We would like to acknowledge the Department of Pathology at the University of Illinois at Chicago for financial support. Histology and visible imaging services were provided by the Research Resources Center - Research Histology and Tissue Imaging Core at the University of Illinois at Chicago established with the support of the Vice Chancellor of Research, in particular we would like to thank Ryan Deaton and Andy Hall for their expertise. We would also like to thank Agilent Technologies, in particular Frank Weston for support and loaning of additional IR lens.

1.建立一个FT-IR显微镜和获取组织图像<ol><li>部分中，在4微米厚的福尔马林固定，石蜡包埋的组织块上使用一个切片机的IR兼容幻灯片。另外，部分液氮冷冻的组织在4微米的厚度上使用低温恒温器的IR兼容的幻灯片。 注意:通常情况下，一个序列段也将在载玻片上获得的，并用一特殊（如苏木精和曙红（H＆E））或免疫组织化学染色。此外，培养的细胞系，可生长在红外兼容幻灯片。 IR兼容的幻灯片可以IR反射幻灯片反射成像模式（例如，MirrIR幻灯片，金）或IR透明幻灯片传输模式成像（如曝气生物滤池2，氟化钙）。 </li><li>清除的FT-IR显微镜和分光计使用任一干燥空气或干燥氮气从系统除去大气中的水。等待至少45分钟前成像，以确保彻底purg即</li><li>凉爽两个焦平面检测器（79 K）和在使用液氮的FT-IR显微镜内部的MCT检测器。冷却该探测器大约每6小时。 注意！液态氮是一种低温流体，并可能导致冷烧伤，冻伤，在密闭空间窒息。 </li><li>安装在显微镜舞台上的FT-IR成像样本幻灯片。 </li><li>确保可见光为"ON"，然后或者使用望远镜或样品捕获程序（它提供了实时的组织的可视图像），聚焦在样​​品上。 </li><li>打开捆绑软件包，例如决议亲，然后点击"收藏"，然后单击"诊断"，然后选择"对齐光谱仪"。确保该光束路径到光谱仪检测器内部不受阻碍。 </li><li>点击"成像设置"。 </li><li>在"电子"选项卡中，选择相应的"速度"，"在萨mpling比（UDR）"和"根据系统的过滤器"的设置。 注:在本例中，设置速度= 2.5千赫，UDR = 4和过滤器=无。 </li><li>在"光学"选项卡上确保"中红外源"，"打开光圈"和"100％光束衰减器"被选中。 </li><li>校准系统，在"光学"选项卡，选择"检测"为"地"，"显微镜检测"为"左"，然后选择"透光"或"反射"下"光学模式"根据类型的滑动被使用。 </li><li>单击设置，这将打开"蓝瑟控制"窗口。 注:对于反射模式按照1.12和1.13和传输模式按照1.14说明1.16指令。继续从1.17为反射和传输。 </li><li> 对于反射模式 ，在＆＃8216;蓝瑟控制"，点击"原始"。通过平台控制操纵杆和观看FT-IR干涉图像（右上图）的实时取景，移动到一个干净的地方在幻灯片上。 </li><li>调整积分时间约8000计数。接着，移动台找到一个片组织与结构，组织的优选的边缘，并完善了图像的焦点。变化"温暖"和"对比"选项，以帮助形成图像，以改善聚焦，这将会对IR数据没有影响。继续从1.18。 </li><li> 对于传输模式 ，在"蓝瑟控制"，点击"原始"。通过平台控制操纵杆和观看FT-IR干涉图像（右上图）的实时取景，移动到幻灯片的洁净区。 </li><li>调整积分时间约8000计数。 </li><li>调整底部聚焦/冷凝器目标计数的数量增加至其maximu米。观看在瑟控制右下方图像的形状，以确保它是高斯在外观和相对均匀的。再次调整积分时间约8000计数。 注意:如果任何在FT-IR干涉图像的像素的变白，减少积分时间。 </li><li>移动台找到一个片组织与结构，组织的优选的边缘和完善的图像的焦点。 （可选）更改的温暖和对比度选项，以帮助形成图像，以提高重点，但对IR数据没有影响。 </li><li>使用阶段控制操纵杆，移动到滑动件的清洁区。按"校准"按钮。确保"超出了范围（OOR）"值小于50的"高通量"和"低流量"数之间的差异是至少有4000计数。 </li><li>选择"OK"两次。在光学标签中选择; &#39;探测器&#39;=&#39;的MCT&#39;，&#39;微指令应对探测器&#39;=&#39;右&#39;，然后单击"设置"。在这一点上会有在屏幕上的FT-IR干涉图。点击"查找Centerburst"。点击"好"。 </li><li>在"光学"标签，重新选择&#39;检测&#39;=&#39;地&#39;，&#39;显微镜检测&#39;=&#39;左&#39;，然后选择"设置"。 </li><li>在蓝瑟控制 - 确保图像仍然在一个干净的地方，然后再次单击"校准"。点击"好"。 </li><li>接着，采集背景的FT-IR图像以校正从大气，系统和滑动吸光度。进入"电子"标签，然后选择合适的光谱分辨率，通常为4厘米-1或8 -1的组织。 </li><li>转到"背景"选项卡，然后输入128"扫描共同添加"。选择"新建文件..."按钮，并放置在适当的褶皱背景文件呃。检查"映像"选项卡，以确保拼接为1 1。点击"背景"，等待扫描完成并确认在哪里保存文件。单击背景FT-IR图像上的区域，并检查频谱。 </li><li>取样品的大型马赛克图像，选择"映像"选项卡，然后插入X和Y的帧数为你想获得马赛克的大小。 </li><li>点击"设置"，并在蓝瑟控制使用现场IR的视角以寻找感兴趣的区域。如果采取了马赛克，居中活饲料在感兴趣的区域的中间。点击"好"。进入"电子"选项卡，并输入扫描次数共同加入（通常是值2和16扫描之间的组织）。点击"扫描"。 </li><li>在屏幕上查看所收集的FT-IR图像，以确保它看起来聚焦。点击图片，从该位置弹出一个红外光谱，并检查它看起来有一个可以接受的信噪比。阿奎重新采样的可视图像。 </li><li>保存图像，并将其导出到需要的格式，如ENVI-IDL。 </li></ol> 2.适应的FT-IR显微镜的高清晰度功能注意:大多数的FT-IR系统都配备有一个客观的大约15X的放大倍率和0.5数值孔径（NA）。到图像中的高清晰度模式时，一个IR兼容36X或74X物镜可用于给衍射受限的成像能力。 <ol><li>按逆时针方向拧开卸下系统的15X目标。 </li><li>拧上的36X或74X在其位的目标。调整使用前的目标。使用镜头延长管带来的客观足够接近的样本。 </li><li>将组织在新的目标和重点使用或者使用双筒望远镜或样品采集的程序可见光。 注意:高清晰度成像必须在传输模式来完成。的非常紧密的工作距离SE目标（1-2毫米）和重点很浅景深需要重点慢慢地，小心地抽出时间来降低目标不接触样本。 </li><li>按照说明，从1.6到1.11。 注:由于存在显著更难以集中和少得多的光到达检测器，以下方案是基于1.14调整。 </li><li>在传输模式，单击"原始"。通过平台控制操纵杆和观看FT-IR干涉图像（右上图）的实时取景，移动到组织和重点（最好在边缘）。 注意:这可能是困难的，因此调整了"温情"和"对比"选项，以帮助形成图像，以提高对焦（这些选项都对IR的数据没有影响）。 </li><li>一旦集中，使用阶段的控制操纵杆和现场IR视图移动到幻灯片的洁净区。调整积分时间约8000计数。调整底部的重点目标增加计数数目到其最大值。再次调整积分时间约8000计数。 </li><li>移动台找到一个片组织与结构，组织的优选的边缘，并完善了图像的焦点。 </li><li>如果镶嵌在高分辨率的图像，调整阶段，以允许跨越的距离所需的精确拼接正确运动。要更改舞台的距离设置，请在蓝瑟控制"设置舞台"，调整的水平和垂直对齐设置，以便正确的镶嵌。 </li><li>使用阶段控制操纵杆移动到滑动件的清洁区。按"校准"按钮。确保超出了范围（OOR）值小于50为36X和74X的目标。确保了高通量和低通量值之间的差至少为1000的计数为74X物镜和2000计数的36X物镜。 </li><li>继续从1.19到1.27。扫描在1.23的数量和1.25将需要由于降低的信号，以用于背景图象约256次扫描和16到128的扫描的组织图像进行调整。 </li></ol> 3，可视化和分类红外光谱数据集注:在本节中，我们将讨论如何以可视化和提取使用的地理空间图像处理和分析软件如ENVI + IDL光谱图像数据，但是这个过程是非常相似的任何替代的软件，如MATLAB，免费软件如CytoSpec或仪器开发者自己的软件。