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。 660 - 685 nm的带通滤波器被定位在用于CARS成像的光电倍增管（PMT）的前面<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54262/54262fig1large.jpg" target="_blank">，请点击此处查看该图的放大版本。</a> 1.激光系统的启动<ol><li>验证该钛蓝宝石波长设定到800nm​​或定义此波长的钛宝石电源控制器。打开从待机状态的关键在于在Ti切换:蓝宝石激光器。 </li><li>打开OPO激光在OPO控制器的背面，并打开钛:在Ti蓝宝石快门:蓝宝石电源控制器。 </li><li>切换平板电脑上打气的OPO。点击平板电脑上的OPO连接和远程连接的图标。等待30 - 40分钟的热身。 </li><li>切换显微镜计算机上打开"显微镜组件"开关。通过双击启动该软件在桌面上的图标。 </li><li>进入软件采购选项卡，在安装管理器打开激光工具从软件操作两个激光器。选择钛宝石激光器在和OPO激光开 。检查光学激光功率值（在800 nm的3700毫瓦的典型值，700兆瓦在1000纳米）。 </li><li>配置的光束路径和激光器，打开设置管理器工具组在光路中的工具和复选框第一光电倍增管 （PMT）。 </li><li>要检查钛:在目标的输出蓝宝石激光光斑，打开采集参数工具组中的通道工具 。选择钛低值（约1％）蓝宝石功率，降低增益为0（需要在这个阶段没有图像），并单击连续按钮开始扫描过程，通过显微镜物镜发射激光束。通过在空气显微镜物镜（10X）的输出处的IR激光器的观看卡定位检查红斑通过直接观察的存在。 </li><li>要检查OPO激光光斑，停止钛的扫描:通过点击停止按钮蓝宝石激光器。在该通道窗口低值选择OPO电源 ，然后单击在Continuous按钮。 </li></ol> 2.显微镜设置<ol><li>手动放置分色镜与在目标管口上方的无限空间的侧口滑块760纳米的截止波长，以向上以从样品760纳米发射的光进入在非退检测（NDD）模式的光电倍增管。 </li><li>设置在660窄带通滤波器 - 685纳米的NDD反射立方体在PMT1面前只有CARS在670 nm信号重现这部作品呈现的结果进行记录。 </li><li>放置一个窄带滤波器500至在PMT3的前面为髓鞘的荧光观察的NDD反射立方体550纳米。放置一个窄带滤波器范围从565到在PMT4面前的SHG观察反光镜立方体610纳米。 </li><li>要在软件中选择与专案带通滤波器在探测器上的信号的记录，打开设置管理器菜单 光路径工具 </STRONG&gt;在取样选项卡 。激活所需PMT（复选框），然后选择此通道的颜色。在这项工作中，被选为汽车，红色荧光和品红色的SHG绿色。 </li></ol> 3.时间同步注意:两个激光束从相同的钛起源:当产生它，以便当它们到达显微镜的两束光束在时间上不同步的蓝宝石激光，但OPO光束被延迟。这里的目标是延迟两个光束之一将它们在时间上它们到达显微镜之前重新同步。 <ol><li>与BNC电缆连接示波器电BNC激光输出（同步，输出）的输入通道CH1。连接输入通道示波器光电二极管的CH2，按触发菜单 ，然后在主菜单按钮源，然后对应于选定的C通道侧面菜单按钮，选择CH1通道作为触发通道H1。 </li><li>位置和除去目标后，用光学安装柱固定在空气显微镜物镜（10X），或在显微镜的光束路径的焦平面的光电二极管。注意:如果需要，除去冷凝器及其载体。 </li><li>在渠道工具（ 采集参数工具组），定义了钛在低功率830 nm的蓝宝石激光波长（即全功率小于1％）。在采集模式工具 ，扫描面积减少到一个点，以照亮与最小的光束的光电二极管。通过点击按钮连续开关上的激光扫描。 </li><li>示波器前面板上按自动设置和手动移动光电二极管的位置，以获得屏幕上的脉冲串。按RUN / STOP按钮冻结显示。 <ol><li>要保存示波器显示的副本，插入一个3.5寸软盘软盘驱动器或连接后面板到计算机上的GPIB端口。然后按住Shift键HARDCOPY MENU，按FORMAT（主）选择TIFF图像格式，并在端口菜单中指定的输出通道。按HARDCOPY键录制钛的脉冲序列的示波器显示:蓝宝石激光器。 </li></ol></li><li>通过点击停止按钮蓝宝石激光扫描:关断Ti。通过点击工具通道在定义纳米1107，低功耗的OPO信号。交换机上的OPO激光扫描并记录示波器上的OPO激光的脉冲序列。切断激光OPO扫描。 </li><li>比较钛之间的时间转变:OPO信号蓝宝石和。 注:时移牛逼转移给它具有以下公式要实现延时线L 延迟线的长度:L 延迟线 = C5;吨移位其中​​c是光的速度。 </li><li>选择激光线之一。 注:在这项工作中，钛宝石激光器线被选中，是因为自由空间是这样的激光线附近使用。此外，这种选择允许实现与可见激光光线的激光线的重新对准。 </li><li>通过在那里的延迟线将实施的位置除去保护管打开激光线。 注意！佩戴合适的护目镜和删除链手镯或手腕上观看。 </li><li>为了选择在可见光范围内的波长，以便能够（在软件的频道的工具 ，例如700纳米，在低功率）容易观察的激光束。交换机上的激光扫描。 </li><li>处，并用光学安装柱沿着开放激光线两条光阑设置。在延迟线路的出口位置的一个光圈，并把其他的虹膜在潜望镜的入口。 注:PE由软件的激光束的入口的角度驾驶到激光扫描显微镜的扫描头两艘机动镜riscope控制。 </li><li>减小可变光阑孔径调整振动板的位置，以适应的激光束的路径。解决这些问题的光学平台。调整第三移动可变光阑的垂直位置，以检查激光光束的高度，同时连续地定位所述延迟线的四个反射镜。 注意:这些光阑将作为展示走的一条路重新调整过程的控制。 </li><li>放置M1装在一个紧凑的运动反射镜的反射镜在延迟线的入口安装（ 如图1），并调整它的位置和它的方向，以保持与使用移动可变光阑的光束的高度。地方反映M2和M3在90°（也安装在紧凑型可调镜座），在其上会被放置在MIDCO翻译阶段URSE。定位它们作为先前计算以适应延迟线长度。 </li><li>调整M2和M3的方向与所用的移动可变光阑。设置的M4在延迟线的出口（也固定在一个紧凑的安装）（仅有虹膜我1之前， 如图1所示），并仔细地调整其位置和角度，通过两个固定光阑以适合的激光束的路径。 </li><li>激光收视卡在显微镜物镜的输出位置，并通过点击连续打开激光扫描检查激光光束轮廓。观察一个统一的光明磁盘。如果需要的话，稍微调整M4的方向。 </li><li>再次位置在显微镜的样品聚焦平面上的激光束下的快速光电二极管。观察钛之间的时间转变:示波器上的OPO光束蓝宝石激光束。 注意:如果需要，通过移动整个系统的M2改变延迟线长度，M3安装在平移台（不改变平移台调谐）到两个脉冲同步。可以根据需要几厘米的变化。 </li></ol> 4.梁的空间重叠注意:为了产生CARS信号时，需要两个激光束的空间重叠。在整个染色用两种不同的荧光染料在同一珠粒两个光束的交替照明可用于指示空间偏移。那么后视镜位置进行微调可以最小化的转变。 <ol><li>使用预安装的荧光微球。或者，如下所述安装悬浮在干净的显微镜载玻片微球: <ol><li>采样之前，混合物（在皮质混合器或通过超声处理）的珠粒溶液以确保珠均匀悬浮。 </li><li>应用5微升珠悬浮液的一个滑的表面，并用吸管尖端传播。等待滴干，然后申请5微升坐骑ING介质，例如甘油，水或浸油以上的珠的干样品。盖上盖玻片样品和密封用快干胶水或熔化的石蜡盖玻片。 </li></ol></li><li>将固定在20X水目标下载玻片荧光聚苯乙烯珠。加水几滴浸泡的目标。 </li><li>为了实现焦点的珠，打开在软件中找到标签，从激光扫描模式切换到与眼睛样品的直接观察，按联机按钮。打开眼工具选择专案过滤器 ，并通过点击图标上的卤素灯切换。 </li><li>手动删除分色镜中的侧孔滑块在无限空间，并使用显微镜的聚焦驱动器通过观察用目镜珠集中样品平面。更换分色镜。 </li><li>在里面定位标签中 ，通过按离线按钮切换到激光扫描方式。转到取样选项卡定义为扫描参数:选择帧大小为512个像素，9个扫描速度，1平均化，8比特的比特深度，并增加扫描区域到最大。 </li><li>在采集卡的渠道工具 ，如果加上尚未创建一个路轨（1）。选择在830nm的波长和低功率的钛蓝宝石激光束勾选颜色为绿色从频道窗口，并在PMT3或从光路窗口的PMT4框轨1框。 </li><li>在采集卡的渠道工具 ，添加第二个轨道（轨道2）。在1107 nm和低功耗的OPO激光束的选择波长。勾选颜色为红色从通道窗口，并从该PMT3盒轨道2盒<strong&gt;的光路窗口。 </li><li>调整两个轨道600的增益。然后，通过点击连续顺序地应用该两个光束的扫描到样品。 </li><li>观察屏幕区域中的图像到二维视图。在显示视图选项控制块，调整显示亮度。 注意:如果需要，稍微移动聚焦驱动找到珠的焦点平面。调整作物和在一个单一的珠子或一组相邻的珠缩放图像。 </li><li>使用潜望镜控制器在xy平面重叠的光束。在软件中，打开维护标签 。点击系统选项并显示电动潜望镜工具窗口。使用钛的潜望镜镜的粗，细的调整:蓝宝石激光束，以便在太空中两个图像同步。 </li><li>对于潜望镜操作，使用前调节杆垂直和塞科次一个用于激光束的水平运动。移动光束与输入镜直到图像略微可见，然后通过点击"输入"和"输出"补偿与潜望镜的输出镜的激光强度。 </li><li>为了垂直地重叠的光束，在保持标签上 ，打开准直器工具和调整的Ti的焦距的值:蓝宝石激光束。 </li><li>轻轻移动目标垂直位置，检查两个图像焦点的差异。或者，拿样品的Z堆栈在收购选项卡中的Z-Stack工具打开和选择不同的参数（范围，片数）。按正交的图像的屏幕区域看到在轴向截面的光束。通过执行相同的步骤数次最大化Z-重叠。 </li></ol> 5.最后的调整和相干反斯托克斯拉曼散射（CARS）信号观察橄榄油博士oplets <ol><li>放橄榄油的液滴在玻璃板上，并用玻璃盖玻片覆盖。加水几滴浸泡20倍水浸泡的目的。集中在通过使用目镜盖玻片的边缘（如在4.2如前所述）。 </li><li>在采集卡的渠道工具 ，选择在田径1日为830钛波长:蓝宝石激光束，并在为OPO 1107纳米。蜱在田径1两种激光来获得两种激光的同时扫描。在一开始低值设定权力。 </li><li>在光路窗口中 ，选择PMT1。通过点击按钮连续开关上的激光扫描。轻微移动焦点到激光输送进油薄层。 </li><li>如果有必要，同时增加激光器的光功率。调整显示视图选项控制块的显示亮度。缓慢移动延迟线的平移台直到信号变为签名着地增强。 </li><li>优良的路线完成后，检查它是否真的是一个信号CARS:轻微移动翻译阶段;的信号的强度必须成为弱。和/或关闭激光束中的一个，或者钛蓝宝石激光器或OPO。再次，必须有强度的衰减强相比，CARS信号。 </li><li>为了达到最大的CARS信号，选择对软件的选项，以提供整个图像的平均强度的值（在屏幕区域标签的HISTO视图）。调节波长（几个纳米），然后将x，y，z中的聚焦光束的位置，以最大限度地提高平均强度值。 </li></ol> 6.盒延迟线的光路<ol><li>由于最终系统专用于非物理学家，则会以管或外壳盒延迟线的光路，以避免有害的非可见光高的峰值功率的激光束的直接访问。照顾以提供平移台的接入旋钮。 </li></ol> 7.波长调谐为CARS <ol><li>用公式<img alt="式（1）" src="/files/ftp_upload/54262/54262eq1.jpg" />调谐激光波长为所需的拉曼振动。 蓝宝石 = 830 nm和λ= OPO波长1095:要重现在这项工作中图像CARS给出的结果不必延伸3015 -1，选择λ 钛振动CH键信号。 注:生物样品，例如水在观察到的拉曼特征振动频率，CH键可以在Evans 等人 13或在Ellis 等 29中找到。 </li><li>用公式<img alt="公式（2）" src="/files/ftp_upload/54262/54262eq2.jpg" />确定CARS信号的发射波长。对于由CARS CH键的成像，因为在λCARS 670纳米选择窄带滤波器=激光波长670纳米的7.1介绍。 注:手机应用是AVailable从计算λCARS λP和λS值（见参考文献30）。 </li></ol> 8.坐骨神经削减观察CARS信号与彩色髓鞘注意:所有动物实验均按照体制法规进行。 <ol><li>准备在显微镜载玻片的轴向和纵向坐骨神经削减作为Ozcelik酒店等人 31提交。 </li><li>通过稀释原液300倍到PBS制备fluoromyelin红染色溶液。洪水神经切口与在RT 20分钟的染色溶液。除去溶液和洗涤3次，每次10分钟，用PBS。 </li><li>定位20X水浸泡的目的下的削减。放置盖玻片。添加的PBS几滴浸入目标和调整物镜的聚焦以获得通过目镜切口的清晰图像（如先前在4.2详述）。 </li> &lt;li&gt;在轨道1，选择钛宝石和OPO激光器和分别定义它们的波长830纳米和1095纳米 。在光路窗口中 ，选择PMT1和绿色。 </li><li>在轨道2，选择OPO激光只（在1095波长 ）。在光路窗口中 ，选择PMT4和红色。 </li><li>对于这两种激光器，选择低功耗和设置增益为600的一个开始。开关上的激光扫描和调整以下参数，以提高车和荧光信号的对比:功率值，平移台旋钮（非常轻微），波长（几个纳米），显示强度。 </li><li>以高分辨率记录最终图像，在采集模式工具选择以下参数:1,024像素帧大小，7扫描速度，4.单击记录一个图像捕捉按钮的平均。保存图像中的专有格式在记录图像和全采集参数。 </li></ol> 9.观察车和SHG信号从坐骨神经割伤<ol><li>准备坐骨神经在Ozcelik酒店等 31提出。 </li><li>操作与部分8解释通过目镜来获得图像，并选择CARS信号参数（第1道）。 </li><li>在轨道2，选择OPO激光只（1095波长 ）。在光路窗口中 ，选择PMT3和品红色的颜色。 </li><li>按照该过程，在部分8说明切换在激光扫描并保存高分辨率的图像。 </li></ol>

