Simple Gaas And Inp Colloidal Quantum Dots Synthesis Using Laser Ablation

In this work we will present a simple synthesis route for obtaining both GaAs and InP colloidal quantum dots using the same laser ablation assembly. © 2010 OSA/FiO/LS 2010

Characterization Of Third-degree Burned Skin By Nonlinear Microscopy Technique

Nonlinear microscopy imaging technique enable take both images of collagen fibers in dermis through second harmonic generation (SHG) signal and elastic fibers by two-photon emission fluorescence microscopy (TPEFM). These techniques are the most commonly used technique for turbid and thick tissue imaging and also to image biological samples which presents highly ordered structural proteins without any exogenous label. The objective of this study is characterizing dermis of third-degree burned skin by TPEFM and SHG technique. The modelocked laser (Spectra Physics) source used in this study with pulse width of approximately 100 fs at 80 MHz was directed into a multiphoton microscope using a laser scanning unit (Olympus Fluoview 300), mounted on an inverted confocal system microscope (Olympus IX81), with focusing objective (40x, NA = 1.30). The samples were obtained from Wistar rats, male, adult. One dorsum area was submitted to burn caused by vapour exposure. The biopsies obtained were cryosectioned in slices of 20 ?m width. Selected area of interface between the injured and healthy subdermal burned skin were imaged by TPEFM and SHG technique. Two different autofluorescence signals are observed as a function of excitation wavelength. The autofluorescence observed at 760 nm and 690 nm suggest components of extracellular matrix at differents depths. In SHG images, collagen fibers are visible. According to the images obtained, these methodologies can be used to characterize dermis of burned tissue as its healing process with reduced out-of-plane photobleaching and phototoxicity. © 2011 SPIE.7903The Society of Photo-Optical Instrumentation Engineers (SPIE),Becker and Hickl GmbH,Boston Electronics,Chroma Technology,Coherent, Inc.Broughton II, G., Janis, J.E., Attinger, C.E., Basic science of wound healing (2006) Plast Reconstr Surg., 117 (7), pp. 12S-34SGloster Jr., H.M., (2008) Complications in Cutaneous Surgery, pp. 77-85. , Springer Science and Business MediaCampagnola, P.J., Millard, A.C., Terasaki, M., Hoppe, P.E., Malone, C.J., Mohler, W.A., Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues (2002) Biophysical Journal, 82 (1), pp. 493-508Birks, J.B., (1970) Photophysics of Aromatic Molecules, , Wiley InterscienceNakamura, O., Fundamentals of two-photon microscopy (1999) Microscopy Research and Technique, 47, pp. 165-171Alberto, D., Giuseppe, C., Maddalena, C., Two-photon fluorescence excitation and related techniques in biological microscopy (2005) Quarterly Reviews of Biophysics, 38 (2), pp. 97-166Van Zuijlen, P.P.M., Ruurda, J.J.B., Van Veen, H.A., Van Marle, J., Van Trier, A.J.M., Groenevelt, F., Kreis, R.W., Middelkoop, E., Collagen morphology in human skin and scar tissue: No adaptations in response to mechanical loading at joints (2003) Burns, 29 (5), pp. 423-431. , DOI 10.1016/S0305-4179(03)00052-4Zhu, X., Zhuo, S., Zheng, L., Lu, K., Jiang, X., Chen, J., Lin, B., Quantified characterization of human cutaneous normal scar using multiphoton microscopy (2010) Journal of Biophotonics, 3 (1-2), pp. 108-116. , JanCampagnola, P.J., Clark, H.A., Mohler, W.A., Lewis, A., Loew, L.M., Second-harmonic imaging microscopy of living cells (2001) Journal of Biomedical Optics, 6 (3), pp. 277-286. , DOI 10.1117/1.1383294Bianchini, P., Diaspro, A., Three-dimensional (3D) backward and forward second harmonic generation (SHG) microscopy of biological tissues (2008) J. Biophoton, 1 (6), pp. 443-450Gartner, L.P., Hiatt, J.L., Color textbook of histology (2001) Saunders, , 2 edition, Chap. 4Pankov, R., Yamada, K.M., Fibronectin at a glance (2002) Journal of Cell Science, 115 (20), pp. 3861-3863. , DOI 10.1242/jcs.0005

Multimodal Nonlinear Optical Microscopy Used To Discriminate Epithelial Ovarian Cancer

