Experimental setup of a nonlinear optical microscopic system.

<p>CCD: charge-coupled device, AOM: acoustic-optic modulator, T₁: telescope, SP: short-pass filter, BP: band-pass filter, LP: long-pass filter, NDD: Non Descanned Detector. The SHG (red lines) and TPEF (green lines) are collected in a transmitted light configuration.</p

Best methods for quantitative analysis in fresh and fixed skin samples.

<p>Representative 3D maximum projection (40 images at intervals of 1 µm) of SHG images from fresh skin biopsies, A: normal skin, B: OI Type III (Patient E) and C: OI Type IV (Patient D). D: depth-dependent decay of the SHG signal analyzed to determine the collagen density in normal (black circle) and severe OI (Type III and IV -red circle). White asterisk indicate the selected regions where the plot was calculated as a function of depth. Each circle represent the mean value and standard deviation of three values corresponding to selected region. Texture analysis (E, F), using the gray-level co-occurrence matrix (GLCM). Energy values was calculated in dermis tissues versus distances pixels; ranging from 1 to 50 pixels (0.35 µm - 17.30 µm) in 0, 45, 90 and 135 deg directions of image (E: n = 12 normal, n = 3 mild OI, and n = 12 severe OI; and F: n = 12 normal, n = 3 OI Type I, n = 9 OI Type IV, n = 3 OI Type III). Pat: patients.</p

Texture analysis of skin dermis.

<p>One representative image (354.30×354.30 µm) of the eight images from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069186#pone-0069186-g003" target="_blank">figure 3</a>. Normal skin (A, B, C, D), OI-Type I (E, F, G, H) and OI-Type III (I, J, K, L). Figure shows H&E-stained samples (A, E, I), TPEF images in green (B, F, J), SHG images in red (C, G, K) and merge TPEF+SHG images (D, H, L). Epithelial/stromal interface is indicated (white outline). Ep: Epithelium, D: Dermis. Scale bars = 100 µm. Texture analysis (M, N, L, O) using the gray-level co-occurrence matrix (GLCM). Correlation (M), Contrast (N), Energy (L), and Homogeneity (O) values in dermis tissues versus distances pixels; ranging from 1 to 50 pixels (0.35 µm–17.30 µm) in 0, 45, 90 and 135 deg directions of image. (n = 12 normal, n = 3 mild OI, and n = 9 severe OI). Black line (normal), red line (mild OI), and blue line (severe OI).</p

Quantification of collagen and elastic tissues.

<p>Montage of SHG (A, C, E) red and TPEF+SHG (B, D, F) green and red images. Representative images of fixed normal skin from control (A, B), Type I OI patient (C, D) and Type III OI patients (E, F). Scale bar = 200 µm. Montage A and B shows the 12 ROI (200×200 µm) selected to perform the quantification of SHG intensity (G) and SHG autofluorescence aging index of dermis (SAAID) (H). These same ROI positioning was used in all images. In G and H, each bar represents the mean ± S.D. of independent measurements. The total number of ROI from which these parameters were extracted was (normal: 85 ROI for 10 images, mild OI: 19 ROI for 3 images, and severe OI: 54 ROI for 8 images). Asterisks indicate a significant (*, p<0.05) difference from normal samples. ROI mainly occupying the dermis area were used.</p

Patients clinical characteristic and molecular analysis.

<p>Pat.: Patients, T.: Type, Bone Def.: Bone Deformity, Sta.: Stature, N.: Normal, E.: Exon, N.A.: Not Available.</p

Postnatal and SHG diagnosis of OI.

<p>N.: Normal, C. Fractures: Congenital Fractures, Bone D.: Bone Deformity, N.A.: Not Available, DI: Dentinogenesis Imperfecta, DDD: Depth-dependent decay, SAAID: SHG autofluorescence aging index of dermis, GLCM: Gray-level co-occurrence matrix, <i>d</i>: pixel distance.</p

SHG signal in function of skin depth.

<p>Representative SHG images from fresh skin biopsies. A, B: normal skin from a control and C,D: Type III OI patient. A and C: gallery view of 40 images at intervals of 1 μm. (1: –20 µm depth, 20: in focus and 40: +20 µm depth). Scale bar = 35 µm. B and D: 3D maximum projection of A and C, respectively. E: depth-dependent decay (DDD) of the SHG signal analyzed to determine the collagen density in normal (black circle) and severe OI-Type III (red circle). The white asterisks indicate the selected regions where the plot was calculated as a function of depth. Each circle represents the mean value and standard deviation of three values corresponding to selected region.</p

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

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

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