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://www-jove-com.remotexs.ntu.edu.sg/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的光束的峰位移。这是需要的CARS信号的两个光束的空间重叠是类似于对于这种激光扫描显微镜系统中的正常光束对准。 延迟线的实施可以在数周内通过具有在实验一基本背景生物学家可实现人光学或会同物理学家。特别小心，必须采取反射镜的选择以最小化反射损失。如果建延迟线大部分材料是可以承受的，这是需要示波器是不是一个标准的示波器。因此，由于几天都足以完成时间同步过程，从一个物理实验室借用示波器似乎是最好的选择。空间重叠可能是耗时，因为它需要多个反射镜和透镜（其可以根据激光扫描系统的机动化）的调谐。此前CARS激光系统基于两个电子同步掺钛蓝宝石激光器曾与长期（几十分钟到几小时）时间脉冲重叠的问题。这个问题已与OPO具有独特的激光源20,21泵浦被完全解决。通过这样的激光系统，和使用牢牢夹住光学，泵和斯托克斯之间没有时间走离梁观察到在运行几个月。 汽车技术的一个主要限制是不谐振分子的存在（从溶剂或散射体）11，就可能出现的CARS信号。此非共振背景可以限制的灵敏度。方法来减少这种背景包括检测（向前或向后检测）的结构中，激光器的特性（飞秒或皮秒），用于浸泡的目的或图像处理溶液的选择。一个落后的检测允许，尽量减少非共振的背景11，因为它是在这项工作中（ 图1）使用。飞秒钛蓝宝石激光器提供高的峰值强度为非线性光学过程非常需要的。然而，在频域中，大多数的拉曼线的宽度比飞秒脉冲的光谱宽度小，而它符合皮秒脉冲的光谱宽度。因此，使用皮秒激光器的增加比O˚F谐振信号以非共振的背景。在脂质CH键，因为键的高密度，中，非谐振背景是微不足道的飞秒激光器。以降低背景的效果，根据非共振记录的参考图像的相减也可应用于（对于此工作未完成）。 尽管近红外激光束，允许在组织深穿透的利用（200 - 300微米），在神经成像获得的实际穿透深度大约为50微米，在这项工作中所进行的实验。但是该参数在很大程度上依赖成像的生物组织的和，因为所有有髓纤维的对准，并且集中在一个很小的空间，外周神经可能不构成最好样品用于评估该光穿透。此外，需要几秒钟来记录高分辨率的图像（1,024×1,024像素），它可以限制在体内的可视化</em&gt;样品因为即使麻醉，呼吸可诱发轻微移动。 CARS显微允许特定化学成像的选择性，分辨率接近衍射极限。该技术不需要任何标签，并且由于钛蓝宝石激光器是可调谐从700至1000纳米和1100 OPO的激光至1,300nm，它覆盖在生物系统中的分子振动的整个频谱区（从500到7000厘米-1）。因此，它是适用的，高度判别的DNA，蛋白质，脂质，或水，而较强的信号从所述脂质CH伸缩键产生。光损伤也降低由于使用相对于第二谐波产生成像以产生CARS信号的激光功率水平低。因为CARS成像能以低的激光功率和无侵入性标记来实现，因此它是选择用于体内研究的工具。 激光扫描显微镜无线TH飞秒钛蓝宝石激光源与OPO组合在许多生物实验室已成为主要可用于双光子显微镜。由于空间同步在这些系统已经实现，在该工作中描述的两个光束的时间重叠过程带来显微镜的不昂贵的附加设备的额外技术。因此，修改后的设置允许细胞同时成像或第二或第三谐波（SHG，THG），双光子荧光和CARS活体组织。 CARS技术已经证明了活的动物，允许动态过程的研究（无污渍或基因操作）的体内成像的巨大潜力。有前景的应用包括在体内无标记的非侵入性的脂质成像，例如用于在活脊髓组织36的神经元髓鞘，用于研究与脂质相关的疾病（肥胖的实时成像，脱髓鞘和cardiovascular疾病）37，人类皮肤渗透机制22或皮肤病38。我们相信，未来的改进将关注使用特殊物镜，比如GRIN微目标或微型客观上提升了CARS信号的穿透深度。

