Name: implementation of a coherent anti-stokes raman scattering (cars) system on a ti:sapphire and opo laser based standard laser scanning microscope
Uploaded: 2016-07-17T14:00:00
Description: Watch this Scientific Journal Video about Implementation of a Coherent Anti-Stokes Raman Scattering CARS System on a Ti:Sapphire and OPO Laser Based Standard Laser Scanning Microscope at JoVE.com

The authors want to thank Dr. Philippe Combette (IES, UM, Montpellier, France) for the loan of the fast oscilloscope and acknowledge financial supports from Montpellier RIO Imaging (MRI). HR acknowledges ANR grants France Bio Imaging (ANR-10-INSB-04-01) and France Life Imaging (ANR-11-INSB-0006) infrastructure networks for coherent Raman imaging developments. This work was mainly supported by an European Research Council grant (FP7-IDEAS-ERC 311610) and an INSERM - AVENIR grant to NT.

<img alt="图1" src="/files/ftp_upload/54262/54262fig1.jpg" /> 图1.一般的建立的示意图它包括钛蓝宝石 （680 - 1080 nm），而OPO（1050 - 1300纳米）激光器，用4个反射镜的延迟线（M 1至M 4），快速示波器，光电二极管和两个固定光阑I 1和I 2。镜M 2和M 3被固定在一个线性平移台使改变延迟线长度用千分尺的分辨率?...