We used human specimens of epithelial ovarian cancer (serous type) to test the feasibility of nonlinear imaging as complementary tools for ovarian cancer diagnosis. Classical hematoxylin-and-eosin stained sections were applied to combining two-photon excitation fluorescence (TPEF), second (SHG), and third (THG) harmonic microscopy within the same imaging platform. We show that strong TPEF + SHG + THG signals can be obtained in fixed samples stained with Hematoxylin & Eosin (H&E) stored for a very long time and that H&E staining enhanced the THG signal. We demonstrate using anisotropy and morphological measurements, that SHG and THG of stained optical sections allow reproducible identification of neoplastic features such as architectural alterations of collagen fibrils at different stages of the neoplastic transformation and cellular atypia. Taken together, these results suggest that, with our viable imaging system, we can qualitatively and quantitatively assess endogenous optical biomarkers of the ovarian tissue with SHG and THG microscopy. This imaging capability may prove to be highly valuable in aiding to determine structural changes at the cellular and tissue levels, which may contribute to the development of new diagnostic techniques. © 2011 SPIE-OSA.8086The Society of Photo-Optical Instrumentation Engineers (SPIE),The Optical Society of America(2006) United States Cancer Statistics: 2003 Incidence and Mortality, , U.S. Cancer Statistics Working Group. Atlanta: US Department of Health and Human Services, Centers for Disease Control and Prevention and National Cancer Institute;Roland, I.H., Yang, W.-L., Yang, D.-H., Daly, M.B., Ozols, R.F., Hamilton, T.C., Lynch, H.T., Xu, X.-X., Loss of Surface and Cyst Epithelial Basement Membranes and Preneoplastic Morphologic Changes in Prophylactic Oophorectomies (2003) Cancer, 98 (12), pp. 2607-2623. , DOI 10.1002/cncr.11847Bhoola, S., Hoskins, W.J., Diagnosis and management of epithelial ovarian cancer (2006) Obstetrics and Gynecology, 107 (6), pp. 1399-1410. , DOI 10.1097/01.AOG.0000220516.34053.48, PII 0000625020060600000029Provenzano, P.P., Eliceiri, K.W., Keely, P.J., Multiphoton microscopy and fluorescence lifetime imaging microscopy (FLIM) to monitor metastasis and the tumor microenviroment (2009) Clin. Exp. Metastasis, 26 (4), pp. 357-370Provenzano, P.P., Rueden, C.T., Trier, S.M., Yan, L., Ponik, S.M., Inman, D.R., Keely, P.J., Eliceiri, K.W., Nonlinear optical imaging and spectral-lifetime computacional analysis of endogenous and exogenous fluorophores in breast cancer (2008) J. Biomed. Opt, 13 (3), pp. 1-9Provenzano, P.P., Eliceiri, K.W., Campbell, J.M., Inman, D.R., White, J.G., Keely, P.J., Collagen reorganization at the tumor-stromal interface facilitates local invasion (2006) BMC Med, 4 (1), pp. 1-16Hooper, S., Marshall, J.F., Sahai, E., Tumor cell migration in three dimensions (2006) Methods in Enzymology, 406, pp. 625-643. , DOI 10.1016/S0076-6879(06)06049-6, PII S0076687906060496, 49, Regulators and Effectors of Small GTPases: Rho FamilySahai, E., Wyckoff, J., Philippar, U., Segall, J.E., Gertler, F., Condeelis, J., Simultaneous imaging of GFP, CFP and collagen in tumors in vivo using multiphoton microscopy (2005) BMC Biotechnol, 5 (14), pp. 1-9Campagnola, P.J., Loew, L.M., Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms (2003) Nature Biotechnology, 21 (11), pp. 1356-1360. , DOI 10.1038/nbt894Plotnikov, S.V., Millard, A.C., Campagnola, P.J., Mohler, W.A., Characterization of the myosin-based source for second-harmonic generation from muscle sarcomeres (2006) Biophys. J, 90 (2), pp. 693-703Campagnola, P.J., Millard, A.C., Terasaki, M., Hoppe, P.E., Malone, C.J., Mohler, W.A., Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues (2002) Biophysical Journal, 82 (1), pp. 493-508Debarre, D., Supatto, W., Pena, A.-M., Fabre, A., Tordjmann, T., Combettes, L., Schanne-Klein, M.-C., Beaurepaire, E., Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy (2006) Nature Methods, 3 (1), pp. 47-53. , DOI 10.1038/nmeth813, PII N813Elie, N., Plancoulaine, B., Signolle, J.-P., Herlin, P., A Simple Way of Quantifying Immunostained Cell Nuclei on the Whole Histologic Section (2003) Cytometry Part A, 56 (1), pp. 37-45Tuer, A., Tokarz, D., Prent, N., Cisek, R., Alami, J., Dumont, D.J., Bakueva, L., Barzda, V., Nonlinear multicontrast microscopy of hematoxylin-and-eosin-stained histological sections (2010) J. Biomed. Opt, 15 (2), pp. 1-9Kirkpatrick, N.D., Brewer, M.A., Utzinger, U., Endogenous optical biomarkers of ovarian cancer evaluated with multiphoton microscopy (2007) Cancer Epidemiology Biomarkers and Prevention, 16 (10), pp. 2048-2057. , http://cebp.aacrjournals.org/cgi/reprint/16/10/2048, DOI 10.1158/1055-9965.EPI-07-0009Scully, R.E., Young, R.H., Clement, P.B., (1998) Atlas of Tumor Pathology: Tumors of the Ovary, Maldeveloped Gonads, Fallopian Tubes and Broad Ligament, , 3rd ed, Washington, D.C.: Armed Forces Institute of PathologyWilliams, R.M., Flesken-Nikitin, A., Ellenson, L.H., Connolly, D.C., Hamilton, T.C., Nikitin, A.Y., Zipfel, W.R., Strategies for high-resolution imaging of epithelial ovarian cancer by laparoscopic nonlinear microscopy (2010) Transl. Oncol, 3 (3), pp. 181-19