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）的记录脉冲串和从钛:未经延时线（B）的蓝宝石激光束并以调整后的延迟线（℃ ）。的脉冲串因而在时间上与实施延迟线如图2A和2C大体同步。 PG"/&gt; 在与OPO束（A）的10X物镜的显微镜样品平面图2 激光脉冲串记录的时间同步记录脉冲串，使Ti:无（B）的蓝宝石激光束和延迟线路（℃ ）。每个图形绘制的信号从在Ti背面的BNC连接直接记录的顶部曲线:蓝宝石激光（同步输出接口）。示波器触发调整了这个信号。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54262/54262fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 两个光束的空间重叠可以通过固定在显微镜载玻片上（ 图3A）的顶部的荧光聚苯乙烯珠的可视化来获得。 图3B示出了由给定的附近的三个小珠的图像在1107纳米（红色假色）和OPO激光器钛:在830nm蓝宝石激光器（绿色假彩色）。两个激光束之间的空间偏移已被补偿以下先前描述（ 图3C）的空间同步的过程。 <img alt="图3" src="/files/ftp_upload/54262/54262fig3.jpg" /> 图 3. 梁的空间重叠。荧光微球（A）的一般看法。放大3相邻微球。的珠在830和1107的照明波长示出了胎圈图像的空间偏移（伪色）（B）。 X，Y，Z的调整后，珠合并图像（C）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54262/54262fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 最后步骤来实现精确时间调整来激活CARS可以通过成像橄榄油滴（含有众多CH键32）和通过移动延迟线的略微的反射镜，完成在两个激光脉冲序列的时间同步来容易地实现。 图4示出了从钛递送橄榄油液滴的图片:蓝宝石激光照射只（ 图4A），并从OPO照明只（ 图4B）。 3100厘米-1 -同时施加两个光束在670nm处，其对应于一个CARS从固有碳氢（CH）的伸缩振动带2800的拉曼振动范围信号诱导的明确信号增强（ 图4C）。当一个两个光束被关闭CARS信号（C）消失。自橄榄油含有天然荧光团33，特别是叶绿素分子的范围内发射650 - 670纳米，在（A）和（B）中的弱信号是穆尔tiphoton荧光过程。这些信号的强度的数量级比CARS信号低几个数量级。因此，它不会扰乱CARS信号的测量。 <img alt="图4" src="/files/ftp_upload/54262/54262fig4.jpg" /> 对于CARS图4.最终精细时间调整信号爆发。橄榄油液滴在时间上与测微驱动器的线性平移阶段调节延迟线路长度用于调整两个光束。在（A）830 nm激光只，并在光气（B）1107纳米的激光只显示一个微弱的信号。下同时照明（C）中 ，明确信号增强观察，对应于CARS信号。比例尺是100微米。在3015厘米拉曼振动峰-1。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54262/54262fig4large.jpg" target="_blank">请点击她的</a>E要查看此图的放大版本。 髓鞘是由包装和冷凝围绕在中央和周围神经系统34的神经元的轴突其质膜特定细胞产生的生物结构。此护套被高度富集不同脂质。我们在这里所使用的小鼠的坐骨神经的髓磷脂鞘。横向和纵向坐骨神经横截面段具有在1095纳米被染色髓鞘的双光子荧光成像分别如图5B和图5E。一个fluoromyelin红色染料，这对髓鞘选择性使用35。这些样品都在830和1095纳米的同时被照射，观察到（ 图5A和图5D）的CARS信号。在图5A-C中 ，圆圈对应于轴突周围的髓鞘（在空的空间侧横向切环）。如在图5C和5F所示，而重叠CARS和荧光图像相同的结构被发现。我们的结论是的细节相似水平可与CARS，因此无需使用荧光标记来获得。 <img alt="图5" src="/files/ftp_upload/54262/54262fig5.jpg" /> 图5.车和坐骨神经削减染色髓鞘成像。（A）横向和髓鞘的无标记CARS成像（D）纵向切割。 （B）的横向和（E）在1095纳米下双光子荧光照明由fluoromyelin红色染料染色的相同的样品的纵向切口。两个图像（C）和横向（F）纵向切割重叠。比例尺是10微米。平均光功率为5毫瓦在830nm和16毫瓦的CARS信号1095纳米。平均光功率是在双光子荧光成像1095毫微米8毫瓦。 2915 -1拉曼振动峰。 CARS信号通过停止一个激光扫描或通过移动平移台出拉曼共振的消失。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54262/54262fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 此外到CARS成像，可用显微镜系统允许在坐骨神经的外表面，以同时产生从胶原纤维的二次谐波。这个附加图象只需要使用一个不同的检测器中的，因为在被用于CARS信号记录670纳米的窄带通滤波器550纳米（OPO的波长的一半）来记录的信号; 图6示出了对比度的图像假绿色描绘髓鞘（CARS信号ONLY在图6A中）通过在假洋红色所示胶原纤维环绕（SHG仅在图6B）。 CARS信号用在830nm和4毫瓦的平均光功率13毫瓦1095处实现，而它需要50毫瓦1095处，以获得在SHG信号。因此低得多的光学能量需要获得CARS相比倍频或三倍频（未示出）提供了生物样品中的信号的信号。 <img alt="图6" src="/files/ftp_upload/54262/54262fig6.jpg" /> 图6.车和鼠标坐骨神经SHG成像。（A）CARS成像，（B）SHG成像和（C）同时髓鞘由CARS成像（绿色假彩色）通过SHG成像（洋红色），可视化和胶原蛋白对小鼠坐骨神经。平均光功率分别为830 nm的4毫瓦和13毫瓦的CARS 1095纳米。平均光功率为50毫瓦1095纳米的SHG。比例尺为10微米。拉曼振动在2915厘米高峰-1。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54262/54262fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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 ...

