This material is based on work supported by the National Institutes of Health (1R03-CA182052, 1R15-CA202662), the National Science Foundation Graduate Research Fellowship Program (G.G., DGE-1450079), the Arkansas Biosciences Institute, and the University of Arkansas Doctoral Academy Fellowship. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the acknowledged funding agencies.

在阿肯色大学从人类受试者的研究项目，获得了本次研究的方方面面机构审查委员会的批准（IRB＃15-09-149）。所描述的方法是在按照批准的指导方针进行，并从所有参与者获得知情同意书。 1.高分辨率荧光显微内窥镜成像设备的组装注意:对于高分辨率荧光显微内窥镜形态的组件的概述的步骤可以在图2中被可视化。 <ol><li>将一个470 nm的分色镜内30毫米笼式立方体。 <ol><li>获得30毫米笼式立方体和删除二向色滤光片安装。 </li><li>放置一个470nm的分色镜的分色滤光器装入。 </li><li>重新插入并确保二向色滤光片装回笼子里面的立方体。 </li></ol></li><li>安装笼式支杆至30毫米笼式立方体。 <ol><li>安全4节1.5英寸的笼式支杆的笼式立方体的前面。 </li><li>安全4 3.0英寸的笼式支杆的笼式立方体的右侧。 </li><li>安全双2.0英寸的笼式支杆斜着笼式立方体的左侧。 </li></ol></li><li>构建笼板/镜头管组件。 <ol><li>获得一个1.0英寸的螺纹30毫米笼板，并使用所提供的螺纹应力自由卡环固定在笼板的内侧。 </li><li>拧一个1.0英寸镜头管无应力卡环。 </li><li>将第二个1.0英寸螺纹30毫米笼板的1.0英寸镜筒和调整标准卡环使两笼板齐平。 </li></ol></li><li> 1.0英寸笼板/镜头管组件滑到30毫米笼式立方体的左侧。 </li><li>构建直角镜座组件。 <ol><li>获取一个直角镜座和一个1.0英寸紫外增强铝镜。 </li><li>放置1.0 INC^ h紫外增强铝镜入镜安装并拧紧。 </li><li>安全4 2.0英寸的笼式支杆到镜子前安装</li><li>安全双2.0英寸的笼式支杆斜着笼式立方体的右侧。 </li></ol></li><li>连接直角反射镜组件贴装到1.0英寸笼板/透镜管组件的左侧由通过30毫米笼板的各自的开口放置在相对保持架组件杆。 </li><li>螺纹z轴平移通过3.0英寸的笼式支杆安装在组件的右侧。 </li><li>附加10X消色差物镜z轴平移安装。 </li><li>建立一个1.0英寸光纤适配器板/ XY轴平移透镜安装组件。 <ol style="margin-left: 40px;"><li>获取XY轴平移安装和一个1.0英寸光纤适配器板。 </li><li>固定1.0英寸光纤适配器板进入XY轴平移镜头卡口。 </li></ol></li><li>幻灯ŧ他1.0英寸光纤适配器/ XY轴平移透镜在物镜的前方安装组件。 </li><li>获得两个0.5英寸长，1.0英寸直径的镜筒，一是四十零分之四百四十○纳米带通滤波器（激发滤光片）和一个三十六分之五百二十五纳米带通滤波器（发射滤波器）。 </li><li>将每个过滤器的0.5英寸长，1.0英寸直径的透镜管内，使得在过滤器的外部的箭头朝向透镜管的侧面与外螺纹。 </li><li>装上过滤器组装。 <ol><li>获得两个标准的护环。 </li><li>固定0.5英寸长，1.0英寸直径的镜片管道内的过滤器与标准的护环。 </li><li>螺杆与所述激发滤光片至30毫米笼立方体的前镜筒并与所述发射滤波器的直角反射镜安装镜头筒螺丝。 </li><li>螺杆与所述发射滤波器的直角镜座前部的0.5英寸透镜管。 </li></ol></li><li> Obtain两个1.0英寸的螺纹30毫米笼板，并将其放置在包含过滤器的0.5英寸长，1.0英寸直径的镜片管的前面。 </li><li>使用环氧树脂或粘结力强，附加一个455纳米的LED连接到激发滤光片笼板。 </li><li>获取一个0.5英寸长，1.0英寸直径的透镜管和1.0英寸消色差双管透镜为50毫米焦距。 </li><li>放置透镜管这样，关于透镜的外侧的箭头朝向与外螺纹的镜筒的侧面内侧的管透镜。 </li><li>拧在管镜头的装配。 <ol><li>获取一个标准挡圈。 </li><li>固定0.5英寸长，1.0英寸直径的镜头管内的镜头与标准挡圈。 </li><li>与管透镜到最左边的笼板安装镜头筒。 </li></ol></li><li>将30毫米笼板的0.5英寸长，1.0英寸口径镜头包含镜头筒管的前面。 </li><li>附加<html>强调自由的扣环到30毫米笼板的内部。 </li><li>将USB黑白摄像机与无应力卡环笼板。 </li><li>构建光后安装设备。 <ol><li>获得4 0.5英寸杆支架，四0.5英寸光学员额，四人安装基座。 </li><li>固定0.5英寸杆架内的0.5英寸光学职位。 </li><li>固定0.5英寸杆支架上安装底座。 </li></ol></li><li>螺钉在四个光后安装装置至位于30毫米笼立方体下的螺丝孔，直角反射镜支架，连接到LED笼板，和连接到摄像机的笼板。 </li><li>在四个螺杆光后安装装置到任何一个光学平台或光表完成高分辨率荧光显微内窥镜形态的构造。 </li></ol>ftp_upload / 54564 / 54564fig2.jpg"/&gt; 图2:高分辨率荧光显微内窥镜形态的组装高分辨率荧光显微内窥镜形态可以通过构建1.0英寸直径大小的组件的外壳构成，与在处理分色镜采取特别的照顾，物镜，激发/发射滤波器，和管透镜。这些组件的玻璃表面必须用镜头纸小心处理。 <a href="http://ecsource.jove.com/files/ftp_upload/54564/54564fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 2.小​​组漫反射光谱形态的大会注意:对于子漫反射光谱形态的组件的概述的步骤可以在图3中被可视化。 <ol><li>得到钨卤素光源和，使用环氧树脂或强粘合剂，确保一个1.0英寸threadeð30毫米笼板推上前台。 </li><li>安全4 3.0英寸的笼式支杆的笼板。 </li><li>附加的z轴平移安装到笼式支杆。 </li><li>拧在20X消色差物镜z轴平移装入。 </li><li>建立一个光纤适配器板/ XY轴平移透镜安装组件。 <ol><li>获取XY轴平移安装和1.0英寸光纤适配器板。 </li><li>固定光纤适配器板进入XY轴平移镜头卡口。 </li></ol></li><li>滑动1.0英寸光纤适配器/ XY平移在物镜的前方安装组件。 </li><li>构建电机臂总成。 <ol><li>获取定制的铝合金电机臂和一个SMA光纤适配器板。 </li><li>拧在光纤适配器板（带外螺纹）进入铝壳电机臂（带内螺纹）。 </li><li>有四个＃4-400.5英寸螺丝将定制铝合金电机手臂适配器连接到电机的手臂。 </li></ol&gt; </li><li>构建电机/马达臂/电机壳组件。 <ol><li>获得定制铝马达壳体和400步的步进电机。 </li><li>排队的步进电机和电机壳体的螺钉孔，然后固定有四个＃4-40 0.5英寸螺钉。 </li><li>通过电动机臂组件的开口进给步进电机的旋转电机杆和紧固铝马达臂适配器上的锁紧螺钉。 </li></ol></li><li>构建光开关总成。 <ol><li>获取定制铝光开关和三个1.0英寸光纤适配器板。 </li><li>螺纹接合板到光开关的螺纹孔。 </li><li>有四个＃4-40 0.5英寸螺丝将定制铝光开关荧光屏上的光开关。 </li></ol></li><li>通过T的中心孔进给步进电机的旋转电动机杆连接马达/电动机臂/马达壳体组件到光开关他光开关。 </li><li>获取电路板及步进电机驱动器，然后将步进电机驱动程序在线路板的中心凹槽。 </li><li>观察的电连接示意图（ 图3，2.12）的步进电机驱动器，12伏电源和步进电机。 </li><li>如在电路图（ 图3，2.12）指定完成电动光学开关的结构连接步进电机驱动器，12伏电源和步进电机。 </li><li>在光开关组件和卤钨灯光源拧到光学面包板或光学平台附近，以前构造（ 图2，1.24）高分辨率荧光显微内窥镜组装。 </li><li>附加一个550微米的一端，0.22 NA跳线到马达臂组件的1.0英寸光纤适配器板。 </li><li>附上550微米的另一端，0.22 NA贴片电缆光纤连接或USB光谱仪。 </li><li>在五个远侧探针电缆拧到在仪器的各1.0英寸光纤适配器板，以完成多峰高分辨率成像和分漫反射光谱纤维束显微内窥镜的完成。 <ol><li>拧在中央直径1毫米的图像光缆在步骤1.9.2中提到的1.0英寸光纤适配器板。 </li><li>拧在最左边的200μm多模光纤电缆在步骤2.6中提到的1.0英寸光纤适配器板。 </li><li>在第2 次 200微米的多模光纤电缆螺丝附着于在步骤2.9.2中提到的钨-卤素灯最左边的1.0英寸光纤适配器。 </li><li>第3 届 200μm多模光纤电缆螺丝步骤2.9.2中提到的中间1.0英寸光纤适配器板。 </li><li>在第4 次 200μm多模光纤电缆螺丝步骤2.9.2中提到的最右边的1.0英寸光纤适配器板。 </li></OL&gt; </li></ol><img alt="图3" src="/files/ftp_upload/54564/54564fig3.jpg" /> 图3:子漫反射光谱形态的装配可以使用耦合到物镜碱性钨-卤素灯通过200微米的多模传输光纤将光聚焦来构造副漫反射光谱形态，和一个分光计。此外，定制的电动光学开关可以将灯泡纤维光谱仪路径内被构造为每SDS之间切换。使用多个光谱仪从多个科学数据采集可以绕过光开关器件调查。 <a href="http://ecsource.jove.com/files/ftp_upload/54564/54564fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 3.小组漫反射光谱成像设备校准注:在Following步骤（第3节）之前，必须光谱数据采集（第4）完成。 <ol><li>打开仪器的所有部件，包括455毫微米的LED，宽带钨卤素灯，CMOS摄像头，USB分光计，步进电机，和电机控制电路板。确保对钨 - 卤素灯快门打开。 </li><li>关闭所有环境光。 </li><li>打开自定义的数据采集软件。 </li><li>保持设备运行的灯30分钟达到适当的温度和用于从分光计固有噪声，以稳定。 </li><li>放置定制，3D印刷校准标准设备的底部开口内的20％的漫反射率标准。 </li><li>放置定制的最左边的槽内的光纤探头，3D印刷光纤保持器，在图4中展示。最左边的槽固定为2.1毫米，这是从光纤探头尖端到反射率标准的垂直距离运timum距离在到达光谱仪的信号最大化的374微米的第一SDS。 </li><li>调节电动光学切换到最左侧的位置，使得分光计被连接到374微米的第一SDS。 </li><li>设置积分时间为500毫秒。这种整合时间必须选择为不饱和光谱仪，但维持一个较低的实际积分时间。 </li><li>通过点击"获得频谱"中的软件获得的频谱，R最大374μm。 </li><li>关闭快门的钨-卤素灯和记录的频谱中，R 暗，374μm，背景噪声，通过点击"获取谱"的软件。一旦收购，再次打开快门。 </li><li>放置光纤探针的定制的最右端的插槽内，3D印刷光纤保持器，在图4中展示。最右边的槽固定从纤维-邻的垂直距离PTIC探针尖端到3.9毫米的反射率标准，这是在其中到达分光计的信号被最大化为730微米的第二SDS的最佳距离。 </li><li>调节电动光学开关至中间位置，使得分光计被连接到730微米的第二SDS。 </li><li>通过点击"获得频谱"中的软件获得的频谱，R最大730μm。 </li><li>关闭快门的钨-卤素灯和记录的频谱中，R 暗，730μm，背景噪声，通过点击"获取谱"的软件。 </li><li>再次打开快门。 </li></ol><img alt="图4" src="/files/ftp_upload/54564/54564fig4.jpg" /> 图4:该子漫反射光谱方式的校准对于实验性预校准，光纤探针尖端必须放置在不同从依赖于SDS的20％的漫反射标准垂直距离。达到一致在所有实验这些垂直距离，校正标准的设备被设计为保持在从20％的漫反射标准精确距离的探针（（a）中所示的装置的横截面）。在这种特定的光纤探头的设置，从钨-卤灯的光通过光开关在源-检测分离（b）所示374微米和（c）730微米（以从光路中除去电机和电机臂为清楚起见）。 （ 四 ）距离2.1毫米为374微米SDS，及（e）3.9毫米为需要校准的730微米SDS。 <a href="http://ecsource.jove.com/files/ftp_upload/54564/54564fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 4. 体内数据习得n和光学特性提取人体皮肤在本节中，多峰显微内窥镜技术将在体内对人皮肤来证明。 <ol><li>打开自定义数据采集软件，并通过点击"积分时间"调整分光计积分时间并进行设置，以便它是相同的校准，这是在这种情况下，（步骤3.8）500毫秒期间。 </li><li>确定皮肤在其中获取数据的区域，其可以在研究者的应用程序不同。在这种情况下，前臂的皮肤薄被选为一个示范。 </li><li>如果皮肤区域包含毛，去除毛用一次性无菌剃须刀。 </li><li>获得一个黄色荧光笔，其中含有羟基芘磺酸墨水，轻轻标记选择的皮肤面积。 </li><li>打开455nm的LED和关闭快门的钨 - 卤素灯。 </li><li>把探头与皮肤轻柔接触。 </li><li>移动探头轮上的组织的染色区域来查看软件的观察窗上心尖角化结构的现场高分辨率饲料。 </li><li>选择适当的曝光时间和增益，150毫秒，在此壳体10 dB的增益，以避免图像的饱和度，通过点击"曝光时间"和"增益"，在适当的值输入，然后点击"应用设置"的软件接口。 </li><li>通过点击软件界面"获取图像"获取图像。 </li><li>同时保持探针在相同的图像部位，关掉455nm的LED和打开快门的钨 - 卤素灯。 </li><li>调节电动光学切换到左侧位置，使得分光计被连接到374微米的第二SDS。 </li><li>获取的光谱，R 组织，374μm，点击"获取光谱"的软件界面。 </li><li>调节电动光学开关至中间位置，使得第在分光计被连接到730微米的第二SDS。 </li><li>获取的光谱，R 组织，730μm，点击"获取光谱"的软件界面。 </li><li>打开自定义后处理软件。 </li><li>通过点击"运行"从其中当由软件提示数据被保存在文件夹运行后处理软件，并选择高分辨率荧光图像，四校准光谱，和两个在体内光谱。 注:定制软件获得使用以下公式真绝对反射（R 腹肌，374μm和R 腹肌，730μm）。 <img alt="式（1）" src="/files/ftp_upload/54564/54564eq1.jpg" /> <img alt="公式（2）" src="/files/ftp_upload/54564/54564eq2.jpg" /> 后处理代码，如先前描述的，计算的漫反射光谱的拟合曲线（等式1和2），然后DETErmines组织的生理参数，包括（[血红蛋白]，[梅尔]，和圣保罗2）。11,22,24 </li></ol>