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 © 2014 WILEY-VCH Verlag GmbH & Co. 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Combined Nonlinear Laser Imaging (two-photon Excitation Fluorescence, Second And Third-harmonic Generation, And Fluorescence Lifetime Imaging Microscopies) In Ovarian Tumors

We applied Two-photon Excited Fluorescence (TPEF), Second/Third Harmonic Generation (SHG and THG) and Fluorescence Lifetime Imaging (FLIM) Non Linear Optics (NLO) Laser-Scanning Microscopy within the same imaging platform to evaluate their use as a diagnostic tool in ovarian tumors. We assess of applicability of this multimodal approach to perform a pathological evaluation of serous and mucinous tumors in human samples. The combination of TPEF-SHG-THG imaging provided complementary information about the interface epithelium/stromal, such as the transformation of epithelium surface (THG) and the overall fibrillar tissue architecture (SHG). The fact that H&E staining is the standard method used in clinical pathology and that the stored samples are usually fixed makes it important a re-evaluation of these samples with NLO microscopy to compare new results with a library of already existing samples. FLIM, however, depends on the chemical environment around the fluorophors that was completely changed after fixation; therefore it only makes sense in unstained samples. Our FLIM results in unstained samples demonstrate that it is possible to discriminate healthy epithelia from serous or mucinous epithelia. Qualitative and quantitative analysis of the different imaging modalities used showed that multimodal nonlinear microscopy has the potential to differentiate between cancerous and healthy ovarian tissue. © 2012 Copyright Society of Photo-Optical Instrumentation Engineers (SPIE).8226The Society of Photo-Optical Instrumentation Engineers (SPIE),Becker and Hickel GmbH,Boston Electronics Corp.,Carl Zeiss MicroImaging GmbH,Chroma Technologies Corp.Gubbels, J.A.A., Claussen, N., Kapur, A.K., Connor, J.P., Patankar, M.S., The detection, treatment, and biology of epithelial ovarian cancer (2010) J. Ovarian. Res., 3, pp. 1-11Kosaka, N., Ogawa, M., Longmire, M.R., Choyke, P.L., Kobayashi, H., Multi-targeted multi-color in vivo optical imaging in a model of disseminated peritoneal ovarian cancer (2009) J. Biomed. Opt., 14 (1), p. 014023Sheth, R.A., Upadhyay, R., Stangenberg, L., Sheth, R., Weissleder, R., Mahmood, U., Improved detection of ovarian cancer metastases by intraoperative quantitative fluorescence protease imaging in a pre-clinical model (2009) Gynecol. Oncol., 112 (3), pp. 616-622Provenzano, P.P., Eliceiri, K.W., Keely, P.J., Multiphoton microscopy and fluorescence lifetime imaging microscopy (FLIM) to monitor metastasis and the tumor microenviroment (2009) Clin. Exp. Metastasis, 26 (4), pp. 357-370Bird, D.K., Eliceiri, K.W., Fan, C.H., White, J.G., Simultaneous two-photon spectral and lifetime fluorescence microscopy (2004) Appl. Opt., 43 (27), pp. 5173-5182Williams, R.M., Zipfel, W.R., Webb, W.W., Interpreting second-harmonic generation images of collagen I fibrils (2005) Biophys. J., 88 (2), pp. 1377-1386Campagnola, P.J., Dong, C.Y., Second harmonic generation microscopy: Principles and applications to disease diagnosis (2011) Laser. Photonics. Rev., 5 (1), pp. 13-26Débarre, D., Supatto, W., Pena, A.M., Fabre, A., Tordjmann, T., Combettes, L., Schanne-Klein, M.C., Beaurepaire, E., Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy (2006) Nat. Methods, 3 (1), pp. 47-53Tai, S.P., Lee, W.J., Shieh, D.B., Wu, P.C., Huang, H.Y., Yu, C.H., Sun, C.K., In vivo optical biopsy of hamster oral cavity with epi-third-harmonic- generation microscopy (2006) Opt. Express, 14 (13), pp. 6178-6187Cicchi, R., Sestini, S., De Giorgi, V., Carli, P., Massi, D., Pavone, F.S., Basal cell carcinoma imaging and characterization by multiple nonlinear microscopy techniques (2007) Biophys. J. Supplement: S, pp. 157aMeyer, T., Bergner, N., Bielecki, C., Krafft, C., Akimov, D., Romeike, B.F., Reichart, R., Popp, J., Nonlinear