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The authors declare that they have no competing financial interests.

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><tbody><tr><td>Oscilloscope</td><td>Tektronix</td><td>TDS 520D</td><td>500 MHz&amp;nbsp;</td></tr><tr><td>Photodetector</td><td>Thorlabs</td><td>DET08C/M, T4290</td><td>5 GHz InGaAs, 800 - 1,700 nm</td></tr><tr><td>Ti:Sapphire laser Chameleon Ultra Family II</td><td>Coherent</td><td></td><td></td></tr><tr><td>Optical parametric oscillator OPO Compact Family</td><td>APE Berlin</td><td></td><td></td></tr><tr><td>Axio Examiner microscope LSM 7 MP</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Motorized periscope</td><td>Newport</td><td></td><td></td></tr><tr><td>Objective W Plan-Apochromat 20X/1.0</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Beam combiner</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Acousto-optic modulator</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>OPO power attenuator</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Photomultiplier tube</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>ZEN software</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Bandpass filters</td><td>Carl Zeiss</td><td>LSM BiG 1935-176</td><td>400 - 480 nm; 500 - 550&amp;nbsp;nm; 465 - 610&amp;nbsp;nm</td></tr><tr><td>Dichroic mirror</td><td>Carl Zeiss</td><td></td><td>Cutoff wavelength 760&amp;nbsp;nm</td></tr><tr><td>Silver mirrors</td><td>Newport</td><td>10D20ER.2&amp;nbsp;</td><td>&amp;lambda;/10, 480 - 20,000&amp;nbsp;nm, Quantity 4</td></tr><tr><td>Single-axis translation stage with standard micrometer</td><td>Thorlabs</td><td>PT1/M</td><td>Quantity 1</td></tr><tr><td>Aluminium breadboard</td><td>Thorlabs</td><td>MB1015/M&amp;nbsp;</td><td>Quantity 1</td></tr><tr><td>Mirror mount</td><td>Thorlabs</td><td>KMSS/M&amp;nbsp;</td><td>Quantity 4</td></tr><tr><td>Mirror holder for &amp;Oslash;1&quot; Optics&amp;nbsp;</td><td>Thorlabs</td><td>MH25</td><td>Quantity 4</td></tr><tr><td>Iris diaphragms&amp;nbsp;</td><td>Thorlabs</td><td>ID8/M</td><td>Quantity 3</td></tr><tr><td>Protective box</td><td>Thorlabs</td><td>TB4, XE25L900/M, T205-1.0, RM1S</td><td>Quantity 1</td></tr><tr><td>Optical posts</td><td>Thorlabs</td><td>TR40/M, PH50/M, PH75/M, BA2/M</td><td>Quantity 8 (lengths depending on the set-up)</td></tr><tr><td>661 - 690 nm bandpass filter</td><td>Semrock</td><td>676/29 nm BrightLine&amp;reg; single-band bandpass filter</td><td>Quantity 1</td></tr><tr><td>Fluorescent beads</td><td>ThermoFisher</td><td>TetraSpeck&amp;trade; Fluorescent Microspheres Size Kit</td><td></td></tr><tr><td>Laser viewing card</td><td>Thorlabs</td><td></td><td>IR laser viewing card</td></tr><tr><td>Laser safety glass</td><td>Newport</td><td>LV-F22.P5L07&amp;nbsp;</td><td></td></tr><tr><td>FluoroMyelin&amp;trade; Red Fluorescent Myelin Stain&amp;nbsp;</td><td>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）的记录脉冲串...

