArgentinaFil: Erbes, Luciana Ariadna. 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: Cesar, Carlos L.. Universidade Federal do Ceará; Brasil. National Institute of Science and Technology on Photonics Applied to Cell Biology; BrasilFil: Andrade, Liliana A. L. A.. Universidade Estadual de Campinas; BrasilFil: Carvahlo, Hernades F.. Universidade Estadual de Campinas; Brasil. National Institute of Science and Technology on Photonics Applied to Cell Biology; BrasilFil: Bottcher Luiz, Fátima. Universidade Estadual de Campinas; Brasil. National Institute of Science and Technology on Photonics Applied to Cell Biology; BrasilFil: Casco, Victor Hugo. Universidad Nacional de Entre Ríos. Facultad de Ingeniería; ArgentinaFil: Adur, Javier Fernando. 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; Argentina. Universidad Nacional de Entre Ríos. Facultad de Ingeniería; Argentin

Feminist Materialisms

We know how much matter and materiality influences, shapes and manipulates our becoming. Therefore how can we be anything but interested in feminist theories of materialism? We are interested in theorizing that opens a redefinition of how matter and materiality may be perceived. It is especially interesting when this theorizing also embraces the queerness in the world. This volume is a tribute to a new way of thinking about materiality, and represents a feminist voice in the ‘material turn’ that appears to be taking place in the social and human sciences

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.Fil: Adur, Javier Fernando. Universidad Nacional de Entre Ríos. Facultad de Ingeniería; Argentina. Universidade Estadual de Campinas; Brasil. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Pelagati, Vitor B.. Universidade Estadual de Campinas; BrasilFil: de Thomaz, Andre A.. Universidade Estadual de Campinas; BrasilFil: Baratti, Mariana O.. Universidade Estadual de Campinas; BrasilFil: Andrade, Liliana A. L. A.. Universidade Estadual de Campinas; BrasilFil: Carvalho, Hernandes F.. Universidade Estadual de Campinas; BrasilFil: Bottcher Luiz, Fátima. Universidade Estadual de Campinas; BrasilFil: Lenz Cesar, Carlos. Universidade Estadual de Campinas; Brasi

Optical Biomarkers Of Serous And Mucinous Human Ovarian Tumor Assessed With Nonlinear Optics Microscopies.

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. 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. 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.7e4700

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

<div><h3>Background</h3><p>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.</p> <h3>Methodology/Principal Findings</h3><p>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.</p> <h3>Conclusions/Significance</h3><p>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.</p> </div

Fluorescence lifetime quantification in the ovarian epithelium.

<p>(A) Multiphoton intensity and FLIM images of endogenous fluorescence resulting from excitation at 890 nm of healthy and tumor ovarian tissues. The color map in (A) represents the weighted average of the two-term model components (τ_m = (<i>a</i>₁τ₁+<i>a</i>₂τ₂)/(<i>a</i>₁+<i>a</i>₂)) using the equation shown in the text. Scale bar = 10 µm (B) Quantitative analysis of fluorescent lifetime weighted mean component (τ_m) calculated only in the epithelium (white dotted line). 15 pixels in different epithelial cells from each image (15×3 = 45 measurements) were used to calculate lifetime values for tumor epithelial cells. † indicates comparison with normal tissues. ††, ** indicates a statistically very significant (p<0.01) difference following ANOVA analysis. N.: normal, Ade. : adenoma, Bord.: borderline, Adecar.: Adenocarcioma. (C) C1. Digital camera image of H&E stained ovarian tumor. Adenoma to borderline transformation is indicated. C2. Color maps of the fluorescence lifetime (τ_m), which illustrate the relatively longer lifetime values in malignant cells when compared to benign epithelium. Scale bar = 20 µm. C.3 Histogram plot (pixel frequency vs. τ_m) of the measures for the range of lifetime values of the two ROIs drawn in (C1) reveals the shift to longer lifetimes in malignant (red line) cells compared to benign epithelium (white line). Cells with mucin are indicated with white arrowhead. Ep: epithelium, St: stromal.</p

Anisotropy and texture quantification in the ovarian stromal region.

<p>(A) Representative SHG images (obtained from H&E-stained samples) of the different 150×150 pixel side yellow squared ROI in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047007#pone-0047007-g004" target="_blank">figure 4</a> and corresponding FFT intensity images obtained after 2D-DFT. Scale bar = 15 µm (B) Results of the aspect ratio (each bar represents the mean ±S.D. of independent measurements) of ovarian samples averaged on all ROI examined: normal (n = 45), serous (adenoma: n = 36, borderline: n = 27, adenocarcinoma: n = 99) and mucinous (adenoma: n = 36, borderline: n = 27, adenocarcinoma: n = 36). Comparisons with normal tissues are indicated with †. †,* indicates a statistically significant (p<0.05) difference and ††, ** indicates a statistically very significant (p<0.01) difference following ANOVA. (C) Correlation values in serous tumor versus pixel separation distance; the correlation for distances ranging from 1 to 18 pixels (0.35–6.0 µm) in the horizontal direction of 200 x 200 pixel ROI of interest was calculated (n = 36). Border: borderline, Adenocar: adenocarcinoma.</p

Multimodal nonlinear optical microscopy platform.

<p>Schematic representation of the multimodal platform based on an inverted microscope Olympus IX-81 and an Olympus FV300 confocal scanning head used to capture TPEF, SHG, THG, FLIM and H&E images. HWP: half wave plate, PBS: polarizing beam splitter, L1–L2: telescope lens, DM: dichroic mirror, G1–G2: galvanometer mirrors, L3: collecting lens, PMT: photomultiplier tubes, BP: bandpass filter, SP: short pass filter, LP: long pass filter. The SHG (red lines) and THG (blue lines) are collected in a transmitted light configuration. The TPEF (green lines) and FLIM (black line) are collected in back-scattering configuration.</p