microscopy, infrared, and Raman microspectroscopy for brain tumor analysis (2011) J. Biomed. Opt., 16 (2), p. 021113Kirkpatrick, N.D., Brewer, M.A., Utzinger, U., Endogenous optical biomarkers of ovarian cancer evaluated with multiphoton microscopy (2007) Cancer. Epidemiol. Biomarkers. Prev., 16 (10), pp. 2048-2057Nadiarnykh, O., LaComb, R.B., Brewer, M.A., Campagnola, P.J., Alterations of the extracellular matrix in ovarian cancer studied by Second Harmonic Generation imaging microscopy (2010) BMC. Cancer, 10 (94), pp. 1-14Williams, R.M., Flesken-Nikitin, A., Ellenson, L.H., Connolly, D.C., Hamilton, T.C., Nikitin, A.Y., Zipfel, W.R., Strategies for high-resolution imaging of epithelial ovarian cancer by laparoscopic nonlinear microscopy (2010) Transl. Oncol., 3 (3), pp. 181-194Rueden, C.T., Conklin, M.W., Provenzano, P.P., Keely, P.J., Eliceiri, K.W., Nonlinear optical microscopy and computational analysis of intrinsic signatures in breast cancer (2009) Conf. Proc. IEEE Eng. Med. Biol. Soc., 2009, pp. 4077-4080Chernyavskiy, O., Vannucci, L., Bianchini, P., Difato, F., Saieh, M., Kubínová, L., Imaging of mouse experimental melanoma in vivo and ex vivo by combination of confocal and nonlinear microscopy (2009) Microsc. Res. Tech., 72 (6), pp. 411-423Provenzano, P.P., Eliceiri, K.W., Yan, L., Ada-Nguema, A., Conklin, M.W., Inman, D.R., Keely, P.J., Nonlinear optical imaging of cellular processes in breast cancer (2008) Microsc. Microanal., 14 (6), pp. 532-548Adur, J., Pelegati, V.B., Costa, L.F.L., Pietro, L., De Thomaz, A.A., Almeida, D.B., Bottcher-Luiz, F., Cesar, C.L., Recognition of serous ovarian tumors in human samples by multimodal nonlinear optical microscopy (2011) J. Biomed. Opt., 16 (9), p. 096017Cicchi, R., Kapsokalyvas, D., De Giorgi, V., Maio, V., Van Wiechen, A., Massi, D., Lotti, T., Pavone, F.S., Scoring of collagen organization in healthy and diseased human dermis by multiphoton microscopy (2010) J. Biophotonics, 3 (1-2), pp. 34-43Bird, D.K., Yan, L., Vrotsos, K.M., Eliceiri, K.W., Vaughan, E.M., Keely, P.J., White, J.G., Ramanujam, N., Metabolic Mapping of MCF10A Human Breast Cells via Multiphoton Fluorescence Lifetime Imaging of the Coenzyme NADH (2005) Cancer. Res., 65 (19), pp. 8766-8773Skala, M.C., Riching, K.M., Bird, D.K., Gendron-Fitzpatrick, A., Eickhoff, J., Eliceiri, K.W., Keely, P.J., Ramanujam, N., In vivo multiphoton fluorescence lifetime imaging of protein-bound and free nicotinamide adenine dinucleotide in normal and precancerous epithelia (2007) J. Biomed. Opt., 12 (2), p. 02401

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. © 2013 SPIE.8588The Society of Photo-Optical Instrumentation Engineers (SPIE),Becker and Hickel GmbH,Boston Electronics Corporation,Coherent, Inc.,Intelligent Software Solutions, Inc.Jemal, A., Bray, F., Center, M.M., Ferlay, J., Ward, E., Forman, D., Global cancer statistics (2011) C.A. Cancer. J. Clin., 61 (2), pp. 69-90Carriles, R., Schafer, D.N., Sheetz, K.E., Field, J.J., Cisek, R., Barzda, V., Sylvester, A.W., Squier, J.A., Imaging techniques for harmonic and multiphoton absorption fluorescence microscopy (2009) Rev. Sci. Instrum., 80 (8), p. 081101Tadrous, P.J., Methods for imaging the structure and function of living tissues and cells: 2. Fluorescence lifetime imaging (2000) J. Pathol., 191 (3), pp. 229-234Tadrous, P.J., Siegel, J., French, P.M., Shousha, S., Lalani, El.-N., Stamp, G.W., Fluorescence lifetime imaging of unstained tissues: Early results in human breast cancer (2003) J. Pathol., 199 (3), pp. 309-317Meyer, T., Bergner, N., Bielecki, C., Krafft, C., Akimov, D., Romeike, B.F., Reichart, R., Popp, J., Nonlinear microscopy, infrared, and Raman microspectroscopy for brain tumor analysis (2011) J. Biomed. Opt., 16 (2), p. 021113Cicchi, R., Massi, D., Sestini, S., Carli, P., De Giorgi, V., Lotti, T., Pavone, F.S., Basal cell carcinoma imaging and characterization by multiple nonlinear microscopy techniques (2007) Biophys. J. Sup:157AAdur, J., Pelegati, V.B., Costa, L.F., Pietro, L., De Thomaz, A.A., Almeida, D.B., Bottcher-Luiz, F., Cesar, C.L., Recognition of serous ovarian tumors in human samples by multimodal nonlinear optical microscopy (2011) J. Biomed. Opt., 16 (9), p. 096017Adur, J., Pelegati, V.B., De Thomaz, A., D'Souza-Li, L., Assunção, M.C., Bottcher-Luiz, F., Andrade, L.A.L.A., Cesar, C.L., Quantitative changes in human epithelial cancers and osteogenesis imperfecta disease detected using