Vitor B. Pelegati

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.

Recognition of serous ovarian tumors in human samples by multimodal nonlinear optical microscopy.

We used a multimodal nonlinear optics microscopy, specifically two-photon excited fluorescence (TPEF), second and third harmonic generation (SHG∕THG) microscopies, to observe pathological conditions of ovarian tissues obtained from human samples. We show that strong TPEF + SHG + THG signals can be obtained in fixed samples stained with hematoxylin and eosin (H&E) stored for a very long time, and that H&E staining enhanced the THG signal. We then used the multimodal TPEF-SHG-THG microscopies in a stored file of H&E stained samples of human ovarian cancer to obtain complementary information about the epithelium∕stromal interface, such as the transformation of epithelium surface (THG) and the overall fibrillary tissue architecture (SHG). This multicontrast nonlinear optics microscopy is able to not only differentiate between cancerous and healthy tissue, but can also distinguish between normal, benign, borderline, and malignant specimens according to their collagen disposition and compression levels within the extracellular matrix. The dimensions of the layers of epithelia can also be measured precisely and automatically. Our data demonstrate that optical techniques can detect pathological changes associated with ovarian cancer.

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

Background 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. Methodology/Principal Findings 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. Conclusions/Significance 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.

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.

Harmonic Optical Microscopy and Fluorescence Lifetime Imaging Platform for Multimodal Imaging

In this work, we proposed and built a multimodal optical setup that extends a commercially available confocal microscope (Olympus VF300) to include nonlinear second harmonic generation (SHG) and third harmonic generation (THG) optical (NLO) microscopy and fluorescence lifetime imaging microscopy (FLIM). We explored all the flexibility offered by this commercial confocal microscope to include the nonlinear microscopy capabilities. The setup allows image acquisition with confocal, brightfield, NLO/multiphoton and FLIM imaging. Simultaneously, two‐photon excited fluorescence (TPEF) and SHG are well established in the biomedical imaging area, because one can use the same ultrafast laser and detectors set to acquire both signals simultaneously. Because the integration with FLIM requires a separated modulus, there are fewer reports of TPEF+SHG+FLIM in the literature. The lack of reports of a TPEF+SHG+THG+FLIM system is mainly due to difficulties with THG because the present NLO laser sources generate THG in an UV wavelength range incompatible with microscope optics. In this article, we report the development of an easy‐to‐operate platform capable to perform two‐photon fluorescence (TPFE), SHG, THG, and FLIM using a single 80 MHz femtosecond Ti:sapphire laser source. We described the modifications over the confocal system necessary to implement this integration and verified the presence of SHG and THG signals by several physical evidences. Finally, we demonstrated the use of this integrated system by acquiring images of vegetables and epithelial cancer biological samples. Microsc. Res. Tech. 2012.

Analysis and control of energy transfer processes and luminescence across the visible spectrum in PFO:P3OT blends

A systematic study of luminescence from blends made of poly (9, 9-dioctylfluorene-2, 7-diyl) (PFO) and poly (3-octylthiophene-2, 5-diyl) (P3OT), and its photophysics was carried out. Acceptor concentration influence and sample preparation conditions was analyzed, particularly regarding the solvent, in order to control emission through the entire visible spectrum, and to understand the physical processes involved. An additional emission band observed in PFO:P3OT films with low concentration of P3OT was studied through confocal luminescence microscopy and was attributed to a decrease of energy transfer between P3OT molecules leading to an emission through chains with smaller conjugation length. The extra emission was also separated from the others by Time Resolved Emission Spectroscopy due to the fact that its lifetime is longer than those of the other emissions. Balance control of the emission through PFO (blue), low conjugation chains of P3OT (green) and P3OT aggregates (red) was possible changing the solvent and the way to prepare samples, that causes a greater or lesser amount of β phase in PFO. The study of the energy processes involved was also performed.