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The authors declare that they have no competing financial interests.

<html>多峰高分辨率成像和分漫反射光谱纤维束显微内窥镜此处报告可以被优化并用于各种应用，包括内窥镜或手持使用用于人类或动物研究中使用由调查员。因此，它提供了用于在体内根尖组织微结构可视化与来自两个不同的组织深度的血红蛋白浓度，黑色素浓度和组织氧饱和度的测量值的灵活的方法。本文介绍了用于光纤探针的说明书，所述的协议用于组装高分辨率成像系统和分漫反射?...

纤维束显微内窥镜技术通常使用两种成像技术或分光技术的组合分析体内组织中 1-3一种这样的成像技术，高分辨率荧光显微内窥镜，可以形象根尖组织微架构具有亚细胞分辨率的小，微尺度场的图，利用局部造影剂如原黄素，荧光素，或羟基芘磺酸油墨。1,3-11该成像模态已示于定性实时低区分患病的和健康上皮组织有前途的临床表现间观察员变异8偶尔，调查人员将使用高分辨率荧光显微镜的数据中提取定量的功能，如细胞与核的大小或腺区，但这仍然瞄准往可视化的组织形态的主要定性技术。1,3,8- 10在另一方面，光谱技术等作为漫反射光谱，正朝着提供功能组织信息并显示在多个器官定量识别病变癌变看好的临床表现有针对性的。2,12-15 因此，有必要对掺入两种类型的模式，从而可能进一步降低观察者间变异性，维持组织微架构的实时可视化，并提供组织健康的一个更完整的分析的装置。为了实现这个目标，在基于探针的多峰仪器构建结合在一个单一的光纤探针两种模式:高分辨率荧光显微内窥镜和分漫反射光谱11此方法共寄存器定性心尖的高清晰度图像组织形态（结构特性）与来自两个不同的组织深度定量光谱信息（功能性），包括局部血红蛋白浓度（[血红蛋白]），黑色素浓度（[梅尔]）和氧饱和度（SAO 2）。11,12,16这个特定副漫反射光谱形态使用两个源-检测器分离（SDSS）采样两个独特的组织深度，以提供通过采样到地下室膜和皮下组织间质组织健康更全面的了解。11 纤维探头由具有大约50,000 4.5微米直径的纤维元件，1.1mm的包层直径和1.2mm的整体涂层直径的中央毫米直径的1个图像的纤维。图像纤维是通过用220微米的包层直径5 200微米直径的纤维包围。每200μm多模光纤位于中心到中心的距离864微米从图像光纤的中心了。每200微米的多模光纤的相隔25°。使用最左边的200μm多模光纤为"源"的纤维，以及附加次稀土元素200μm多模光纤为"收藏"的纤维，这种几何必然创建三个中心到中心的科学数据集的374微米，730微米，1051微米，1323微米。纤维尖端封闭在保持纤维常数之间的距离的筒状金属外壳。圆筒形金属外壳的直径为3毫米。前端（朝向光纤探针尖端）的光纤探针的为2英尺长。探针然后分离成在近端（朝向仪表）的六个相应的各个纤维是额外的2英尺长，对于4英尺总长度。 图1示出了光纤探针的表示。 <img alt="图1" src="/files/ftp_upload/54564/54564fig1.jpg" /> 图1:光纤探头设计的光纤探针包括一个毫米直径的1图像纤维和四个200微米的多模光纤。这个图中显示的表示（a）该金属端盖这限制了纤维的几何形状在探头尖端，得到的安全数据表374，730，和1 051微米相对于最左边的200微米的多模光纤（比例尺≈1毫米）， （b）该纤维被约束在金属盖内，示出了纤维芯，光纤包层，和纤维涂层（比例尺≈1毫米），（C）周围的纤维保护聚酰胺护套（比例尺≈1毫米），（ 四 ）成品远侧尖端的探头，与金属手指抓握和含有所有纤维单黑线（比例尺≈4毫米），和（e）的探针（比例尺≈为4mm远侧尖端的一个图片）。 <a href="http://ecsource.jove.com/files/ftp_upload/54564/54564fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 这种多模式的仪器和相关TECHNI阙是一个探头内的这些方式的第一组合，但也存在其他组合结构/功能技术，结合不同的方式。例如，高光谱成像结合定量血红蛋白和黑色素特性，17,18和其它技术已经开发了与组织蛋白表达的分析，19结合光学相干断层扫描（OCT），仅举几宽视场成像。对使用的一般的光纤探针可用于各种目的，包括在口腔中的下胃肠道和食道或作为手持式探头内窥镜用途的使用进行优化的紧凑和易于实施的仪器设置本文报告和外部皮肤位置。11,20 该仪器的硬件既需要自定义的数据采集和处理后的代码来获得漫反射光谱，然后提取所产生的volumE-平均组织的生理参数，包括[血红蛋白]，[梅尔]和血氧饱和。定制数据获取代码的建立是为了允许来自摄像头的同时采集（高分辨率荧光显微镜）和一个分光计（对于漫反射光谱）。驱动程序通常可从制造商的网站以允许与各种编程语言的融合。定制后处理代码出口先验吸收值的体内 [血红蛋白]和[梅尔] 21，然后利用创建该光谱的拟合曲线一个先前开发的非线性优化拟合过程22的拟合曲线是通过最小化建本身和原光谱和之间χ2值从拟合曲线并具有最低χ2值22的代码确定该组织的生理参数（[血红蛋白]，[梅尔]和血氧饱和2）可修改成包括吸收来自其他发色为好，如这里使用的外源性羟基芘磺酸墨水，使目标生理参数均不受影响。 组织健康的生理指标，如[血红蛋白]，[梅尔]和血氧饱和，可作为肿瘤对治疗的反应的报告或本地血管和血管生成的指标。14,23包括一个高分辨率荧光显微内窥镜形态有助于引导探头的位置，并提供与调查上皮组织结构和功能之间的关系更完整的画面。在这篇文章中，建设和多式联运显微内窥镜的应用说明。11

