Article

Quantitative changes in human epithelial cancers and osteogenesis imperfecta disease detected using nonlinear multicontrast microscopy

State University of Campinas (UNICAMP), "Gleb Wataghin" Institute of Physics, Optics and Photonics Research Center, Biomedical Lasers Application Laboratory, BrazilbNational University of Entre Ríos (UNER), School of Bioengineering, Microscopy Laboratory Applied to Molecular and Cellular Studies, Argentina.
Journal of Biomedical Optics (Impact Factor: 2.86). 08/2012; 17(8):81407-1. DOI: 10.1117/1.JBO.17.8.081407
Source: PubMed

ABSTRACT

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.

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    • "Under the chosen MPM conditions (excitation 950 nm), we observed no overlap between the SHG (emission at 475 nm) and two-photon autofluorescence (emission at 515–560 nm) images, which indicates the specificity of the signals obtained [8]. Similar MPM modalities, i.e., 940 nm excitation, have been applied successfully to detect collagen and elastin in ex vivo breast tissues [28]. In several other tissues, e.g., skin, arteries, or brain, other wavelength ranges have been used for specific SHG imaging of collagen (excitation 780–860 nm, emission 390–430 nm) and two-photon autofluorescence of elastin (excitation 760–850 nm, emission 435–697 nm) [4, 20, 22, 24, 29]. "
    [Show abstract] [Hide abstract] ABSTRACT: INTRODUCTION AND HYPOTHESIS: Multiphoton microscopy (MPM) is a nonlinear, high-resolution laser scanning technique and a powerful approach for analyzing the spatial architecture within tissues. To demonstrate the potential of this technique for studying the extracellular matrix of the pelvic organs, we aimed to establish protocols for the detection of collagen and elastin in the vagina and to compare the MPM density of these fibers to fibers detected using standard histological methods. METHODS: Samples of the anterior vaginal wall were obtained from nine patients undergoing a hysterectomy or cystocele repair. Samples were shock frozen, fixed with formaldehyde or Thiel's solution, or left untreated. Samples were imaged with MPM to quantify the amount of collagen and elastin via second harmonic generation and autofluorescence, respectively. In six patients, sample sections were also histologically stained and imaged with brightfield microscopy. The density of the fibers was quantified using the StereoInvestigator and Cavalieri software. RESULTS: With MPM, collagen and elastin could be visualized to a depth of 100 μm, and no overlap of signals was detected. The different tissue processing protocols used did not result in significantly different fiber counts after MPM. MPM-based fiber quantifications are comparable to those based on conventional histological stains. However, MPM provided superior resolution, particularly of collagen fibers. CONCLUSIONS: MPM is a robust, rapid, and label-free method that can be used to quantify the collagen and elastin content in thick specimens of the vagina. It is an excellent tool for future three-dimensional studies of the extracellular matrix in patients with pelvic organ prolapse.
    Full-text · Article · May 2014 · International Urogynecology Journal
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    • "All images were acquired with a custom multimodal NLO platform built at the Laboratory for Biophotonics of the University of Campinas, which was recently described in detail [7], [20], [26]. The system was built around an inverted microscope IX-81, equipped with an Olympus FV300 scanner (Olympus, Tokyo, Japan), as shown in figure 1. "
    [Show abstract] [Hide abstract] ABSTRACT: 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.
    Full-text · Article · Oct 2012 · PLoS ONE
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    [Show abstract] [Hide abstract] ABSTRACT: Colon cancer is one of the most diffused cancers in the Western World, ranking third worldwide in frequency of incidence after lung and breast cancers. Even if it is curable when detected and treated early, a more accurate premature diagnosis would be a suitable aim for both cancer prognostic and treatment. Combined multimodal nonlinear optical (NLO) microscopies, such as two-photon excitation fluorescence (TPEF), second-harmonic generation (SHG), third harmonic generation (THG), and fluorescence lifetime imaging microscopy (FLIM) can be used to detect morphological and metabolic changes associated with stroma and epithelial transformation in colon cancer disease. NLO microscopes provide complementary information about tissue microstructure, showing distinctive patterns between normal and malignant human colonic mucosa. Using a set of scoring methods significant differences both in the content, distribution and organization of stroma collagen fibrils, and lifetime components of NADH and FAD cofactors of human colon mucosa biopsies were found. Our results provide a framework for using NLO techniques as a clinical diagnostic tool for human colon cancer, and also suggest that the SHG and FLIM metrics could be applied to other intestinal disorders, which are characterized by abnormal cell proliferation and collagen assembly.
    Full-text · Article · Feb 2013 · Proceedings of SPIE - The International Society for Optical Engineering
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