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Nanyang Technological University

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

Laser Capture Microdissection of Mammalian Tissue

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or formalin-fixed, paraffin-embedded tissue sections. LMD techniques rely on a thermo labile membrane placed either on top of, or underneath, the tissue section. In one method, focused laser energy is used to melt the membrane onto the underlying cells, which can then be lifted out of the tissue section. In the other, the laser energy vaporizes the foil along a path "drawn" on the tissue, allowing the selected cells to fall into a collection device. Each technique allows the selection of cells with a minimum resolution of several microns. DNA, RNA, protein, and lipid samples may be isolated and analyzed from micro-dissected samples. In this video, we demonstrate the use of the Leica AS-LMD laser microdissection instrument in seven segments, including an introduction to the principles of LMD, initializing the instrument for use, general considerations for sample preparation, mounting the specimen and setting up capture tubes, aligning the microscope, adjusting the capture controls, and capturing tissue specimens. Laser-capture micro-dissection enables the investigator to isolate samples of pure cell populations as small as a few cell-equivalents. This allows the analysis of cells of interest that are free of neighboring contaminants, which may confound experimental results.

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

Glaucoma is one of the major causes of blindness in the world. Elevated intraocular pressure is a major risk factor. Laser photocoagulation induced ocular hypertension is one of the well established animal models. This video demonstrates how to induce ocular hypertension by Argon laser photocoagulation in rat.

Glaucoma is one of the major causes of blindness in the world. Elevated intraocular pressure is a major risk factor. Laser photocoagulation induced ocular hypertension is one of the well established animal models. This video demonstrates how to induce ocular hypertension by Argon laser photocoagulation in rat.

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)

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 ...

Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated precursors. Researchers have employed various methods of lineage labeling, such as DiI labeling1 and pressure injection of traceable enzymes2 to ascertain cell fate at later stages of development in model systems. The first fate maps in zebrafish (Danio rerio) were assembled by iontophoretic injection of fluorescent dyes, such as rhodamine dextran, into single cells in discrete regions of the embryo and tracing the labeled cell's fate over time3-5. While effective, these methods are technically demanding and require specialized equipment not commonly found in zebrafish labs. Recently, photoconvertable fluorescent proteins, such as Eos and Kaede, which irreversibly switch from green to red fluorescence when exposed to ultraviolet light, are seeing increased use in zebrafish6-8. The optical clarity of the zebrafish embryo and the relative ease of transgenesis have made these particularily attractive tools for lineage labeling and to observe the migration of cells in vivo7. Despite their utility, these proteins have some disadvantages compared to dye-mediated lineage labeling methods. The most crucial is the difficulty we have found in obtaining high 3-D resolution during photoconversion of these proteins. In this light, perhaps the best combination of resolution and ease of use for lineage labeling in zebrafish makes use of caged fluorescein dextran, a fluorescent dye that is bound to a quenching group that masks its fluorescence9. The dye can then be "uncaged" (released from the quenching group) within a specific cell using UV light from a laser or mercury lamp, allowing visualization of its fluorescence or immunodetection. Unlike iontophoretic methods, caged fluorescein can be injected with standard injection apparatuses and uncaged with an epifluorescence microscope equipped with a pinhole10. In addition, antibodies against fluorescein detect only the uncaged form, and the epitope survives fixation well11. Finally, caged fluorescein can be activated with very high 3-D resolution, especially if two-photon microscopy is employed 12,13. This protocol describes a method of lineage labeling by caged fluorescein and laser uncaging. Subsequenctly, uncaged fluorescein is detected simultaneously with other epitopes such as GFP by labeling with antibodies.