<table><tbody><tr><td>30 mm Cage Cube with Dichroic Filter Mount</td><td>Thorlabs, Inc.</td><td>CM1-DCH</td><td></td></tr><tr><td>470 nm Dichroic Mirror (Beam Splitter)</td><td>Chroma Corporation</td><td>T470lpxr</td><td></td></tr><tr><td>Cage Assembly Rod, 1.5&quot;, 4-Pack</td><td>Thorlabs, Inc.</td><td>ER1.5-P4</td><td></td></tr><tr><td>Cage Assembly Rod, 3.0&quot;, 4-Pack</td><td>Thorlabs, Inc.</td><td>ER3-P4</td><td></td></tr><tr><td>Cage Assembly Rod, 2.0&quot;, 4-Pack</td><td>Thorlabs, Inc.</td><td>ER2-P4</td><td></td></tr><tr><td>SM1-Threaded 30 mm Cage Plate</td><td>Thorlabs, Inc.</td><td>CP02</td><td></td></tr><tr><td>SM1 Series Stress-Free Retaining Ring</td><td>Thorlabs, Inc.</td><td>SM1PRR</td><td></td></tr><tr><td>SM1 Lens Tube, 1.00&quot; Thread Depth</td><td>Thorlabs, Inc.</td><td>SM1L10</td><td></td></tr><tr><td>Right-Angle Kinematic Mirror Mount</td><td>Thorlabs, Inc.</td><td>KCB1</td><td></td></tr><tr><td>1&quot; UV Enhanced Aluminum Mirror</td><td>Thorlabs, Inc.</td><td>PF10-03-F01</td><td></td></tr><tr><td>Z-Axis Translation Mount</td><td>Thorlabs, Inc.</td><td>SM1Z</td><td></td></tr><tr><td>10X Olympus Plan Achromatic Objective</td><td>Thorlabs, Inc.</td><td>RMS10X</td><td></td></tr><tr><td>XY Translating Lens Mount</td><td>Thorlabs, Inc.</td><td>CXY1</td><td></td></tr><tr><td>SMA Fiber Adapter Plate with SM1 Thread</td><td>Thorlabs, Inc.</td><td>SM1SMA</td><td></td></tr><tr><td>SM1 Lens Tube, 0.50&quot; Thread Depth</td><td>Thorlabs, Inc.</td><td>SM1L05</td><td></td></tr><tr><td>440/40 Bandpass Filter (Excitation)</td><td>Chroma Corporation</td><td>ET440/40x</td><td></td></tr><tr><td>525/36 Bandpass Filter (Emission)</td><td>Chroma Corporation</td><td>ET525/36m</td><td></td></tr><tr><td>Quick Set Epoxy</td><td>Loctite</td><td>1395391</td><td></td></tr><tr><td>455 nm LED Light Housing Kit - 3-Watt</td><td>LED Supply</td><td>ALK-LH-3W-KIT</td><td></td></tr><tr><td>1&quot; Achromatic Doublet, f = 50 mm</td><td>Thorlabs, Inc.</td><td>AC254-050-A</td><td></td></tr><tr><td>Flea 3 USB Monochrome Camera</td><td>Point Grey, Inc.</td><td>FL3-U3-32S2M-CS</td><td></td></tr><tr><td>0.5&quot; Post Holder, L = 1.5&quot;</td><td>Thorlabs, Inc.</td><td>PH1.5</td><td></td></tr><tr><td>0.5&quot; Optical Post, L = 4.0&quot;</td><td>Thorlabs, Inc.</td><td>TR4</td><td></td></tr><tr><td>Mounting Base, 1&quot; x 2.3&quot; x 3/8&quot;</td><td>Thorlabs, Inc.</td><td>BA1S</td><td></td></tr><tr><td>Long Lifetime Tungsten-Halogen Light Source (Vis-NIR)</td><td>Ocean Optics</td><td>HL-2000-LL</td><td></td></tr><tr><td>20X Olympus Plan Objective</td><td>Edmund Optics, Inc.</td><td>PLN20X</td><td></td></tr><tr><td>Custom-Built Aluminum Motor Arm</td><td>N/A</td><td>N/A</td><td>Custom designed and built</td></tr><tr><td>Custom-Built Aluminum Motor Arm Adaptor</td><td>N/A</td><td>N/A</td><td>Custom designed and built</td></tr><tr><td>Custom-Built Aluminum Motor Housing</td><td>N/A</td><td>N/A</td><td>Custom designed and built</td></tr><tr><td>Stepper Motor - 400 steps/revolution</td><td>SparkFun Electronics</td><td>ROB-10846</td><td>Multiple suppliers</td></tr><tr><td>Custom-Built Aluminum Optical Fiber Switch</td><td>N/A</td><td>N/A</td><td>Custom designed and built</td></tr><tr><td>Custom-Built Aluminum Optical Fiber Switch Face-Plate</td><td>N/A</td><td>N/A</td><td>Custom designed and built</td></tr><tr><td>Arduino Uno - R3</td><td>SparkFun Electronics</td><td>DEV-11021</td><td>Multiple suppliers</td></tr><tr><td>Electronic Breadboard - Self-Adhesive</td><td>SparkFun Electronics</td><td>PRT-12002</td><td>Multiple suppliers</td></tr><tr><td>EasyDriver - Stepper Motor Driver</td><td>Sparkfun Electronics</td><td>ROB-12779</td><td></td></tr><tr><td>12 V, 229 mA Power Supply</td><td>Phihong</td><td>PSM03A</td><td>Multiple suppliers</td></tr><tr><td>Enhanced Sensitivity USB Spectrometer (Vis-NIR)</td><td>Ocean Optics</td><td>USB2000+VIS-NIR-ES</td><td></td></tr><tr><td>550 &amp;micro;m, 0.22 NA, SMA-SMA Fiber Patch Cable</td><td>Thorlabs, Inc.</td><td>M37L01</td><td></td></tr><tr><td>Custom-Built Fiber-Optic Probe</td><td>Myriad Fiber Imaging</td><td>N/A</td><td></td></tr><tr><td>20% Spectralon Diffuse Reflectance Standard</td><td>Labsphere, Inc.</td><td>SRS-20-010</td><td></td></tr><tr><td>Standard Yellow Highlighter</td><td>Sharpie</td><td>25005</td><td>Multiple suppliers, proflavine or fluorescein can be substituted</td></tr></tbody></table>