nonlinear multicontrast microscopy (2012) J. Biomed. Opt., 17 (8), p. 081407Skala, M.C., Riching, K.M., Gendron-Fitzpatrick, A., Eickhoff, J., Eliceiri, K.W., White, J.G., Ramanujam, N., In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia (2007) Proc. Natl. Acad. Sci. U S A., 104 (49), pp. 19494-19499Provenzano, P.P., Eliceiri, K.W., Campbell, J.M., Inman, D.R., White, J.G., Keely, P.J., Collagen reorganization at the tumor-stromal interface facilitates local invasion (2006) BMC Medicine, 4 (1), p. 38Skala, M.C., Riching, K.M., Bird, D.K., Gendron-Fitzpatrick, A., Eickhoff, J., Eliceiri, K.W., Keely, P.J., Ramanujam, N., In vivo multiphoton fluorescence lifetime imaging of protein-bound and free nicotinamide adenine dinucleotide in normal and precancerous epithelia (2007) J. Biomed. Opt., 12 (2), p. 024014Schneckenburger, H., Wagner, M., Weber, P., Strauss, W.S., Sailer, R., Autofluorescence lifetime imaging of cultivated cells using a UV picosecond laser diode (2004) J. Fluoresc., 14 (5), pp. 649-654Konig, K., Riemann, I., High-resolution multiphoton tomography of human skin with subcellular spatial resolution and picosecond time resolution (2003) J. Biomed. Opt., 8 (3), pp. 432-439Zhuo, S., Zhu, X., Wu, G., Chen, J., Xie, S., Quantitative biomarkers of colonic dysplasia based on intrinsic second-harmonic generation signal (2011) J. Biomed. Opt., 16 (12), p. 120501Provenzano, P.P., Inman, D.R., Eliceiri, K.W., Trier, S.M., Keely, P.J., Contact guidance mediated threedimensional cell migration is regulated by Rho/ROCK-dependent matrix reorganization (2008) Biophys. J., 95 (11), pp. 5374-538

Quantitative Second-harmonic Generation Imaging To Detect Osteogenesis Imperfecta In Human Skin Samples

Osteogenesis Imperfecta (OI) is a genetic disorder that leads to bone fractures due to mutations in the Col1A1 or Col1A2 genes that affect the primary structure of the collagen I chain with the ultimate outcome in collagen I fibrils that are either reduced in quantity or abnormally organized in the whole body. A quick test screening of the patients would largely reduce the sample number to be studied by the time consuming molecular genetics techniques. For this reason an assessment of the human skin collagen structure by Second Harmonic Generation (SHG) can be used as a screening technique to speed up the correlation of genetics/phenotype/OI types understanding. In the present work we have used quantitative second harmonic generation (SHG) imaging microscopy to investigate the collagen matrix organization of the OI human skin samples comparing with normal control patients. By comparing fibril collagen distribution and spatial organization, we calculated the anisotropy and texture patterns of this structural protein. The analysis of the anisotropy was performed by means of the two-dimensional Discrete Fourier Transform and image pattern analysis with Gray-Level Co-occurrence Matrix (GLCM). From these results, we show that statistically different results are obtained for the normal and disease states of OI. © 2012 Copyright Society of Photo-Optical Instrumentation Engineers (SPIE).8226The Society of Photo-Optical Instrumentation Engineers (SPIE),Becker and Hickel GmbH,Boston Electronics Corp.,Carl Zeiss MicroImaging GmbH,Chroma Technologies Corp.Campagnola, P.J., Dong, C.Y., Second harmonic generation microscopy: Principles and applications to disease diagnosis (2011) Laser Photonics Rev., 5 (1), pp. 13-26Nadiarnykh, O., LaComb, R.B., Brewer, M.A., Campagnola, P.J., Alterations of the extracellular matrix in ovarian cancer studied by second harmonic generation imaging microscopy (2010) BMC Cancer, 10 (94), pp. 1-14Bianchini, P., Diaspro, A., Three-dimensional (3D) backward and forward second harmonic generation (SHG) microscopy of biological tissues (2008) J Biophotonics, 1 (6), pp. 443-450. , DecReviewCampagnola, P.J., Millard, A.C., Terasaki, M., Hoppe, P.E., Malone, C.J., Mohler, W.A., Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues (2002) Biophys. J., 82, pp. 493-508Sillence, D.O., Senn, A., Danks, D.M., Genetic heterogeneity in osteogenesis imperfect (1979) J. Med. Genet., 16 (2), pp. 101-116Gajko-Galicka, Mutations in type I collagen genes resulting in osteogenesis imperfecta in humans (2008) Acta. Biochim. Pol., 49 (2), pp. 433-441Canuto, H.C., Fishbein, K.W., Huang, A., Doty, S.B., Herbert, R.A., Peckham, J., Pleshko, N., Spencer, R.G., Characterization of skin abnormalities in