<ol>
	<li>Valeur, B., Berberan-Santos, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Fluorescence: Principles and Applications. , (2012).</li><li>Denk, W., Strickler, J. H., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+laser+scanning+fluorescence+microscopy.">Two-photon laser scanning fluorescence microscopy.</a> Science. 248 (4951), 73-76 (1990).</li><li>Moreaux, L., Sandre, O., Mertz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+imaging+by+second-harmonic+generation+microscopy.">Membrane imaging by second-harmonic generation microscopy.</a> JOSA B. 17 (10), 1685-1694 (2000).</li><li>Zoumi, A., Yeh, A., Tromberg, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+cells+and+extracellular+matrix+in+vivo+by+using+second-harmonic+generation+and+two-photon+excited+fluorescence.">Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence.</a> Proc. Natl. Acad. Sci. USA. 99 (17), 11014-11019 (2002).</li><li>Yelin, D., Silberberg, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+scanning+third-harmonic-generation+microscopy+in+biology.">Laser scanning third-harmonic-generation microscopy in biology.</a> Opt. Express. 5 (8), 169-175 (1999).</li><li>Campagnola, P. J., Millard, A. C., Terasaki, M., Hoppe, P. E., Malone, C. J., Mohler, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+high-resolution+Second-Harmonic+Generation+imaging+of+endogenous+structural+proteins+in+biological+tissues.">Three-dimensional high-resolution Second-Harmonic Generation imaging of endogenous structural proteins in biological tissues.</a> Biophys. J. 81 (1), 493-508 (2002).</li><li>Olivier, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+lineage+reconstruction+of+early+zebrafish+embryos+using+label-free+nonlinear+microscopy.">Cell lineage reconstruction of early zebrafish embryos using label-free nonlinear microscopy.</a> Science. 329 (5994), 967-971 (2010).</li><li>Farrar, M. J., Wise, F. W., Fetcho, J. R., Schaffer, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+myelin+in+the+vertebrate+central+nervous+system+using+third+harmonic+generation+microscopy.">In vivo imaging of myelin in the vertebrate central nervous system using third harmonic generation microscopy.</a> Biophys. J. 100 (5), 1362-1371 (2011).</li><li>Lim, H., Sharoukhov, D., Kassim, L., Zhang, Y., Salzer, J. L., Melendez-Vasquez, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+imaging+of+Schwann+cell+myelination+by+third+harmonic+generation+microscopy.">Label-free imaging of Schwann cell myelination by third harmonic generation microscopy.</a> Proc. Natl. Acad. Sci. U.S.A. 111 (50), 18025-18030 (2014).</li><li>Strupler, M., Pena, A. M., Hernest, M., Tharaux, P. L., Martin, J. L., Beaurepaire, E., Schanne-Klein, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Second+harmonic+imaging+and+scoring+of+collagen+in+fibrotic+tissues.">Second harmonic imaging and scoring of collagen in fibrotic tissues.</a> Opt. Express. 15 (7), 4054-4065 (2007).</li><li>Cheng, J. X., Xie, X. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coherent+anti-Stokes+Raman+scattering+microscopy:+Instrumentation,+theory,+and+applications.">Coherent anti-Stokes Raman scattering microscopy: Instrumentation, theory, and applications.</a> J. Phys. Chem. B. 108 (3), 827-840 (2004).</li><li>Volkmer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vibrational+imaging+and+microspectroscopies+based+on+coherent+anti-Stokes+scattering+microscopy.">Vibrational imaging and microspectroscopies based on coherent anti-Stokes scattering microscopy.</a> J. Phys. D: Appl. Phys. 38, R59-R81 (2005).</li><li>Evans, C. L., Xie, X. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coherent+anti-Stokes+Raman+scattering+microscopy:+chemical+imaging+for+biology+and+medicine.">Coherent anti-Stokes Raman scattering microscopy: chemical imaging for biology and medicine.</a> Annu. Rev. Anal. Chem. 1, 883-909 (2008).</li><li>Mukamel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles of nonlinear optical spectroscopy. , (1995).</li><li>Duncan, M. D., Reintjes, J., Manuccia, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scanning+coherent+anti-Stokes+Raman+microscope.">Scanning coherent anti-Stokes Raman microscope.</a> Opt. Lett. 7 (8), 350-352 (1982).</li><li>Zumbusch, A., Holtom, G. R., Xie, X. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+vibrational+imaging+by+coherent+anti-Stokes+Raman+scattering.">Three-dimensional vibrational imaging by coherent anti-Stokes Raman scattering.</a> Phys. Rev. Lett. 82 (20), 4142-4145 (1999).</li><li>Folick, A., Min, W., Wang, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+imaging+of+lipid+dynamics+using+Coherent+Anti-Stokes+Raman+Scattering+(CARS)+and+Stimulated+Raman+Scattering+(SRS)+microscopy.">Label-free imaging of lipid dynamics using Coherent Anti-Stokes Raman Scattering (CARS) and Stimulated Raman Scattering (SRS) microscopy.</a> Curr. Opin. Genet. Dev. 21 (5), 585-590 (2011).</li><li>Wang, P., Liu, B., Zhang, D., Belew, M. Y., Tissenbaum, H. A., Cheng, J. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+lipid+metabolism+in+live+Caenorhabditis+elegans+using+fingerprint+vibrations.">Imaging lipid metabolism in live Caenorhabditis elegans using fingerprint vibrations.</a> Angew. Chem. Int. Ed. Engl. 53 (44), 11787-11792 (2014).</li><li>Min, W., Freudiger, C. W., Lu, S., Xie, X. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coherent+nonlinear+optical+imaging:+beyond+fluorescence+microscopy.">Coherent nonlinear optical imaging: beyond fluorescence microscopy.</a> Annu. Rev. Phys. Chem. 62, 507-530 (2011).</li><li>Cheng, J. X., Jia, Y. K., Zheng, G., Xie, X. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-scanning+coherent+anti-Stokes+Raman+scattering+microscopy+and+applications+to+cell+biology.">Laser-scanning coherent anti-Stokes Raman scattering microscopy and applications to cell biology.