This protocol delineates a way to label and trace the fate of small groups of cells zebrafish embryos using UV-uncaging of caged fluorescein, followed by whole mount immunolabeling to amplify the signal from the uncaged fluorescein.

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

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 renal failure1. AKI can be caused by a number of factors including ischemia, drug-based toxicity, or obstructive injury1. This results in an inability to maintain fluid and electrolyte homeostasis. While AKI has been observed for decades, effective clinical therapies have yet to be developed. Intriguingly, some patients with AKI recover renal functions over time, a mysterious phenomenon that has been only rudimentally characterized1,2. Research using mammalian models of AKI has shown that ischemic or nephrotoxin-injured kidneys experience epithelial cell death in nephron tubules1,2, the functional units of the kidney that are made up of a series of specialized regions (segments) of epithelial cell types3. Within nephrons, epithelial cell death is highest in proximal tubule cells. There is evidence that suggests cell destruction is followed by dedifferentiation, proliferation, and migration of surrounding epithelial cells, which can regenerate the nephron entirely1,2. However, there are many unanswered questions about the mechanisms of renal epithelial regeneration, ranging from the signals that modulate these events to reasons for the wide variation of abilities among humans to regenerate injured kidneys.
The larval zebrafish provides an excellent model to study kidney epithelial regeneration as its pronephric kidney is comprised of nephrons that are conserved with higher vertebrates including mammals4,5. The nephrons of zebrafish larvae can be visualized with fluorescence techniques because of the relative transparency of the young zebrafish6. This provides a unique opportunity to image cell and molecular changes in real-time, in contrast to mammalian models where nephrons are inaccessible because the kidneys are structurally complex systems internalized within the animal. Recent studies have employed the aminoglycoside gentamicin as a toxic causative agent for study of AKI and subsequent renal failure: gentamicin and other antibiotics have been shown to cause AKI in humans, and researchers have formulated methods to use this agent to trigger kidney damage in zebrafish7,8. However, the effects of aminoglycoside toxicity in zebrafish larvae are catastrophic and lethal, which presents a difficulty when studying epithelial regeneration and function over time. Our method presents the use of targeted cell ablation as a novel tool for the study of epithelial injury in zebrafish. Laser ablation gives researchers the ability to induce cell death in a limited population of cells. Varying areas of cells can be targeted based on morphological location, function, or even expression of a particular cellular phenotype. Thus, laser ablation will increase the specificity of what researchers can study, and can be a powerful new approach to shed light on the mechanisms of renal epithelial regeneration. This protocol can be broadly applied to target cell populations in other organs in the zebrafish embryo to study injury and regeneration in any number of contexts of interest.

Acute kidney injury (AKI) in humans is a common clinical problem caused by damage to the epithelial cells that comprise kidney nephrons, and AKI is associated with high mortality rates of 50-70%1. Following epithelial cell destruction, nephrons have a limited ability to regenerate, though the mechanisms and limitations that guide this phenomenon remain poorly understood. In this video article, we describe our technique for targeted laser ablation of kidney nephron cells in the zebrafish embryo kidney, or pronephros. Our new method can be used to complement nephrotoxicity-induced models of AKI and gain a high-resolution understanding of the cell and molecular alterations that are associated with epithelial regeneration in the kidney nephron.