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 ...

implementation-coherent-anti-stokes-raman-scattering-cars-system-on

Universidad de Cartagena UDEC

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

11.1K 次观看.  INSERM U1051, Institut des Neurosciences de Montpellier (INM), Université de Montpellier. 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。 

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

Implementation of a Nonlinear Microscope Based on Stimulated Raman Scattering

Stimulated Raman scattering (SRS) microscopy uses near-infrared excitation light; therefore, it shares many multi-photon microscopic imaging properties. SRS imaging modality can be obtained using commercial laser-scanning microscopes by equipping with a non-descanned forward detector with proper bandpass filters and lock-in amplifier (LIA) detection scheme. A schematic layout of a typical SRS microscope includes the following: two pulsed laser beams, (i.e., the pump and probe directed in a scanning microscope), which must be overlapped in both space and time at the image plane, then focused by a microscope objective into the sample through two scanning mirrors (SMs), which raster the focal spot across an x-y plane. After interaction with the sample, transmitted output pulses are collected by an upper objective and measured by a forward detection system inserted in an inverted microscope. Pump pulses are removed by a stack of optical filters, whereas the probe pulses that are the result of the SRS process occurring in the focal volume of the specimen are measured by a photodiode (PD). The readout of the PD is demodulated by the LIA to extract the modulation depth. A two-dimensional (2D) image is obtained by synchronizing the forward detection unit with the microscope scanning unit. In this paper, the implementation of an SRS microscope is described and successfully demonstrated, as well as the reporting of label-free images of polystyrene beads with diameters of 3 &#181;m. It is worth noting that SRS microscopes are not commercially available, so in order to take advantage of these characteristics, the homemade construction is the only option. Since SRS microscopy is becoming popular in many fields, it is believed that this careful description of the SRS microscope implementation can be very useful for the scientific community.

In this manuscript, the implementation of a stimulated Raman scattering (SRS) microscope, obtained by the integration of an SRS experimental set-up with a laser scanning microscope, is described. The SRS microscope is based on two femtosecond (fs) laser sources, a Ti-Sapphire (Ti:Sa) and synchronized optical parametric oscillator (OPO).

基于刺激拉曼散射的非线性显微镜的实现

Stimulated Raman scattering (SRS) microscopy uses near-infrared excitation light; therefore, it shares many multi-photon microscopic imaging properties. ...

科研

工程学

Ultrafast Time-resolved Near-IR Stimulated Raman Measurements of Functional &#960;-conjugate Systems

Femtosecond time-resolved stimulated Raman spectroscopy is a promising method of observing the structural dynamics of short-lived transients with near infrared (near-IR) transitions, because it can overcome the low sensitivity of spontaneous Raman spectrometers in the near-IR region. Here, we describe technical details of a femtosecond time-resolved near-IR multiplex stimulated Raman spectrometer that we have recently developed. A description of signal generation and optimization, measurement, data acquisition, and calibration and correction of recorded data is provided as well. We present an application of our spectrometer to analyze the excited-state dynamics of &#946;-carotene in toluene solution. A C=C stretch band of &#946;-carotene in the second lowest excited singlet (S2) state and the lowest excited singlet (S1) state is clearly observed in the recorded time-resolved stimulated Raman spectra. The femtosecond time-resolved near-IR stimulated Raman spectrometer is applicable to the structural dynamics of &#960;-conjugate systems from simple molecules to complex materials.

Details of signal generation and optimization, measurement, data acquisition, and data handling for a femtosecond time-resolved near-IR stimulated Raman spectrometer are described. A near infrared stimulated Raman study on the excited-state dynamics of &#946;-carotene in toluene is shown as a representative application.

超快时间解析近红外刺激拉曼测量功能 +偶联系统

Femtosecond time-resolved stimulated Raman spectroscopy is a promising method of observing the structural dynamics of short-lived transients with near ...