</a> Biophys J. 83 (1), 502-509 (2002).</li><li>Chiu, W. S., Belsey, N. A. N., Garrett, L., Moger, J., Delgado-Charro, M. B., Guy, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+diffusion+in+the+human+nail+measured+by+stimulated+Raman+scattering+microscopy.">Molecular diffusion in the human nail measured by stimulated Raman scattering microscopy.</a> Proc Natl. Acad. Sci. U.S.A. 112, 7725-7730 (2015).</li><li>Chen, X., Gr&#233;goire, S., Formanek, F., Galey, J. -. B., Rigneault, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+3D+molecular+cutaneous+absorption+in+human+skin+using+label+free+nonlinear+microscopy.">Quantitative 3D molecular cutaneous absorption in human skin using label free nonlinear microscopy.</a> J. of Control. Release. 200, 78-86 (2015).</li><li>Kano, H., Hamaguchi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+multi-nonlinear+optical+imaging+of+a+living+cell+using+a+supercontinuum+light+source+generated+from+a+photonic+crystal+fiber.">In vivo multi-nonlinear optical imaging of a living cell using a supercontinuum light source generated from a photonic crystal fiber.</a> Opt. Express. 14 (7), 2798-2804 (2006).</li><li>Brustlein, S., Ferrand, P., Walther, N., Brasselet, S., Billaudeau, C., Marguet, D., Rigneault, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+parametric+oscillator-based+light+source+for+coherent+Raman+scattering+microscopy:+practical+overview.">Optical parametric oscillator-based light source for coherent Raman scattering microscopy: practical overview.</a> J. Biomed. Opt. 16 (2), 021106 (2011).</li><li>Chen, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multimodal+platform+for+nonlinear+optical+microscopy+and+microspectroscopy.">A multimodal platform for nonlinear optical microscopy and microspectroscopy.</a> Opt. Express. 17 (3), 1282-1290 (2009).</li><li>Yue, S., Slipchenko, M. N., Cheng, J. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multimodal+nonlinear+optical+microscopy.">Multimodal nonlinear optical microscopy.</a> Laser Photonics Rev. 5 (4), 496-512 (2011).</li><li>Sun, Q., Li, Y., He, S., Situ, C., Wu, Z., Qu, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+multimodal+nonlinear+optical+microscopy+reveals+fundamental+insights+of+skeletal+muscle+development.">Label-free multimodal nonlinear optical microscopy reveals fundamental insights of skeletal muscle development.</a> Biomed Opt Express. 5 (1), 158-166 (2013).</li><li>Le, T. T., Langohr, I. M., Locker, M. J., Sturek, M., Cheng, J. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+molecular+imaging+of+atherosclerotic+lesions+using+multimodal+nonlinear+optical+microscopy.">Label-free molecular imaging of atherosclerotic lesions using multimodal nonlinear optical microscopy.</a> J. Biomed. Opt. 12 (5), 054007 (2007).</li><li>Ellis, D. I., Cowcher, D. P., Ashton, L., O'Hagana, S., Goodacre, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Illuminating+disease+and+enlightening+biomedicine:+Raman+spectroscopy+as+a+diagnostic+tool.">Illuminating disease and enlightening biomedicine: Raman spectroscopy as a diagnostic tool.</a> Analyst. 138, 3871-3884 (2013).</li><li>Oz&#231;elik, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pals1+is+a+major+regulator+of+the+epithelial-like+polarization+and+the+extension+of+the+myelin+sheath+in+peripheral+nerves.">Pals1 is a major regulator of the epithelial-like polarization and the extension of the myelin sheath in peripheral nerves.</a> J Neurosci. 30 (11), 4120-4131 (2010).</li><li>Heinrich, C., Hofer, A., Ritsch, A., Ciardi, C., Bernet, S., Ritsch-Marte, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+imaging+of+saturated+and+unsaturated+lipids+by+wide-field+CARS-microscopy.">Selective imaging of saturated and unsaturated lipids by wide-field CARS-microscopy.</a> Opt. Express. 16 (4), 2699-2708 (2008).</li><li>Kyriakidis, N. B., Skarkalis, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+spectra+measurement+of+olive+oil+and+other+vegetable+oils.">Fluorescence spectra measurement of olive oil and other vegetable oils.</a> J. AOAC Int. 83 (6), 1435-1439 (2000).</li><li>King, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscopic+anatomy:+normal+structure.">Microscopic anatomy: normal structure.</a> Handb. Clin. Neurol. 115, 7-27 (2013).</li><li>Monsma, P. C., Brown, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FluoroMyelin+Red+is+a+bright,+photostable+and+non-toxic+fluorescent+stain+for+live+imaging+of+myelin.">FluoroMyelin Red is a bright, photostable and non-toxic fluorescent stain for live imaging of myelin.</a> J. Neurosci. Methods. 209 (2), 344-350 (2012).</li><li>Wang, H., Fu, Y., Zickmund, P., Shi, R., Cheng, J. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coherent+anti-stokes+Raman+scattering+imaging+of+axonal+myelin+in+live+spinal+tissues.">Coherent anti-stokes Raman scattering imaging of axonal myelin in live spinal tissues.</a> Biophys. J. 89 (1), 581-591 (2005).</li><li>Wang, H. W., Fu, Y., Huff, T. B., Le, T. T., Wang, H., Cheng, J. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chasing+lipids+in+health+and+diseases+by+coherent+anti-Stokes+Raman+scattering+microscopy.">Chasing lipids in health and diseases by coherent anti-Stokes Raman scattering microscopy.</a> Vib. Spectrosc. 50 (1), 160-167 (2009).</li><li>Jung, Y., Tam, J., Jalian, H. R., Anderson, R. R., Evans, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal,+3D+in+vivo+imaging+of+sebaceous+glands+by+coherent+anti-stokes+Raman+scattering+microscopy:+normal+function+and+response+to+cryotherapy.">Longitudinal, 3D in vivo imaging of sebaceous glands by coherent anti-stokes Raman scattering microscopy: normal function and response to cryotherapy.</a> J. Invest. Dermatol. 135 (1), 39-44 (2015).</li></ol>