multimodal imaging and spectroscopy fiber-bundle microendoscopy platform for non-invasive, in vivo tissue analysis

最近纤维束显微内窥镜技术使用两种成像技术或分光技术的组合在体内组织的非侵入性的分析。结合成像和光谱学技术成一个单一的光学探针可提供组织健康的更完整的分析。在这篇文章中，两种不同的方式组合，高分辨率荧光显微内窥镜成像和漫反射光谱，成一个单一的光学探头。高分辨率荧光显微内窥镜成像是用于可视化根尖组织微结构的技术，虽然大多是定性的技术，已经证明肿瘤和非肿瘤组织之间的有效实时分化。漫反射光谱是一种技术，它可以提取组织的生理参数，包括当地的血红蛋白浓度，黑色素浓度和血氧饱和度。本文介绍的规格řequired构建光纤探头，如何建立仪器仪表，然后演示了在体内对人体皮肤的技术。这项工作表明该组织的微架构，特别是心尖皮肤角质，可以和其相关的生理参数共同注册。这里介绍的仪器和纤维束探针可以被优化为在各种器官系统的使用任一手持或内窥镜兼容设备。另外的临床研究来测试此技术对于不同的上皮疾病状态的可行性。

按照此协议，研究者将获得组织部位的在焦高分辨率图像与全视场（ 图5）。细胞的轮廓可以看出，如果沾有墨水羟基芘磺酸从一个黄色荧光笔，而单个细胞核，如果能与染料染色，如原黄素可以看出。下列光谱采集，后处理软件使用体内血红蛋白浓度（[血红蛋白]）和黑素浓度（[梅尔]）21的先验知识，以适应子漫反射光谱，并确定为[血...

Watch this Scientific Journal Video about Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis at JoVE.com

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

The assembly and use of a multimodal microendoscope is described which can co-register superficial tissue image data with tissue physiological parameters including hemoglobin concentration, melanin concentration, and oxygen saturation. This technique can be useful for evaluating tissue structure and perfusion, and can be optimized for individual needs of the investigator.

多峰成像和光谱学纤维束显微内窥镜纲要非侵入性，<em&gt;在体内</em&gt;组织分析

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of ...

multimodal-imaging-spectroscopy-fiber-bundle-microendoscopy-platform

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

7.8K 次观看.  University of Arkansas. The assembly and use of a multimodal microendoscope is described which can co-register superficial tissue image data with tissue physiological parameters including hemoglobin concentration, melanin concentration, and oxygen saturation. This technique can be useful for evaluating tissue structure and perfusion, and can be optimized for individual needs of the investigator.

视频: Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ 1. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user.
We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture 2-5, in excised 6 and living animal tissues 2,5, and in human tissues in vivo 2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine 2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) 5, and fluoroscein 6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.

高分辨率光纤内窥镜为原位细胞成像

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution.  ...

科研

生物工程

Dual-mode Imaging of Cutaneous Tissue Oxygenation and Vascular Function

Accurate assessment of cutaneous tissue oxygenation and vascular function is important for appropriate detection, staging, and treatment of many health disorders such as chronic wounds. We report the development of a dual-mode imaging system for non-invasive and non-contact imaging of cutaneous tissue oxygenation and vascular function. The imaging system integrated an infrared camera, a CCD camera, a liquid crystal tunable filter and a high intensity fiber light source. A Labview interface was programmed for equipment control, synchronization, image acquisition, processing, and visualization. Multispectral images captured by the CCD camera were used to reconstruct the tissue oxygenation map. Dynamic thermographic images captured by the infrared camera were used to reconstruct the vascular function map. Cutaneous tissue oxygenation and vascular function images were co-registered through fiduciary markers. The performance characteristics of the dual-mode image system were tested in humans.