化学

Multiplex Chemical Imaging Based on Broadband Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy is a nonlinear optical technique for label-free chemical imaging. This analytical tool delivers chemical maps at high speed, and high spatial resolution of thin samples by directly interrogating their molecular vibrations. In its standard implementation, SRS microscopy is narrowband and forms images with only a single vibrational frequency at a time. However, this approach not only hinders the chemical specificity of SRS but also neglects the wealth of information encoded within vibrational spectra. 
These limitations can be overcome by broadband SRS, an implementation capable of extracting a vibrational spectrum per pixel of the image in parallel. This delivers hyperspectral data that, when coupled with chemometric analysis, maximizes the amount of information retrieved from the specimen. Thus, broadband SRS improves the chemical specificity of the system, allowing the quantitative determination of the concentration of the different constituents of a sample. Here, we report a protocol for chemical imaging with broadband SRS microscopy, based on a home-built SRS microscope operating with a custom differential multichannel-lock-in amplifier detection. It discusses the sample preparation, alignment of the SRS apparatus, and chemometric analysis. By acquiring vibrational Raman spectra, the protocol illustrates how to identify different chemical species within a mixture, determining their relative concentrations.

We present a protocol to acquire chemical images with broadband stimulated Raman scattering (SRS) microscopy. Based on an SRS microscope that operates with differential multichannel-lock-in detection, the protocol describes the sample preparation, adjustment of the SRS apparatus, and chemometrics to disentangle different constituents of chemically heterogeneous samples.

基于宽带受激拉曼散射显微镜的多重化学成像

Stimulated Raman scattering (SRS) microscopy is a nonlinear optical technique for label-free chemical imaging. This analytical tool delivers chemical maps ...

Direct Comparison of Hyperspectral Stimulated Raman Scattering and Coherent Anti-Stokes Raman Scattering Microscopy for Chemical Imaging

Stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) microscopy are the most widely used coherent Raman scattering imaging technologies. Hyperspectral SRS and CARS imaging offer Raman spectral information at every pixel, which enables better separation of different chemical compositions. Although both techniques require two excitation lasers, their signal detection schemes and spectral properties are quite different. The goal of this protocol is to perform both hyperspectral SRS and CARS imaging on a single platform and compare the two microscopy techniques for imaging different biological samples. The spectral focusing method is employed to acquire spectral information using femtosecond lasers. By using standard chemical samples, the sensitivity, spatial resolution, and spectral resolution of SRS and CARS in the same excitation conditions (i.e., power at the sample, pixel dwell time, objective lens, pulse energy) are compared. The imaging contrasts of CARS and SRS for biological samples are juxtaposed and compared. The direct comparison of CARS and SRS performances would allow for optimal selection of the modality for chemical imaging.

This paper directly compares the resolution, sensitivity, and imaging contrasts of stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) integrated into the same microscope platform. The results show that CARS has a better spatial resolution, SRS gives better contrasts and spectral resolution, and both methods have similar sensitivity.

高光谱受激拉曼散射与相干反斯托克斯拉曼散射显微镜在化学成像中的应用的直接比较

Stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) microscopy are the most widely used coherent Raman scattering imaging ...

Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering (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 with high molecular specificity 1. The sensitivity of these techniques, however, is many orders of magnitude weaker than fluorescence, requiring milli-molar molecular concentrations 1,2. Here, we describe a technique that can enable the detection of weak or low concentrations of Raman-active molecules by amplifying their signal with that obtained from strong or abundant Raman scatterers. The interaction of short pulsed lasers in a biological sample generates a variety of coherent Raman scattering signals, each of which carry unique chemical information about the sample. Typically, only one of these signals, e.g. Coherent Anti-stokes Raman scattering (CARS), is used to generate an image while the others are discarded. However, when these other signals, including 3-color CARS and four-wave mixing (FWM), are collected and compared to the CARS signal, otherwise difficult to detect information can be extracted 3. For example, doubly-resonant CARS (DR-CARS) is the result of the constructive interference between two resonant signals 4. We demonstrate how tuning of the three lasers required to produce DR-CARS signals to the 2845 cm-1 CH stretch vibration in lipids and the 2120 cm-1 CD stretching vibration of a deuterated molecule (e.g. deuterated sugars, fatty acids, etc.) can be utilized to probe both Raman resonances simultaneously. Under these conditions, in addition to CARS signals from each resonance, a combined DR-CARS signal probing both is also generated. We demonstrate how detecting the difference between the DR-CARS signal and the amplifying signal from an abundant molecule's vibration can be used to enhance the sensitivity for the weaker signal. We further demonstrate that this approach even extends to applications where both signals are generated from different molecules, such that e.g. using the strong Raman signal of a solvent can enhance the weak Raman signal of a dilute solute.

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

A combination of three single wavelength short-pulsed lasers is used to generate coherent anti-Stokes Raman scattering (CARS) and doubly-resonant CARS (DR-CARS). The difference between these signals provides enhanced sensitivity for otherwise difficult to detect coherent Raman signals, enabling imaging of weak Raman scatterers.