工作中最具挑战性的部分是激光束的时间同步。它需要一个快速光电二极管具有快速示波器组合，但是可以在第一执行只在时间粗略重叠。然后几个厘米的进一步调整是必需的。最后，由线性平移平台微米移动以触发CARS信号允许执行延迟线长度的最终微调。这个信号被维持在一个窄的范围内大约20微米，通过调整平移台测微驱动器作为观察。它对应于约一半的脉冲持续时间， 即，140 FSEC的?...

光学显微镜已经成为在活生物系统具有亚细胞分辨率的动态过程的无损可视的主要技术。荧光显微镜当前在活细胞中使用的最流行 ​​的成像造影由于其高特异性和灵敏度1。荧光探针的大型面板已经出现了（外源性色素，基因编码的蛋白质，半导体纳米粒子）。各种样品照明基于荧光的技术已经蓬勃发展（如共焦或双光子显微镜）来执行3D成像，并减少该技术被漂白2的主要缺点。其它限制包括荧光团标记的要求，因为大多数分子种类并不固有荧光，因此，这些荧光团必须被成像样品中人为地引入的。这种人为操纵可能是破坏性尤其是小分子或诱导锅无穷区间的光毒性。这些原因使得荧光显微镜不能很好地适用于体内的观察。因此，在不使用荧光分子的使用的具有高灵敏度和特异性的分子的对比光学成像技术是在生物医学科学高度期望的。 无标签或染色的几个非线性光学成像技术已经出现，包括二次谐波产生（SHG）3,4和三次谐波产生（THG）5。 SHG显微镜已被用于图像的结构安排在超分子水平，如微管或胶原6。 THG从光学不均匀性产生的诸如水性介质和脂质7之间的界面。 THG也证明图像髓鞘8,9。这两种技术都可以在双光子荧光显微镜来实现，并且只需要一个激光束。不过，他们需要高功率激光强度（一般为50毫瓦对于SHG 10,25 860纳米- 50毫瓦对于THG 9 1180毫微米），它是活样品中的有害，并且不提供其需要明确图像特定生物结构的化学特异性。 化学选择性成像可以基于拉曼散射固有分子振动信号来获得。当一束光事项命中，光子可以被吸收并通过原子或分子散射。大部分的散射光子将具有相同的能量， 即，频率，作为入射的光子。这个过程被称为瑞利散射。然而，光子少数将分散在从入射光子， 即频率不同的光频率，与称为拉曼散射的非弹性散射过程。的能量差，从取决于分子结构和环境振动模式激发起源。因此，自发拉曼散射省化学的IDE选择性成像不同的分子具有特定的振动频率。然而，它是因为它极其微弱信号的限制。共焦拉曼显微镜已经开发，并提供了三维空间分辨率，但它需要高平均功率和长采集时间11。为了克服这些困难，最近在激光技术的进步已经允许非线性光学振动显微镜的上升，特别是相干反斯托克斯拉曼散射（CARS）11,12,13。 CARS是一个三阶非线性光学过程。三个激光束，在频率ωP上泵浦光束组成，在频率ωS A斯托克斯光束和一个探测光束（通常是泵）被聚焦在样品中，并产生在频率ω 的AS =反司托克斯束（ 2ω 对 - ωS）14。在反司托克斯信号可以显著增强时的频率差（ - ω 小号 ωP）的泵和斯托克斯光束被调谐到的拉曼分子振动ΩR =之间。 CARS信号是基于多光子相互作用。因此，它产生要比自发拉曼散射更强的相干信号的订单。 CARS显微镜首先通过实验邓肯等人 15表明。 Zumbusch 等人则改善的技术中，通过使用两个聚焦的近红外飞秒激光束具有高数值孔径的物镜，从而使轿厢的相位匹配条件，并避免了双光子非共振背景16。因此CARS显微镜已作为活细胞和组织成像的有力工具，通过在活细胞19,20化学检测分子，例如脂质（经由CH伸缩振动）17,18，水（经由OH拉伸振动），蛋白质，DNA而且氘代化合物S表示药物21和化妆品应用22。 非线性显微镜的主要限制从复杂性和光学源的成本起源。一个CARS系统需要短脉冲持续时间，并与时间和空间的同步脉冲串双波长可调谐激光器。早期的汽车显微镜是基于两个同步皮秒钛宝石激光器20。蓝宝石激光器产生超连续谱光源23：也从单一的飞秒钛获得CARS成像。最近，一个单一的飞秒钛组成的激光源：蓝宝石激光器泵浦的可调光参量振荡器（OPO）已用于CARS显微镜。这种设置允许内在时间上同步梁频率的泵和斯托克斯光束覆盖整个分子振动光谱24之间的差。此外，根据唱机激光扫描显微镜关键飞秒激光和OPO，主要用于双光子荧光（TPF）现在可用于非物理学家。这样调校的电​​位，可大大提高，而不需要通过其他的非线性光学成像的掺入补充投资，因为每个非线性（NLO）成像模态是将特定结构或分子敏感。因此，多模态的非线性光学成像大写非线性光学显微镜对复杂生物样品25的潜力。这些技术的耦合允许的许多生物的问题调查，特别是对脂质代谢，皮肤或癌症的发展26，骨骼肌发育27，动脉粥样硬化病变28。而且，随着CARS激光束扫描的实现提供高速率成像， 即一个有吸引力的工具来研究体内动力学过程的能力。 这个工作的目的是，以显示每个步骤来实现吨在一个标准的多光子激光扫描显微镜他CARS技术。显微镜是基于一个FSEC钛蓝宝石激光器和OPO（由钛泵浦：蓝宝石激光器）通过生物学家软件操作。通过调整，以便在时间上两个光束同步的激光束路径中的一个的长度进行积分。我们描述了一步一步的实现这种技术只要求在实验光学的基本背景。我们还说明了对啮齿动物的坐骨神经的髓磷脂鞘得到CARS成像，并且我们显示可以与其它的非线性光学成像同时执行这种成像，如标准双光子荧光技术和二次谐波产生。