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

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly using MALDI (matrix&#160;assisted laser desorption/ionization)-based analytical techniques. New matrices for small-molecule MSI, which can improve the analysis of low-molecular weight (MW) compounds, are needed. These matrices should provide increased analyte signals while decreasing MALDI background signals. In addition, the use of ultrahigh-resolution instruments, such as Fourier transform ion cyclotron resonance (FTICR) mass spectrometers, has&#160;the ability to resolve analyte signals from matrix signals, and this can partially overcome many problems associated with the background originating from the MALDI matrix. The reduction in the intensities of the metastable matrix clusters by FTICR MS can also help to overcome some of the interferences associated with matrix peaks on other instruments. High-resolution instruments such as the FTICR mass spectrometers are advantageous as they can produce distribution patterns of many compounds simultaneously while still providing confidence in chemical identifications. Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging. In this work, a protocol for the use of DT for MALDI imaging of endogenous lipids from the surfaces of mammalian tissue sections, by positive-ion MALDI-MS, on an ultrahigh-resolution hybrid quadrupole FTICR instrument has been provided.

Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging of small molecules; protocols for the use of DT for the MALDI imaging of endogenous lipids on the surface of tissue sections&#160;by positive-ion MALDI-MS on an ultrahigh-resolution quadrupole-FTICR instrument are provided here.

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 ...

Real Time In Vivo Tracking of Thymocytes in the Anterior Chamber of the Eye by Laser Scanning Microscopy

The purpose of the method being presented is to show, for the first time, the transplant of newborn thymi into the anterior eye chamber of isogenic adult mice for in vivo longitudinal real-time monitoring of thymocytes&#180; dynamics within a vascularized thymus segment. Following the transplantation, laser scanning microscopy (LSM) through the cornea allows in vivo noninvasive repeated imaging at cellular resolution level. Importantly, the approach adds to previous intravital T-cell maturation imaging models the possibility for continuous progenitor cell recruitment and mature T-cell egress recordings in the same animal. Additional advantages of the system are the transparency of the grafted area, permitting macroscopic rapid monitoring of the implanted tissue, and the accessibility to the implant allowing for localized in addition to systemic treatments. The main limitation being the volume of the tissue that fits in the reduced space of the eye chamber which demands for lobe trimming. Organ integrity is maximized by dissecting thymus lobes in patterns previously shown to be functional for mature T-cell production. The technique is potentially suited to interrogate a milieu of medically relevant questions related to thymus function that include autoimmunity, immunodeficiency and central tolerance; processes which remain mechanistically poorly defined. The fine dissection of mechanisms guiding thymocyte migration, differentiation and selection should lead to novel therapeutic strategies targeting developing T cells.

Real Time In Vivo Tracking of Thymocytes in the Anterior Chamber of the Eye by Laser Scanning Microscopy

The goal of the protocol is to show longitudinal intravital real-time tracking of thymocytes by laser scanning microscopy in thymic implants in the anterior chamber of the mouse eye. The transparency of the cornea and vascularization of the graft allows for continuously recording progenitor cell recruitment and mature T-cell egress.

激光扫描显微术实时跟踪眼球前室胸腺细胞

The purpose of the method being presented is to show, for the first time, the transplant of newborn thymi into the anterior eye chamber of isogenic adult ...

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

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits obesity and many associated diseases including cardiovascular disorders, type II diabetes, and cancer. To advance the current understanding of lipid metabolic regulation, quantitative methods to precisely measure in vivo lipid storage levels in time and space have become increasingly important and useful. Traditional approaches to analyze lipid storage are semi-quantitative for microscopic assessment or lacking spatio-temporal information for biochemical measurement. Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology that enables rapid and quantitative detection of lipids in live cells with a subcellular resolution. As the contrast is exploited from intrinsic molecular vibrations, SRS microscopy also permits four-dimensional tracking of lipids in live animals. In the last decade, SRS microscopy has been widely used for small molecule imaging in biomedical research and overcome the major limitations of conventional fluorescent staining and lipid extraction methods. In the laboratory, we have combined SRS microscopy with the genetic and biochemical tools available to the powerful model organism, Caenorhabditis elegans, to investigate the distribution and heterogeneity of lipid droplets across different cells and tissues and ultimately to discover novel conserved signaling pathways that modulate lipid metabolism. Here, we present the working principles and the detailed setup of the SRS microscope and provide methods for its use in quantifying lipid storage at distinct developmental timepoints of wild-type and insulin signaling deficient mutant C. elegans.

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

Stimulated Raman scattering (SRS) microscopy allows selective, label-free imaging of specific chemical moieties and it has been effectively employed to image lipid molecules in vivo. Here, we provide a brief introduction to the principle of SRS microscopy and describe methods for its use in imaging lipid storage in Caenorhabditis elegans.