a mouse model of osteogenesis imperfecta using high resolution magnetic resonance imaging and Fourier transform infrared imaging spectroscopy (2011) NMR Biomed., , Aug 15. doi: 10.1002/nbm.1732Lacomb, R., Nadiarnykh, O., Campagnola, P.J., Quantitative second harmonic generation imaging of the diseased state osteogenesis imperfecta: Experiment and simulation (2008) Biophys. J., 94 (11), pp. 4504-4514Adur, J., Pelegati, V.B., Costa, L.F., Pietro, L., De Thomaz, A.A., Almeida, D.B., Bottcher-Luiz, F., Cesar, C.L., Recognition of serous ovarian tumors in human samples by multimodal nonlinear optical microscopy (2011) J. Biomed. Opt., 16 (9), p. 096017Pelegati, V.B., Adur, J., De Thomaz, A.A., Almeida, D.B., Baratt, M.O., Andrade, L.A., Bottcher-Luiz, F., Cesar, C.L., Multimodal Optical Setup for Nonlinear and Fluorescence Lifetime Imaging Microscopies: Improvement a Commercial Confocal Inverted Microscope Microsc. Res. Tech., , SubmitedZhuo, S., Chen, J., Wu, G., Xie, S., Zheng, L., Jiang, X., Zhu, X., Quantitatively linking collagen alteration and epithelial tumor progression by second harmonic generation microscopy (2010) Appl. Phys. Lett., 96 (21), p. 213704Cicchi, R., Kapsokalyvas, D., De Giorgi, V., Maio, V., Van Wiechen, A., Massi, D., Lotti, T., Pavone, F.S., Scoring of collagen organization in healthy and diseased human dermis by multiphoton microscopy (2010) J. Biophotonics, 3 (1-2), pp. 34-43Nadiarnykh, O., Plotnikov, S., Mohler, W.A., Kalajzic, I., Redford-Badwal, D., Campagnola, P.J., Second harmonic generation imaging microscopy studies of osteogenesis imperfecta (2007) J. Biomed. Opt., 12 (5), p. 05180

Multiphoton Intravital Microscopy Setup To Visualize The Mouse Mammary Gland

Recently, light microscopy-based techniques have been extended to live mammalian models leading to the development of a new imaging approach called intravital microscopy (IVM). Although IVM has been introduced at the beginning of the last century, its major advancements have occurred in the last twenty years with the development of non-linear microscopy that has enabled performing deep tissue imaging. IVM has been utilized to address many biological questions in basic research and is now a fundamental tool that provide information on tissues such as morphology, cellular architecture, and metabolic status. IVM has become an indispensable tool in numerous areas. This study presents and describes the practical aspects of IVM necessary to visualize epithelial cells of live mouse mammary gland with multiphoton techniques. © 2013 OSA-SPIE.8797The Optical Society,The Society of Photo-Optical Instrumentation Engineers (SPIE)Amornphimoltham, P., Masedunskas, A., Weigert, R., Intravital microscopy as a tool to study drug delivery in preclinical studies (2011) Adv. Drug. Deliv. Rev., 63, pp. 119-128Andresen, V., Alexander, S., Heupel, W.M., Hirschberg, M., Hoffman, R.M., Friedl, P., Infrared multiphoton microscopy: Subcellular-resolved deep tissue imaging (2009) Curr. Opin. Biotechnol., 20 (1), pp. 54-62Kedrin, D., Gligorijevic, B., Wyckoff, J., Verkhusha, V.V., Condeelis, J., Intravital imaging of metastatic behavior through a mammary imaging window (2008) Nat. Methods., 5 (12), pp. 1019-1021Provenzano, P.P., Eliceiri, K.W., Keely, P.J., Multiphoton microscopy and fluorescence lifetime imaging microscopy (FLIM) to monitor metastasis and the tumor microenvironment (2009) Clin. Exp. Metastasis, 26 (4), pp. 357-370Zipfel, W.R., Williams, R.M., Webb, W.W., Nonlinear magic: Multiphoton microscopy in the biosciences (2003) Nat. Biotechnol., 21 (11), pp. 1369-1377Campagnola, P.J., Loew, L.M., Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms (2003) Nat. Biotechnol., 21 (11), pp. 1356-1360Levitt, J.A., Matthews, D.R., Ameer-Beg, S.M., Suhling, K., Fluorescence lifetime and polarization-resolved imaging in cell biology (2009) Curr. Opin. Biotechnol., 20 (1), pp. 28-36Muller, M., Zumbusch, A., Coherent anti-Stokes Raman scattering microscopy (2007) Chemphyschem, 8 (2), pp. 2156-2170Vakoc, B.J., Lanning, R.M., Tyrrell, J.A., Padera, T.P., Bartlett, L.A., Stylianopoulos, T., Munn, L.L., Bouma, B.E., Three-dimensional microscopy of the tumor microenvironment in vivo using optical frequency domain imaging (2009) Nat. Med, 15 (10), pp. 1219-1223Drew, P.J., Shih, A.Y., Driscoll, J.D., Knutsen, P.M., Blinder, P., Chronic optical access through a polished and reinforced thinned skull (2010) Nat. Methods, 7 (12), pp. 