<table border="0" cellpadding="0" cellspacing="0" width="1157">
	<tbody>
		<tr height="23">
			<td height="23" style="height:23px;width:419px;">Oscilloscope</td>
			<td style="width:120px;">Tektronix</td>
			<td style="width:317px;">TDS 520D&#160;</td>
			<td style="width:301px;">500 MHz&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:419px;">Photodetector</td>
			<td style="width:120px;">Thorlabs</td>
			<td>DET08C/M, T4290</td>
			<td>5 GHz InGaAs, 800-1700 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Ti:Sapphire laser Chameleon Ultra Family II</td>
			<td style="width:120px;">Coherent</td>
			<td style="width:317px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Optical parametric oscillator OPO Compact Family</td>
			<td style="width:120px;">APE Berlin</td>
			<td style="width:317px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Axio Examiner microscope LSM 7 MP</td>
			<td style="width:120px;">Carl Zeiss</td>
			<td style="width:317px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Motorized periscope</td>
			<td style="width:120px;">Newport</td>
			<td style="width:317px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:419px;">Objective W Plan-Apochromat 20x/1.0</td>
			<td style="width:120px;">Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Beam combiner</td>
			<td style="width:120px;">Carl Zeiss</td>
			<td style="width:317px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Acousto-optic modulator</td>
			<td style="width:120px;">Carl Zeiss</td>
			<td style="width:317px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">OPO power attenuator</td>
			<td style="width:120px;">Carl Zeiss</td>
			<td style="width:317px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Photomultiplier tube</td>
			<td style="width:120px;">Carl Zeiss</td>
			<td style="width:317px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">ZEN software</td>
			<td style="width:120px;">Carl Zeiss</td>
			<td style="width:317px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Bandpass filters</td>
			<td style="width:120px;">Carl Zeiss</td>
			<td>LSM BiG 1935-176</td>
			<td>400-480 nm ; 500-550 nm ; 465-610 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Dichroic mirror</td>
			<td style="width:120px;">Carl Zeiss</td>
			<td></td>
			<td>Cutoff wavelength 760 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Silver mirrors</td>
			<td style="width:120px;">Newport</td>
			<td>10D20ER.2&#160;</td>
			<td>&#955;/10, 480-20,000 nm , Quantity 4</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:419px;">Single-axis translation stage with standard micrometer</td>
			<td style="width:120px;">Thorlabs</td>
			<td style="width:317px;">PT1/M</td>
			<td>Quantity 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Aluminium breadboard</td>
			<td style="width:120px;">Thorlabs</td>
			<td>MB1015/M&#160;</td>
			<td>Quantity 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Mirror mount</td>
			<td style="width:120px;">Thorlabs</td>
			<td>KMSS/M&#160;</td>
			<td>Quantity 4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mirror holder for &#216;1&#34; Optics&#160;</td>
			<td style="width:120px;">Thorlabs</td>
			<td>MH25</td>
			<td>Quantity 4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Iris diaphragms&#160;</td>
			<td style="width:120px;">Thorlabs</td>
			<td>ID8/M</td>
			<td>Quantity 3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protective box</td>
			<td style="width:120px;">Thorlabs</td>
			<td>TB4, XE25L900/M, T205-1.0, RM1S</td>
			<td>Quantity 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Optical posts</td>
			<td style="width:120px;">Thorlabs</td>
			<td>TR40/M, PH50/M, PH75/M, BA2/M</td>
			<td>Quantity 8 (lengths depending on the set-up)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">661-690 nm bandpass filter</td>
			<td>Semrock</td>
			<td>676/29 nm BrightLine&#174; single-band bandpass filter</td>
			<td>Quantity 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Fluorescent beads</td>
			<td style="width:120px;">ThermoFisher</td>
			<td>TetraSpeck&#8482; Fluorescent Microspheres Size Kit</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Laser viewing card</td>
			<td style="width:120px;">Thorlabs</td>
			<td style="width:317px;"></td>
			<td>IR laser viewing card</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Laser safety glass</td>
			<td style="width:120px;">Newport</td>
			<td style="width:317px;">LV-F22.P5L07&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">FluoroMyelin&#8482; Red Fluorescent Myelin Stain&#160;</td>
			<td style="width:120px;">ThermoFisher</td>
			<td>F34652</td>
			<td></td>
		</tr>
	</tbody>
</table>

implementation of a coherent anti-stokes raman scattering (cars) system on a ti:sapphire and opo laser based standard laser scanning microscope