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 ...

生物学

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy (oSLO) and Optical Coherence Tomography (OCT)

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big challenge with the state-of-the-art retinal imaging modalities because they would require z-stacking to compile a volumetric dataset. Newer optical coherence tomography (OCT) and OCT angiography (OCTA) systems overcome these restrictions to provide three-dimensional (3D) anatomical and vascular images, but the dye-free nature of OCT cannot visualize leakage indicative of vascular dysfunction. This protocol describes a novel oblique scanning laser ophthalmoscopy (oSLO) technique that provides 3D volumetric fluorescence retinal imaging. The setup of the imaging system generates the oblique scanning by a dove tail slider and aligns the final imaging system at an angle to detect fluorescent cross-sectional images. The system uses the laser scanning method, and therefore, allows an easy incorporation of OCT as a complementary volumetric structural imaging modality. In vivo imaging on rat retina is demonstrated here. Fluorescein solution is intravenously injected to produce volumetric fluorescein angiography (vFA).

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy oSLO and Optical Coherence Tomography OCT

Here, we present a protocol to get a large field of view (FOV) three-dimensional (3D) fluorescence and OCT retinal image by using a novel imaging multimodal platform. We will introduce the system setup, the method of alignment, and the operational protocols. In vivo imaging will be demonstrated, and representative results will be provided.

斜扫描激光检眼镜 (奥斯陆) 与光学相干层析成像 (OCT) 的多模态视网膜成像

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big ...

生物工程

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic molecular vibration, thus providing a perfect tool for in vivo study of biological processes at subcellular resolution. By integrating two-photon excited fluorescence (TPEF) imaging into the SRS microscope, the dual-modal in vivo imaging of tissues can acquire critical biochemical and biophysical information from multiple perspectives which helps understand the dynamic processes involved in cellular metabolism, immune response and tissue remodeling, etc. In this video protocol, the setup of a TPEF-SRS microscope system as well as the in vivo imaging method of the animal spinal cord is introduced. The spinal cord, as part of the central nervous system, plays a critical role in the communication between the brain and peripheral nervous system. Myelin sheath, abundant in phospholipids, surrounds and insulates the axon to permit saltatory conduction of action potentials. In vivo imaging of myelin sheaths in the spinal cord is important to study the progression of neurodegenerative diseases and spinal cord injury. The protocol also describes animal preparation and in vivo TPEF-SRS imaging methods to acquire high-resolution biological images.

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows label-free imaging of biomolecules based on their intrinsic vibration of specific chemical bonds. In this protocol, the instrumental setup of an integrated SRS and two-photon fluorescence microscope is described to visualize cellular structures in the spinal cord of live mice.

体内 使用双光子荧光和受激拉曼散射显微镜对生物组织进行成像

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic ...

Real-Time, Two-Color Stimulated Raman Scattering Imaging of Mouse Brain for Tissue Diagnosis

Stimulated Raman scattering (SRS) microscopy has emerged as a powerful optical imaging technique for tissue diagnosis. In recent years, two-color SRS has been shown to be able to provide hematoxylin and eosin (H&amp;E)-equivalent images that allow fast and reliable diagnosis of brain cancer. Such capability has enabled exciting intraoperative cancer diagnosis applications. Two-color SRS imaging of tissue can be done with either a picosecond or femtosecond laser source. Femtosecond lasers have the advantage of enabling flexible imaging modes, including fast hyperspectral imaging and real-time, two-color SRS imaging. A spectral-focusing approach with chirped laser pulses is typically used with femtosecond lasers to achieve high spectral resolution. 
Two-color SRS acquisition can be realized with orthogonal modulation and lock-in detection. The complexity of pulse chirping, modulation, and characterization is a bottleneck for the widespread adoption of this method. This article provides a detailed protocol to demonstrate the implementation and optimization of spectral-focusing SRS and real-time, two-color imaging of mouse brain tissue in the epi-mode. This protocol can be used for a broad range of SRS imaging applications that leverage the high speed and spectroscopic imaging capability of SRS.

Stimulated Raman scattering (SRS) microscopy is a powerful, nondestructive, and label-free imaging technique. One emerging application is stimulated Raman histology, where two-color SRS imaging at the protein and lipid Raman transitions are used to generate pseudo-hematoxylin and eosin images. Here, we demonstrate a protocol for real-time, two-color SRS imaging for tissue diagnosis.

用于组织诊断的小鼠脑实时双色受激拉曼散射成像

Stimulated Raman scattering (SRS) microscopy has emerged as a powerful optical imaging technique for tissue diagnosis. In recent years, two-color SRS has ...

