The Severity of Osteogenesis Imperfecta and Type I Collagen Pattern in Human Skin as Determined by Nonlinear Microscopy: Proof of Principle of a Diagnostic Method

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Background: The confirmatory diagnosis of Osteogenesis Imperfecta (OI) requires invasive, commonly bone biopsy, time consuming and destructive methods. This paper proposes an alternative method using a combination of two-photon excitation fluorescence (TPEF) and second-harmonic generation (SHG) microscopies from easily obtained human skin biopsies. We show that this method can distinguish subtypes of human OI. Methodology/Principal Findings: Different aspects of collagen microstructure of skin fresh biopsies and standard H&E-stained sections of normal and OI patients (mild and severe forms) were distinguished by TPEF and SHG images. Moreover, important differences between subtypes of OI were identified using different methods of quantification such as collagen density, ratio between collagen and elastic tissue, and gray-level co-occurrence matrix (GLCM) image-pattern analysis. Collagen density was lower in OI dermis, while the SHG/autofluorescence index of the dermis was significantly higher in OI as compared to that of the normal skin. We also showed that the energy value of GLCM texture analysis is useful to discriminate mild from severe OI and from normal skin. Conclusions/Significance: This work demonstrated that nonlinear microscopy techniques in combination with image-analysis approaches represent a powerful tool to investigate the collagen organization in skin dermis in patients with OI and has the potential to distinguish the different types of OI. The procedure outlined in this paper requires a skin biopsy, which is almost painless as compared to the bone biopsy commonly used in conventional methods. The data presented here complement existing clinical diagnostic techniques and can be used as a diagnostic procedure to confirm the disease, evaluate its severity and treatment efficacy.87Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP [2011/51591-3]FAPESP [2008/57906-3

Harmonic optical microscopy and fluorescence lifetime imaging platform for multimodal imaging

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)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. (c) 2012 Wiley Periodicals, Inc.751013831394Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)National Institute of Photonics Applied to Cell Biology (INFABIC)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

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. (C) 2011 Society of Photo-Optical Instrumentation Engineers (SPIE). [DOI: 10.1117/1.3626575]16