激光扫描显微镜结合飞秒钛宝石激光器和光学参量振荡器（OPO）复制激光线已经成为可供生物学家。这些系统的设计主要是用于多通道的双光子荧光显微镜。然而，在没有任何修改，补充非线性光学显微镜如二次谐波产生（SHG）或第三谐波（THG）也可以使用这组时执行，使构成分子或含水中期的无标记成像脂质接口。这些技术非常适合于在生物体内的观察，但在化学特异性有限。化学选择性成像可以基于拉曼散射固有振动信号来获得。共焦显微拉曼光谱提供了三维空间分辨率，但它需要高平均功率，长采集时间。为了克服这些困难，最近在激光技术的进步已经允许DEVEL非线性光学振动显微镜opment，特别是相干反斯托克斯拉曼散射（CARS）。因此CARS显微镜已成为生物和活细胞成像的有力工具，通过化学映射脂质（通过CH伸缩振动），水（经由OH拉伸振动），蛋白质或DNA。在这项工作中，我们描述了CARS技术在一个标准的OPO耦合多光子激光扫描显微镜的执行。它是基于通过调节激光束的路径中的一个的长度的两个激光线的在时间同步。我们目前现有的多光子系统上一步一步实现这个技术的。在实验光学的基本背景是有益的，所提出的系统不需要昂贵的配套设备。我们还说明CARS成像于啮齿动物的坐骨神经的髓磷脂鞘获得，并且我们表明，该成像可以同时与其他的非线性光学成像，如标准的T进行WO光子荧光技术和二次谐波产生。

<html>标准的Ti的脉冲串频率：蓝宝石激光器通常约为80兆赫。 OPO的具有相同的频率，因为它是由钛泵浦：蓝宝石激光器。因此，至少有200 MHz的快速示波器是必需的。范围内快速光电二极管，也需要600至1100纳米。蓝宝石和OPO信号被移位的1 /（2×80×10 6）= 6.2纳秒：若Ti发生的最大时移。它对应于1.9微米的最大光束路径移图2示出了从OPO激光束（A）的记录脉...

Watch this Scientific Journal Video about Implementation of a Coherent Anti-Stokes Raman Scattering CARS System on a Ti:Sapphire and OPO Laser Based Standard Laser Scanning Microscope at JoVE.com

Implementation of a Coherent Anti-Stokes Raman Scattering CARS System on a Ti:Sapphire and OPO Laser Based Standard Laser Scanning Microscope

根据分子键固有振动相干反斯托克斯拉曼散射（CARS）显微允许无标记化学选择性活细胞成像。这项工作提出了一种基于飞秒钛互补显微技术在标准的多光子激光扫描显微镜实现：蓝宝石激光器和激光OPO。

蓝宝石OPO基于激光的标准激光扫描显微镜：在TI相干反斯托克斯拉曼散射（CARS）系统的实现

Laser scanning microscopes combining a femtosecond Ti:sapphire laser and an optical parametric oscillator (OPO) to duplicate the laser line have become ...