Increased metabolic activity detected by FLIM in human breast cancer cells with desmoplastic reaction: a pilot study

Introduction: In breast cancer (BC), desmoplastic reaction, assembled primarily by fibroblasts, is associated with unfavorable prognosis, but the reason of this fact remains still unclear. In this context, nonlinear optics microscopy, including Fluorescence Lifetime Imaging Microscopy (FLIM), has provided advancement in cellular metabolism research. In this paper, our purpose is to differentiate BC cells metabolism with or without contact to desmoplastic reaction. Formalin fixed, paraffin embedded samples were used at different points of hematoxylin stained sections. Methodology: Sections from 14 patients with invasive ductal breast carcinoma were analyzed with FLIM methodology to NAD(P)H and FAD fluorescence lifetime on a Confocal Upright LSM780 NLO device (Carl Zeiss AG, Germany). Quantification of the fluorescence lifetime and fluorescence intensity was evaluated by SPC Image software (Becker &Hickl) and ImageJ (NIH), respectively. Optical redox ratio was calculated by dividing the FAD fluorescence intensity by NAD(P)H fluorescence intensity. Data value for FLIM measurements and fluorescence intensities were calculated using Wilcoxon test; p< 0.05 was considered significant. Results: BC cells in contact with desmoplastic reaction presented a significantly lower NAD(P)H and FAD fluorescence lifetime. Furthermore, optical redox ratio was also lower in these tumor cells. Conclusion: Our results suggest that contact of BC cells with desmoplastic reaction increase their metabolic activity, which might explain the adverse prognosis of cases associated with higher peritumoral desmoplastic reaction.

Multimodal Nonlinear Optical Microscopy used to Discriminate Human Colon Cancer

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.

The role of stress in CdTe quantum dot doped glasses

In this work, we unequivocally demonstrate the influence of matrix-related stresses on quantum dots by measuring, side by side, a CdTe quantum dot doped glass and a colloidal sample with similar sizes. We measured the fluorescence spectra and fluorescence lifetime for both samples as a function of the temperature. We show that the expansion coefficient mismatch between CdTe quantum dots and the glass host causes stresses and drastically changes its behavior compared to its colloidal counterpart, even leading to phase transitions. This finding indicates that most experimental data on glass-doped quantum dots used to validate confinement models should be revised, taking stress into account.

Bartonella henselae Biofilm Detected on Catheter of Patient with Persistent Bartonellosis

Bartonella spp. can induce long-lasting bacteremia in mammals (1). Bartonella spp. are causative agents of cat-scratch disease, endocarditis, bacillary angiomatosis, bacillary peliosis, trench fever, neurocognitive dysfunction and regional pain syndrome (2). Chronic Bartonella spp. infection and subsequent manifestations of persistent infection may be the result of the bacteria growing in biofilms. In humans, at least four Bartonella species have been associated with infectious endocarditis, a biofilmassociated infectious disease (3). Biofilms are sessile communities of interacting unicellular organisms that adhere to a surface and to each other and create unique, complex structures, covered with a protective layer of extracellular polymeric substances (EPS). Biofilms and their inherent resistance to antimicrobial agents are often key players in many persistent and chronic bacterial infections (4, 5). We acquired skin biopsies from 5 patients with blood PCR-positive B. henselae. 60-100 micron-thick sections were immunostained with antibodies to B. henselae. Stained sections were imaged using both singleand multi-photon imaging to capture epifluorescence emission and second harmonic generation signal of non-stained collagen I and II (Fig 1). The crystalline triple-helix of native fibrillar collagen (I, II, III and V) generates a strong second harmonic generation (SHG) signal (6). In patients with longstanding bartonellosis, clusters of immuno-reactive B. henselae were found intimately associated with fibrillar dermal collagen (Fig 2). Additionally, we found a Bartonella spp. biofilm on a peripheral intravenous catheter line removed from a patient with persistent Bartonella (Fig 3). Further, B. henselae, cultured 14 or 21 days, on collagen I and II-coated coverslips revealed a B. henselae biofilm matrix (Fig 4). Identification and characterization of Bartonella spp. biofilms in vivo and in situ will lead to a better understanding of persistent Bartonellosis and provide direction in treatment modalities.