981-984Flusberg, B.A., Nimmerjahn, A., Cocker, E.D., Mukamel, E.A., Barretto, R.P., High-speed, miniaturized fluorescence microscopy in freely moving mice (2008) Nat. Methods., 5 (11), pp. 935-938Looney, M.R., Thornton, E.E., Sen, D., Lamm, W.J., Glenny, R.W., Stabilized imaging of immune surveillance in the mouse lung (2011) Nat. Methods., 8 (1), pp. 91-96Weigert, R., Sramkova, M., Parente, L., Amornphimoltham, P., Masedunskas, A., Intravital microscopy: A novel tool to study cell biology in living animals (2010) Histochem. Cell. Biol., 133 (5), pp. 481-491Cohen, M., Georgiou, M., Stevenson, N.L., Miodownik, M., Baum, B., Dynamic filopodia transmit intermittent Delta-Notch signaling to drive pattern refinement during lateral inhibition (2010) Dev. Cell., 19 (3), pp. 78-89Serrels, A., Timpson, P., Canel, M., Schwarz, J.P., Carragher, N.O., Realtime study of E-cadherin and membrane dynamics in living animals: Implications for disease modeling and drug development (2009) Cancer. Res., 69 (7), pp. 2714-2719Adur Pelegati, J.V.B., De Thomaz, A.A., Baratti, M.O., Andrade, L.A., Second harmonic generation microscopy as a powerful diagnostic imaging modality for human ovarian cancer (2012) J. Biophotonics, , doi: 10.1002/jbio.201200108Adur, J., Pelegati, V.B., De Thomaz, A.A., Baratti, M.O., Almeida, D.B., Optical biomarkers of serous and mucinous human ovarian tumor assessed with nonlinear optics microscopies (2012) PLoS One, 7, pp. e47007. , doi: 10.1371/journal.pone.004700

Second-harmonic Generation Microscopy Used To Evaluate The Effect Of The Dimethyl Sulfoxide In The Cryopreservation Process In Collagen Fibers Of Differentiated Chondrocytes

Cartilaginous lesions are a significant public health problem and the use of adult stem cells represents a promising therapy for this condition. Cryopreservation confers many advantages for practitioners engaged in cell-based therapies. However, conventional slow freezing has always been associated with damage and mortality due to intracellular ice formation, cryoprotectant toxicity, and dehydration. The aim of this work is to observe the effect of the usual Dimethyl Sulfoxide (DMSO) cryopreservation process on the architecture of the collagen fiber network of chondrogenic cells from mesenchymal stem cells by Second Harmonic Generation (SHG) microscopy. To perform this study we used Mesenchymal Stem Cells (MSC) derived from adipose tissue which presents the capacity to differentiate into other lineages such as osteogenic, adipogenic and chondrogenic lineages. Mesenchymal stem cells obtained after liposuction were isolated digested by collagenase type I and characterization was carried out by differentiation of mesodermic lineages, and flow cytometry using specific markers. The isolated MSCs were cryopreserved by the DMSO technique and the chondrogenic differentiation was carried out using the micromass technique. We then compared the cryopreserved vs non-cryopreserved collagen fibers which are naturally formed during the differentiation process. We observed that noncryopreserved MSCs presented a directional trend in the collagen fibers formed which was absent in the cryopreserved MSCs. We confirmed this trend quantitatively by the aspect ratio obtained by Fast Fourier Transform which was 0.76 for cryopreserved and 0.52 for non-cryopreserved MSCs, a statistical significant difference. © 2012 Copyright Society of Photo-Optical Instrumentation Engineers (SPIE).8226The Society of Photo-Optical Instrumentation Engineers (SPIE),Becker and Hickel GmbH,Boston Electronics Corp.,Carl Zeiss MicroImaging GmbH,Chroma Technologies Corp.Hunziker, E.B., Articular cartilage repair: Basic science and clinical progress. A review of the current status and prospects (2002) Osteoarthritis Cartilage, 10, pp. 432-463Liu, Y., Shu, X.Z., Prestwich, G.D., Osteochondral defect repair with autologous bone marrow-derived mesenchymal stem cells in an injectable, in situ, cross-linked synthetic extracellular matrix (2006) Tissue Eng, 12, pp. 3405-3416Buckwalter, J.A., Mankin, H.J., Articular cartilage: Degeneration and osteoarthritis, repair, regeneration, and transplantation (1998) Instr Course Lect, 47, pp. 487-504Knutsen, G., Engebretsen, L., Ludvigsen, T.C., Drogset, J.O., Grontvedt, T., Solheim, E., Strand, T., Johansen, O., Autologous chondrocyte implantation compared with microfracture in the knee. A randomized trial (2004) J Bone Joint Surg Am, 86 A, pp. 