The overall goal of this procedure is to implement a complimentary microscopy technique called CARS, which allows label-free, chemically selective, lifestyle imaging on a standard multi-photon laser scanning microscope based on a femto-second titanium-sapphire laser and an optical parameter oscillator. This method can help answer key questions in the field of biology or neuroscience such as understanding of lipid-related diseases, skin penetration mechanisms, or skin disorders. The main advantage of this technique is that it's easy to implement on a standard laser scanning microscopy based on Ti:Sapphire and OPO lasers.Demonstrating the procedure will be Hassan Boukhaddaoui, research associate and head of the imaging platform of the laboratory and Vasyl Mytskaniuk. Turn on the titanium-sapphire and OPO lasers as presented in the text protocol. Switch on the microscope computer.Turn on the microscope component switches. Start the software by double-clicking the icon on the desktop and set it up according to the text protocol. Then, place a dichroic mirror with a cutoff wavelength at 760 nanometers in the side port slider.Locate it in the infinity space above the objective nose piece. Set the narrow band pass filter and the reflector cube in front of PMT1 to record only the CARS signal at 670 nanometers to reproduce the presented results. Then, place specific narrow band filters in front of PMT3 for fluorescence observation of the myelin, and in front of PMT4 for SHG observation.In the software, set the signal on the detector with an ad hoc band pass filter. Open the light path tool in the setup manager menu in the acquisition tab, then activate the desired PMT and select a color for the channel. For example, use green for CARS, and magenta for SHG.The two beams that originate from the same titanium sapphire laser are not in sync when they reach the microscope because the OPO beam is delayed when it is generated. Follow this procedure to resynchronize them by adjusting the length of one of the laser beam pads by incorporating a delay line. The use of a fast photo diode and a fast oscilloscope is required.First, using BNC cables, connect the input channel CH1 of the oscilloscope to the electrical BNC laser output sync out. Next, connect the input channel CH2 to the photo diode. Then, choose the CH1 channel as the trigger by pressing trigger menu, then the main menu button source and then the side menu button that corresponds to the channel selected as CH1.Now position and fix with optical mounting posts to the photo diode in the focal plane of a 10x air microscope objective. In the Channels tool, define the titanium-sapphire laser wavelength at 830 nanometers at low power, which is less than 1%of the full power. In the Acquisition Mode tool, reduce the scan area to one point in order to illuminate the photo diode with the tiniest beam.Then switch on the laser scan by clicking on the continuous button. Press Autoset on the oscilloscope and manually move the photo diode to get a pulse train on the oscilloscope screen. Press the run/stop button to freeze the display.Next, switch off the titanium-sapphire laser scan. In the software, click the Channels tool, deselect the 830 nanometer laser and set the OPO signal to 1107 nanometers and low power. Then switch on the OPO laser scan, record the pulse trains of the OPO laser on the oscilloscope and switch off the OPO laser scan.Now, compare the temporal shift between the titanium-sapphire and the OPO signals. Calculate the length of the delay line, which will be used to position the mirrors. Before proceeding, put on safety goggles and remove any chain bracelets or watches from wrists.Now, open the laser titanium-sapphire line where the delay line will be implemented by removing the protective tubes. Then select a wavelength in the visible range in order to be able to easily observe the laser beam, for example, 700 nanometers at low power, and switch on the laser scan. Now put two iris diaphragms into the open laser line using the optical mounting posts.Position one iris at the exit of the delay line and place the other iris at the entrance of the periscope. Next, decrease the iris diaphragm aperture and adjust the diaphragm positions to fit the laser beam path then fix them on the optical table. Finally, adjust the vertical position of a third mobile iris diaphragm.The size diaphragms will serve as a control for the realignment procedure to check the position of the laser beam while successively positioning the four mirrors of the delay line. Now, place the mirror M1 on a compact kinematic mirror mount at the entrance of the delay line, and adjust its position and its orientation to maintain the beam height with the use of the mobile iris diaphragm. Then place mirrors M2 and M3 at 90 degrees onto the translation stage according to the calculated delay line length and adjust their orientation using the mobile iris diaphragm.Set M4 at the exit f the delay line just before the iris and carefully adjust its position and angle to fit the laser beam path through the two fixed iris diaphragms. Now position the laser viewing card at the output of the microscope objective and check the laser beam profile. If necessary, slightly adjust the orientation of M4 to observe a uniform bright disc.Lastly, reposition the fast photo diode under the laser beam in the sample focus plane. Then observe the temporal shift between the titanium-sapphire laser beam and the OPO beam on the oscilloscope. If necessary, change the delay line length by moving the whole translation stage to synchronize the pulses.The spacial overlap of the two beams needed for a CARS signal is obtained by visualizing fluorescent polystyrene beads fixed to a microscope slide. Focus on the beads using a 20x water objective. Now, in the Channels tool of the acquisition tab, add track one, or use the existing track.Select the wavelength at 830 nanometers and low power for the titanium-sapphire laser beam. Toggle the color to green in the track one box from the Channels window, and the PMT3 or the PMT4 box from the light path window. Next, add track two for the OPO laser beam and set the wavelength at 1107 nanometers and low power.Toggle the color to red in the track two box from the Channels window and in the PMT3 or the PMT4 box from the light path window. Now, select the maximum scan area and adjust the gain of both tracks to 600. Then sequentially apply the scan of the two beams onto the sample by clicking on continuous.Observe the image in the screen area in the 2D view. Increase the power of both lasers. If necessary, slightly move the focusing drive to find the focus plane of the beads.Finally, adjust the crop and zoom the image in on a single bead or on a group of adjacent beads. Next, use the periscope controller to overlap the beams in the XY plane. In the software, open the Maintain tab.Click on the system options and display the motorized periscope tool window. Further details are discussed in the text protocol. After overlapping images from both laser beams, synchronization is achieved.To achieve the precise temporal adjustment to activate CARS, prepare a glass slide with a droplet of olive oil and cover slip. Then using a 20x water immersion objective, focus on the edge of the cover slip. Now, on track one, set the wavelength to 830 nanometrs for the titanium-sapphire laser beam and at 1107 nanometers for the OPO.Toggle both lasers in track one to get a simultaneous scan. Start with both lasers at low power. Next, on the light path window, uncheck PMT4 and select PMT1.Then choose the maximum scan area and switch on the laser scans by clicking on the continuous button. Adjust the gain to 600 and, if necessary, increase the power to both lasers. Next, adjust the display intensity in the display view option control block.Then slowly move the translation stage of the delay line until the signal becomes significantly enhanced. Next, decrease the power to both lasers. Now, check whether the signal is a CARS signal by alternatively switching off one of the two lasers.If the signal intensity becomes weaker or disappears, a CARS signal has been obtained. Since the final system is dedicated to non-physicists, enclose the light path of the delay line with an enclosure box and tubes to avoid direct access to a harmful, nonvisible, high peak powered laser beam. However, do provide access to the translation stage knob.A fluro-myelin red dye, which has selectivity for myelin, was used to observe the myelin sheath. Simultaneous illumination at 830 and at 1095 nanometers provides a CARS signal. The circles correspond to the myelin sheath surround axons in a transversal cut.The same structure is found while overlapping CARS and fluorescence images. The level of detail of CARS and fluorescent labeling is very similar. Potentially obviating the need for dye use.The system can also simultaneously generate a second harmonic signal from the collagen fibers at the outer service of the sciatic nerves. A different detector is used to record a signal at 550 nanometers since a narrow band pass filter at 670 nanometers is used for the CARS signal recording. The fibers are illustrated by a false magenta color.The CARS signal alone clearly shows the myelin sheaths. By simultaneously visualizing both signals, the myelin sheaths in green are seen surrounded by collagen fibers in magenta. A CARS signal requires less energy than SHG or THG signals.CARS signals were achieved with four milliWatts at 830 nanometers and 13 milliWatts at 1095 nanometers, while 50 milliWatts at 1095 nanometers was required to obtain an SHG signal. After watching this video, you should have a good understanding how to modify step by step one of the laser beam paths to temporally synchronize both laser beams. The implementation of the delay line can be achieved in a few weeks by biologists with basic background in experimental optics or in collaboration with physicists.Don't forget that working with high power, invisible laser beams can be extremely hazardous. And precautions, like wearing goggles while performing the procedure and final enclosure of the laser beam path should be taken.