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 ...

Serial Block-Face Scanning Electron Microscopy (SBF-SEM) of Biological Tissue Samples

Serial block-face scanning electron microscopy (SBF-SEM) allows for the collection of hundreds to thousands of serially-registered ultrastructural images, offering an unprecedented three-dimensional view of tissue microanatomy. While SBF-SEM has seen an exponential increase in use in recent years, technical aspects such as proper tissue preparation and imaging parameters are paramount for the success of this imaging modality. This imaging system benefits from the automated nature of the device, allowing one to leave the microscope unattended during the imaging process, with the automated collection of hundreds of images possible in a single day. However, without appropriate tissue preparation cellular ultrastructure can be altered in such a way that incorrect or misleading conclusions might be drawn. Additionally, images are generated by scanning the block-face of a resin-embedded biological sample and this often presents challenges and considerations that must be addressed. The accumulation of electrons within the block during imaging, known as "tissue charging," can lead to a loss of contrast and an inability to appreciate cellular structure. Moreover, while increasing electron beam intensity/voltage or decreasing beam-scanning speed can increase image resolution, this can also have the unfortunate side effect of damaging the resin block and distorting subsequent images in the imaging series. Here we present a routine protocol for the preparation of biological tissue samples that preserves cellular ultrastructure and diminishes tissue charging. We also provide imaging considerations for the rapid acquisition of high-quality serial-images with minimal damage to the tissue block.

Serial Block-Face Scanning Electron Microscopy SBF-SEM of Biological Tissue Samples

This protocol outlines a routine method for using serial block-face scanning electron microscopy (SBF-SEM), a powerful 3D imaging technique. Successful application of SBF-SEM hinges on proper fixation and tissue staining techniques, as well as careful consideration of imaging settings. This protocol contains practical considerations for the entirety of this process.

生物组织样本的串行块面扫描电子显微镜 （SBF-SEM）

Serial block-face scanning electron microscopy (SBF-SEM) allows for the collection of hundreds to thousands of serially-registered ultrastructural images, ...

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

The acute mouse pancreatic tissue slice is a unique in situ preparation with preserved intercellular communication and tissue architecture that entails significantly fewer preparation-induced changes than isolated islets, acini, ducts, or dispersed cells described in typical in vitro studies. By combining the acute pancreatic tissue slice with live-cell calcium imaging in confocal laser scanning microscopy (CLSM), calcium signals can be studied in a large number of endocrine and exocrine cells simultaneously, with a single-cell or even subcellular resolution. The sensitivity permits the detection of changes and enables the study of intercellular waves and functional connectivity as well as the study of the dependence of physiological responses of cells on their localization within the islet and paracrine relationship with other cells. Finally, from the perspective of animal welfare, recording signals from a large number of cells at a time lowers the number of animals required in experiments, contributing to the 3R-replacement, reduction, and refinement-principle.

We present the preparation of acute pancreatic tissue slices and their use in confocal laser scanning microscopy to study calcium dynamics simultaneously in a large number of live cells, over long time periods, and with high spatiotemporal resolution.

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 ...

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

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此视频中的章节

激光捕获显微切割哺乳动物组织

激光诱导SD大鼠慢性高眼压高压模型

差分成像生物结构与双共振相干反斯托克斯拉曼散射（CARS）

天堂斑马鱼的细胞标记与激光Uncagable荧光葡聚糖

激光烧蚀斑马鱼Pronephros研究肾小管上皮再生

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

激光扫描显微术实时跟踪眼球前室胸腺细胞

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

生物组织样本的串行块面扫描电子显微镜（SBF-SEM）

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

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

激光捕获显微切割哺乳动物组织

激光诱导SD大鼠慢性高眼压高压模型

差分成像生物结构与双共振相干反斯托克斯拉曼散射（CARS）

天堂斑马鱼的细胞标记与激光Uncagable荧光葡聚糖

激光烧蚀斑马鱼Pronephros研究肾小管上皮再生

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

激光扫描显微术实时跟踪眼球前室胸腺细胞

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

生物组织样本的串行块面扫描电子显微镜 （SBF-SEM）

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

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

生物组织样本的串行块面扫描电子显微镜（SBF-SEM）