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

Characterization of epithelial/stromal interface using SHG+THG.

<p>Representative SHG (red) and THG (magenta) images of ovarian tissues obtained from H&E-stained samples. Yellow squares, near the epithelium, represent the selected 150×150 pixel side ROI used to perform anisotropy quantification. Insert shows more precisely the morphology of nuclei delimitated by white square. Scale bars = 20 µm.</p