Highly-Multiplexed Tissue Imaging with Raman Dyes

Visualizing a vast scope of specific biomarkers in tissues plays a vital role in exploring the intricate organizations of complex biological systems. Hence, highly multiplexed imaging technologies have been increasingly appreciated. Here, we describe an emerging platform of highly-multiplexed vibrational imaging of specific proteins with comparable sensitivity to standard immunofluorescence via electronic pre-resonance stimulated Raman scattering (epr-SRS) imaging of rainbow-like Raman dyes. This method circumvents the limit of spectrally-resolvable channels in conventional immunofluorescence and provides a one-shot optical approach to interrogate multiple markers in tissues with subcellular resolution. It is generally compatible with standard tissue preparations, including paraformaldehyde-fixed tissues, frozen tissues, and formalin-fixed paraffin-embedded (FFPE) human tissues. We envisage this platform will provide a more comprehensive picture of protein interactions of biological specimens, particularly for thick intact tissues. This protocol provides the workflow from antibody preparation to tissue sample staining, to SRS microscope assembly, to epr-SRS tissue imaging.

Electronic pre-resonance stimulated Raman scattering (epr-SRS) imaging of rainbow-like Raman dyes is a new platform for highly multiplexed epitope-based protein imaging. Here, we present a practical guide including antibody preparation, tissue sample staining, SRS microscope assembly, and epr-SRS tissue imaging.

使用拉曼染料进行高度多重组织成像

Visualizing a vast scope of specific biomarkers in tissues plays a vital role in exploring the intricate organizations of complex biological systems. ...

Biomolecular Imaging of Cellular Uptake of Nanoparticles using Multimodal Nonlinear Optical Microscopy

Probing gold nanoparticles (AuNPs) in living systems is essential to reveal the interaction between AuNPs and biological tissues. Moreover, by integrating nonlinear optical signals such as stimulated Raman scattering (SRS), two-photon excited fluorescence (TPEF), and transient absorption (TA) into an imaging platform, it can be used to reveal biomolecular contrast of cellular structures and AuNPs in a multimodal manner. This article presents a multimodal nonlinear optical microscopy and applies it to perform chemically specific imaging of AuNPs in cancer cells. This imaging platform provides a novel approach for developing more efficient functionalized AuNPs and determining whether they are within vasculatures surrounding the tumor, pericellular, or cellular spaces.

This article presents the integration of a spectral-focusing module and a dual-output pulse laser, enabling rapid hyperspectral imaging of gold nanoparticles and cancer cells. This work aims to demonstrate the details of multimodal nonlinear optical techniques on a standard laser scanning microscope.

使用多模态非线性光学显微镜对纳米颗粒的细胞摄取进行生物分子成像

Probing gold nanoparticles (AuNPs) in living systems is essential to reveal the interaction between AuNPs and biological tissues. Moreover, by integrating ...

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

摘要

探索更多视频

此视频中的章节

基于刺激拉曼散射的非线性显微镜的实现

超快时间解析近红外刺激拉曼测量功能 +偶联系统

基于宽带受激拉曼散射显微镜的多重化学成像

高光谱受激拉曼散射与相干反斯托克斯拉曼散射显微镜在化学成像中的应用的直接比较

差分成像生物结构与双共振相干反斯托克斯拉曼散射（CARS）

斜扫描激光检眼镜 (奥斯陆) 与光学相干层析成像 (OCT) 的多模态视网膜成像

体内使用双光子荧光和受激拉曼散射显微镜对生物组织进行成像

用于组织诊断的小鼠脑实时双色受激拉曼散射成像

使用拉曼染料进行高度多重组织成像

使用多模态非线性光学显微镜对纳米颗粒的细胞摄取进行生物分子成像

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

摘要

探索更多视频

此视频中的章节

基于刺激拉曼散射的非线性显微镜的实现

超快时间解析近红外刺激拉曼测量功能 +偶联系统

基于宽带受激拉曼散射显微镜的多重化学成像

高光谱受激拉曼散射与相干反斯托克斯拉曼散射显微镜在化学成像中的应用的直接比较

差分成像生物结构与双共振相干反斯托克斯拉曼散射（CARS）

斜扫描激光检眼镜 (奥斯陆) 与光学相干层析成像 (OCT) 的多模态视网膜成像

体内 使用双光子荧光和受激拉曼散射显微镜对生物组织进行成像

用于组织诊断的小鼠脑实时双色受激拉曼散射成像

使用拉曼染料进行高度多重组织成像

使用多模态非线性光学显微镜对纳米颗粒的细胞摄取进行生物分子成像

体内使用双光子荧光和受激拉曼散射显微镜对生物组织进行成像