Figure 1.
Multimodal nonlinear optical microscopy platform.
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.
Figure 2.
Multimodal nonlinear images of human ovary.
Multicontrast cross-sectional images of human ovary. The color code for each technique are: in green TPEF; in red SHG; in magenta THG; in blue/green/yellow/orange FLIM. Column 1 represents normal ovary and column 2 and 3 represent serous and mucinous ovarian tumor types, respectively. Row 1 shows representative brightfield H&E-image. Rows 2 and 3 show different merged combinations of NLO techniques obtained from H&E-stained samples excited at 940 nm. At this wavelength TPEF signal is due to eosin fluorescence, SHG is collagen, and THG represent the nuclei. Row 4 shows FLIM images obtained from unstained samples exited at 890 nm. Blue and orange colors represent lower and higher fluorescence lifetime, respectively. SHG is also detected by the FLIM detection channel due to the choice of emission filter. SHG is an instantaneous nonlinear optical process which, therefore, appears as a very fast lifetime component (blue). In contrast, epithelial cells show lifetime values around 0.5 to 2.5 ns (yellow/orange) which match FAD endogenous fluorophor emmision. Inserts in the right of each image represents enlarged images of white square ROI where specific biomarkers could be identified (insert are not presented for mucinous tumor images). Ep: epithelium, St: stromal, N: nucleus, C: collagen fibers. Scale bars = 20 µm.
Figure 3.
Collagen/elastin content ratio quantification in the ovarian stroma.
(A) Representative merges of TPEF (green) and SHG (red) cross-sectional images of ovarian tissues obtained from H&E-stained samples. All scale bars are 20 µm. (B) SAAID index. Each bar represents the mean ±S.D. of independent measurements from stroma regions of image A. The total number of regions from which values were extracted was normal (n = 15), serous (adenoma: n = 12, borderline: n = 9, adenocarcinoma: n = 33) and mucinous (adenoma: n = 12, borderline: n = 9, adenocarcinoma: n = 12). Asterisks indicate a significant increase as compared to the non-tumor tissues (p<0.05, t-test).
Figure 4.
Anisotropy and texture quantification in the ovarian stromal region.
(A) Representative SHG images (obtained from H&E-stained samples) of the different 150×150 pixel side yellow squared ROI in figure 4 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.
Figure 5.
Characterization of epithelial/stromal interface using SHG+THG.
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.
Figure 6.
Fluorescence lifetime quantification in the ovarian epithelium.
(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 (τm = (a1τ1+a2τ2)/(a1+a2)) using the equation shown in the text. Scale bar = 10 µm (B) Quantitative analysis of fluorescent lifetime weighted mean component (τm) 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 (τm), 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. τm) 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.