455-464Bouwmeester, S.J., Beckers, J.M., Kuijer, R., Van Der Linden, A.J., Bulstra, S.K., Long-term results of rib perichondrial grafts for repair of cartilage defects in the human knee (1997) Int Orthop, 21, pp. 313-317Brittberg, M., Lindahl, A., Nilsson, A., Ohlsson, C., Isaksson, O., Peterson, L., Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation (1994) N Engl J Med, 331, pp. 889-895Ferretti, M., Marra, K.G., Kobayashi, K., Defail, A.J., Chu, C.R., Controlled in vivo degradation of genipin crosslinked polyethylene glycol hydrogels within osteochondral defects (2006) Tissue Eng, 12, pp. 2657-2663Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal, R.K., Douglas, R., Mosca, J.D., Moorman, M.A., Marshak, D.R., Multilineage potential of adult human mesenchymal stem cells (1999) Science, 284, pp. 143-147Nasef, A., Ashammakhi, N., Fouillard, L., Immunomodulatory effect of mesenchymal stromal cells: Possible mechanisms (2008) Regen Med, 3, pp. 531-546Ishige, I., Nagamura-Inoue, T., Honda, M.J., Harnprasopwat, R., Kido, M., Sugimoto, M., Nakauchi, H., Tojo, A., Comparison of mesenchymal stem cells derived from arterial, venous, and Wharton's jelly explants of human umbilical cord (2009) Int J Hematol, 90, pp. 261-269Chamberlain, J., Yamagami, T., Colletti, E., Theise, N.D., Desai, J., Frias, A., Pixley, J., Almeida-Porada, G., Efficient generation of human hepatocytes by the intrahepatic delivery of clonal human mesenchymal stem cells in fetal sheep (2007) Hepatology, 46, pp. 1935-1945Campagnoli, C., Roberts, I.A., Kumar, S., Bennett, P.R., Bellantuono, I., Fisk, N.M., Identification of mesenchymal stem/progenitor cells in human first-trimester fetal blood, liver, and bone marrow (2001) Blood, 98, pp. 2396-2402Erices, A., Conget, P., Minguell, J.J., Mesenchymal progenitor cells in human umbilical cord blood (2000) Br J Haematol, 109, pp. 235-242Mello, M.A., Tuan, R.S., Effects of TGF-beta1 and triiodothyronine on cartilage maturation: In vitro analysis using long-term high-density micromass cultures of chick embryonic limb mesenchymal cells (2006) J Orthop Res, 24, pp. 2095-2105Honorati, M.C., Cattini, L., Facchini, A., VEGF production by osteoarthritic chondrocytes cultured in micromass and stimulated by IL-17 and TNF-alpha (2007) Connect Tissue Res, 48, pp. 239-245Gruber, H.E., Chow, Y., Hoelscher, G.L., Ingram, J.A., Zinchenko, N., Norton, H.J., Sun, Y., Hanley Jr., E.N., Micromass culture of human anulus cells: Morphology and extracellular matrix production (2010) Spine (Phila Pa 1976), 35, pp. 1033-1038Wescoe, K.E., Schugar, R.C., Chu, C.R., Deasy, B.M., The role of the biochemical and biophysical environment in chondrogenic stem cell differentiation assays and cartilage tissue engineering (2008) Cell Biochem Biophys, 52, pp. 85-102Xiang, Y., Zheng, Q., Jia, B., Huang, G., Xie, C., Pan, J., Wang, J., Ex vivo expansion, adipogenesis and neurogenesis of cryopreserved human bone marrow mesenchymal stem cells (2007) Cell Biology International, 31, pp. 444-450Ock, S.A., Rho, G.J., Effect of dimethyl sulfoxide (DMSO) on cryopreservation of porcine mesenchymal stem cells (pMSCs) (2011) Cell Transplant, 20, pp. 1231-1239Fleming, K.K., Hubel, A., Cryopreservation of hematopoietic and non-hematopoietic stem cells (2006) Transfus Apher Sci, 34, pp. 309-315Buchanan, S.S., Gross, S.A., Acker, J.P., Toner, M., Carpenter, J.F., Pyatt, D.W., Cryopreservation of stem cells using trehalose: Evaluation of the method using a human hematopoietic cell line (2004) Stem Cells Dev, 13, pp. 295-305Schenke-Layland, K., Riemann, I., Damour, O., Stock, U.A., Konig, K., Two-photon microscopes and in vivo multiphoton tomographs - Powerful diagnostic tools for tissue engineering and drug delivery (2006) Advanced Drug Delivery Reviews, 58, pp. 878-896Kiviranta, P., Rieppo, J., Korhonen, R.K., Julkunen, P., Toyras, J., Jurvelin, J.S., Collagen network primarily controls Poisson's ratio of bovine articular cartilage in compression (2006) J Orthop Res, 24, pp. 690-699Zhang, Z.X., Guan, L.X., Zhang, K., Wang, S., Cao, P.C., Wang, Y.H., Wang, Z., Dai, L.J., Cytogenetic analysis of human bone marrow-derived mesenchymal stem cells passaged in vitro (2007) Cell Biology International, 31, pp. 645-648Cicchi, R., Kapsokalyvas, D., De Giorgi, V., Maio, V., Van Wiechen, A., Massi, D., Lotti, T., Pavone, F.S., Scoring of collagen organization in healthy and diseased human dermis by multiphoton microscopy (2010) Journal of Biophotonics, 3, pp. 34-4