A dual-mode imaging system was developed for non-contact assessment of cutaneous tissue oxygenation and vascular function.

双模式成像，皮肤组织氧合和血管功能

Accurate assessment of cutaneous tissue oxygenation and vascular function is important for appropriate detection, staging, and treatment of many health ...

医学

Universal Hand-held Three-dimensional Optoacoustic Imaging Probe for Deep Tissue Human Angiography and Functional Preclinical Studies in Real Time

The exclusive combination of high optical contrast and excellent spatial resolution makes optoacoustics (photoacoustics) ideal for simultaneously attaining anatomical, functional and molecular contrast in deep optically opaque tissues. While enormous potential has been recently demonstrated in the application of optoacoustics for small animal research, vast efforts have also been undertaken in translating this imaging technology into clinical practice. We present here a newly developed optoacoustic tomography approach capable of delivering high resolution and spectrally enriched volumetric images of tissue morphology and function in real time. A detailed description of the experimental protocol for operating with the imaging system in both hand-held and stationary modes is provided and showcased for different potential scenarios involving functional and molecular studies in murine models and humans. The possibility for real time visualization in three dimensions along with the versatile handheld design of the imaging probe make the newly developed approach unique among the pantheon of imaging modalities used in today&#8217;s preclinical research and clinical practice.

We provide herein a detailed description of the experimental protocol for imaging with a newly developed hand-held optoacoustic (photoacoustic) system for three-dimensional functional and molecular imaging in real time. The demonstrated powerful performance and versatility may define new application areas of the optoacoustic technology in preclinical research and clinical practice.

通用型手持式三维光声成像探测深部组织人力造影和功能的临床前研究中实时

The exclusive combination of high optical contrast and excellent spatial resolution makes optoacoustics (photoacoustics) ideal for simultaneously ...

Three-Dimensional Ultrasonic Needle Tip Tracking with a Fiber-Optic Ultrasound Receiver

Ultrasound is frequently used for guiding minimally invasive procedures, but visualizing medical devices is often challenging with this imaging modality. When visualization is lost, the medical device can cause trauma to critical tissue structures. Here, a method to track the needle tip during ultrasound image-guided procedures is presented. This method involves the use of a fiber-optic ultrasound receiver that is affixed within the cannula of a medical needle to communicate ultrasonically with the external ultrasound probe. This custom probe comprises a central transducer element array and side element arrays. In addition to conventional two-dimensional (2D) B-mode ultrasound imaging provided by the central array, three-dimensional (3D) needle tip tracking is provided by the side arrays. For B-mode ultrasound imaging, a standard transmit-receive sequence with electronic beamforming is performed. For ultrasonic tracking, Golay-coded ultrasound transmissions from the 4 side arrays are received by the hydrophone sensor, and subsequently the received signals are decoded to identify the needle tip&#39;s spatial location with respect to the ultrasound imaging probe. As a preliminary validation of this method, insertions of the needle/hydrophone pair were performed in clinically realistic contexts. This novel ultrasound imaging/tracking method is compatible with current clinical workflow, and it provides reliable device tracking during in-plane and out-of-plane needle insertions.

Accurate and efficient visualization of invasive medical devices is extremely important in many ultrasound-guided minimally invasive procedures. Here, a method for localizing the spatial position of a needle tip relative to the ultrasound imaging probe is presented.

基于光纤超声接收机的三维超声针尖跟踪

Ultrasound is frequently used for guiding minimally invasive procedures, but visualizing medical devices is often challenging with this imaging modality. ...

工程学

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions preclinically. However, valid assessment of pathological alterations often requires histological analysis, and when performed ex vivo, necessitates death of the animal. Therefore in conventional experimental settings, intra-individual follow-up examinations are rarely possible. Thus, development of murine endoscopy in live mice enables investigators for the first time to both directly visualize the gastrointestinal mucosa and also repeat the procedure to monitor for alterations. Numerous applications for in vivo murine endoscopy exist, including studying intestinal inflammation or wound healing, obtaining mucosal biopsies repeatedly, and to locally administer diagnostic or therapeutic agents using miniature injection catheters. Most recently, molecular imaging has extended diagnostic imaging modalities allowing specific detection of distinct target molecules using specific photoprobes. In conclusion, murine endoscopy has emerged as a novel cutting-edge technology for diagnostic experimental in vivo imaging and may significantly impact on preclinical research in various fields.

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Small animal imaging techniques allow serial diagnostic examinations and therapeutic interventions in vivo. Recently, the scope of applications has significantly widened and currently includes assessment of colonic tumor development, wound healing and monitoring of inflammation. This protocol illustrates these diverse potential applications of murine endoscopy.

鼠内镜<em&gt;体内</em&gt;发生和肠道的评价多模式成像伤口愈合和炎症

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions ...