Research

JoVE Journal

Biology

Implementation of a Coherent Anti-Stokes Raman Scattering (CARS) System on a Ti:Sapphire and OPO Laser Based Standard Laser Scanning Microscope

Laser Capture Microdissection of Mammalian Tissue

激光捕获显微切割哺乳动物组织

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or ...

科研

生物学

Laser-Induced Chronic Ocular Hypertension Model on SD Rats

激光诱导SD大鼠慢性高眼压高压模型

Glaucoma is one of the major causes of blindness in the world. Elevated intraocular pressure is a major risk factor. Laser photocoagulation induced ocular ...

Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering (CARS)

Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering CARS

差分成像生物结构与双共振相干反斯托克斯拉曼散射（CARS）

Coherent Raman imaging techniques have seen a dramatic increase in activity over the past decade due to their promise to enable label-free optical imaging ...

Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

天堂斑马鱼的细胞标记与激光Uncagable荧光葡聚糖

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated ...

Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration

激光烧蚀斑马鱼Pronephros研究肾小管上皮再生

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in ...

Dithranol as a Matrix for Matrix Assisted Laser Desorption/Ionization Imaging on a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

地蒽酚作为傅立叶的基质辅助激光解吸/离子化成像矩阵变换离子回旋共振质谱仪

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly ...

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

使用刺激拉曼散射显微镜对 卡诺哈布迪炎电子人 中的脂质存储动力学进行无标签成像

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits ...

Confocal Laser Scanning Microscopy of Calcium Dynamics in Acute Mouse Pancreatic Tissue Slices

急性小鼠胰腺组织切片钙动力学的共聚焦激光扫描显微镜

The acute mouse pancreatic tissue slice is a unique in situ preparation with preserved intercellular communication and tissue architecture that entails ...

Rapid Antimicrobial Susceptibility Testing by Stimulated Raman Scattering Imaging of Deuterium Incorporation in a Single Bacterium

通过受激拉曼散射成像对氘掺入单个细菌进行快速抗菌药敏试验

To slow and prevent the spread of antimicrobial resistant infections, rapid antimicrobial susceptibility testing (AST) is in urgent need to ...

Laser Microdissection for Species-Agnostic Single-Tissue Applications

激光显微切割，用于与物种无关的单组织应用

Single-cell methodologies have revolutionized the analysis of the transcriptomes of specific cell types. However, they often require species-specific ...