使用 VSFG 显微镜进行多模式、快速、高光谱化学分析

Multimodal Nonlinear Hyperspectral Chemical Imaging Using Line-Scanning Vibrational Sum-Frequency Generation Microscopy

Vibrational sum-frequency generation (VSFG), a second-order nonlinear optical signal, has traditionally been used to study molecules at interfaces as a spectroscopy technique with a spatial resolution of ~100 &#181;m. However, the spectroscopy is not sensitive to the heterogeneity of a sample. To study mesoscopically heterogeneous samples, we, along with others, pushed the resolution limit of VSFG spectroscopy down to ~1 &#181;m level and constructed the VSFG microscope. This imaging technique not only can resolve sample morphologies through imaging, but also record a broadband VSFG spectrum at every pixel of the images. Being a second-order nonlinear optical technique, its selection rule enables the visualization of non-centrosymmetric or chiral self-assembled structures commonly found in biology, materials science, and bioengineering, among others. In this article, the audience will be guided through an inverted transmission design that allows for imaging unfixed samples. This work also showcases that VSFG microscopy can resolve chemical-specific geometric information of individual self-assembled sheets by combining it with a neural network function solver. Lastly, the images obtained under brightfield, SHG, and VSFG configurations of various samples briefly discuss the unique information revealed by VSFG imaging.

作者聚焦:揭示 VSFG 显微镜在研究介观异质自组装结构中的潜力 

A multimodal, rapid hyperspectral imaging framework was developed to obtain broadband vibrational sum-frequency generation (VSFG) images, along with brightfield, second harmonic generation (SHG) imaging modalities. Due to the infrared frequency being resonant with molecular vibrations, microscopic structural and mesoscopic morphology knowledge is revealed of symmetry-allowed samples.

基于线扫描振动和频生成显微镜的多模态非线性高光谱化学成像

Vibrational sum-frequency generation (VSFG), a second-order nonlinear optical signal, has traditionally been used to study molecules at interfaces as a ...

化学

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

In cardiac muscle, intracellular Ca2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and geometric&#160;deformation. Our understanding of the internal relationships between these events has been limited because we can neither &#39;see&#39; inside the muscle nor precisely track the spatio-temporal nature of excitation-contraction dynamics. To resolve these problems, we have constructed a device that combines a suite of imaging modalities. Specifically, it integrates a brightfield microscope to measure local changes of sarcomere length and tissue strain, a fluorescence microscope to visualize the Ca2+ transient, and an optical coherence tomograph to capture the tissue&#39;s geometric&#160;changes throughout the time-course of a cardiac cycle. We present here the imaging infrastructure and associated data collection framework. Data are collected from isolated rod-like tissue structures known as trabeculae carneae. In our instrument, a pair of position-controlled platinum hooks hold each end of an ex vivo muscle sample while it is continuously superfused with nutrient-rich saline solution. The hooks are under independent control, permitting real-time control of muscle length and force. Lengthwise translation enables the piecewise scanning of the sample, overcoming limitations associated with the relative size of the microscope imaging window (540 &#181;m by 540 &#181;m) and the length of a typical trabecula (&#62;2000 &#181;m). Platinum electrodes at either end of the muscle chamber stimulate the trabecula at a user-defined rate. We exploit the stimulation signal as a trigger for synchronizing the data from each imaging window to reconstruct the entire sample twitching under steady-state conditions. Applying image-processing techniques to these brightfield imaging data provides tissue displacement and sarcomere length maps. Such a collection of data, when incorporated into an experiment-modeling pipeline, will provide a deeper understanding of muscle contractile homogeneity and heterogeneity in physiology and pathophysiology.

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

This protocol presents a collection of sarcomere, calcium, and macroscopic geometric&#160;data from an actively contracting cardiac trabecula ex vivo. These simultaneous measurements are made possible by the integration of three imaging modalities.

同时布莱特菲尔德， 荧光， 和光学相干断层成像收缩心脏特拉贝库莱 Ex Vivo

In cardiac muscle, intracellular Ca2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and ...

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 ...

多峰成像和光谱学纤维束显微内窥镜纲要非侵入性，

摘要

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此视频中的章节

高分辨率光纤内窥镜为原位细胞成像

双模式成像，皮肤组织氧合和血管功能

通用型手持式三维光声成像探测深部组织人力造影和功能的临床前研究中实时

基于光纤超声接收机的三维超声针尖跟踪

鼠内镜

基于线扫描振动和频生成显微镜的多模态非线性高光谱化学成像

基于线扫描振动和频生成显微镜的多模态非线性高光谱化学成像

基于线扫描振动和频生成显微镜的多模态非线性高光谱化学成像

同时布莱特菲尔德，荧光，和光学相干断层成像收缩心脏特拉贝库莱 Ex Vivo

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

多峰成像和光谱学纤维束显微内窥镜纲要非侵入性，

摘要

探索更多视频

此视频中的章节

高分辨率光纤内窥镜为原位细胞成像

双模式成像，皮肤组织氧合和血管功能

通用型手持式三维光声成像探测深部组织人力造影和功能的临床前研究中实时

基于光纤超声接收机的三维超声针尖跟踪

鼠内镜

基于线扫描振动和频生成显微镜的多模态非线性高光谱化学成像

基于线扫描振动和频生成显微镜的多模态非线性高光谱化学成像

基于线扫描振动和频生成显微镜的多模态非线性高光谱化学成像

同时布莱特菲尔德， 荧光， 和光学相干断层成像收缩心脏特拉贝库莱 Ex Vivo

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

同时布莱特菲尔德，荧光，和光学相干断层成像收缩心脏特拉贝库莱 Ex Vivo

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