有可能在IR数据进行几个不同的光谱处理技术。 <ol><li>开放的地理空间图像处理和分析软件并加载IR数据文件。 </li><li>通过选择"光谱工具"应用基线校正算法，红外数据和向下滚动并单击"吸收光谱"。观察一个弹出菜单，选择"基线CORREction" 注:基线校正将从由于散射数据删除基线斜率补偿</li><li>执行频谱归一化，由一个限定的光谱的区域下的数据之比来在一定的光谱峰值（通常为酰胺I（1650厘米-1）或酰胺二（1550厘米-1））或区域。通过选择"正常谱"下的"吸收光谱"菜单选项做到这一点。 注:光谱归一被执行以使得在光谱的任何差异是真实的生化特性差异，而不是由于厚度或样品之间的密度差异。 </li><li>使用降噪算法，从光谱如有必要，28消除噪声。 注意:有通过该基线校正，谱归一化和噪声降低，可以实现与具有内置自动算法大多数软件可用多种方法此外，还有的办法，将修正为一个新兴的数目。频谱畸变，这已经详细29-42讨论;然而，社会上还没有达成一致，需要上这些。 </li><li>观察图像（典型的光谱范围从900到4000 -1）内收集的所有的IR频率的列表。点击对应于特定的生物分子，观察组织的图像，在所选择的频率的频率。 <ol><li>例如，点击1650厘米-1波段来观察蛋白质的样品中的分布。可替代地点击1080厘米-1频带观察核酸在样品中的分布。用波数1650厘米-1。 </li></ol></li><li>通过计算谱峰高比，峰面积比等，这将允许不同的生物分子部件可视化创建附加的图像。点击"光谱工具"，然后选择"峰高比"或"计算图像区域"来创建图像。 </li><li>扫描的使用独立的整个幻灯片成像系统捕获图像明被染红了相应的邻近组织部分。沿着该IR图像，调出染色的组织的数字图像与可见光图像的程序。 </li><li>右键点击图片，在感兴趣的区域，然后选择&#39;Z曲线"。这会给在所选择的像素的光谱信息。 </li><li>看多个类，如比较细胞类型，疾病状态的多个像素的红外光谱。使用"感兴趣区"（ROI）工具在图像上标记特定的像素。要执行此，右键点击图片，选择"投资回报率的工具"。创建在被标记，例如正常，发育不良，和癌症的类的类。然后选择"ROI类型&#39; - &#39;点&#39;。然后选择类来选择像素和借鉴的红外图像上相应的像素。得到平均光谱˚F或者每个使用"平均投资回报率"工具类。 </li><li>通过绘制在绘图软件进行比较得出的光谱。 </li></ol>

The authors have nothing to disclose.

的FT-IR是一个新兴的方式对组织切片的无标记生化显像，具有在提高诊断的病理当前标准中起重要作用的潜力。目前金标准病理组织需要被活组织检查，在福尔马林中固定，石蜡包埋，切片多次，并用多种污渍。训练有素的病理学家有主观视觉评估组织结构和细胞形态，以确定诊断。这里，我们显示了如何从相同类型的段的收集高分辨率红外图像和讨论的一些计算方法来检查的细胞类型和疾病状态之间的化学差异。 此协议中的关键步骤是，以确保组织被非常仔细地集中并且该系统是公校准，以确保非常高的质量光谱数据。设置系统时，护理尤为criti<html> CAL和高放大倍率的目标工作时。为了帮助排除故障，下面的列表包括遇到的一些潜在的困难; 问题:在反射成像时，低IR强度。解决办法:检查IR幻灯片的方向作为反射涂层可以在幻灯片上的错误的一边。 问题:在蓝瑟控制低信号/红色预警信号。解决方案:酷探测器与LN2。液氮是必需的FPA探测器的功能，并要求定期被突破了。 问题:速度误差/移动的错误。解决方案:重新谱仪和减少振动。振动将导致在干涉仪的移动镜被干扰。 问题:数据水蒸气尖峰。解决方案:增加净化系统，保护样品空气。 问题:无效centerburst。解决方案:查找centerburst了。 e_content"&gt;问题:在发送低通量差，即使集中解决方案:调整底部冷凝器这将发生作为红外光不被聚焦到样品上的一个点。 在本文中，我们把重点放在如何获得在传输或transflectance方式组织高清晰度红外图像。的FT-IR成像的性质，是有可向数据采集多种修改，例如，类型的衬底，固定技术，样品的厚度，光谱分辨率，干涉仪反射镜的速度等这些参数的效果有最近4,5,17,51中广泛详细地进行了讨论。 有许多的修改，即，可以向包括在成像的ATR模式10,24,26和使用纳米级的热的方法52,53，以允许高分辨率红外成像的成像系统。具有高分辨率的红外成像的主要限制是，在Tissues必须小心制备和用于IR足够薄以穿过（一般为4微米厚）。此外，透射率和反射率的FT-IR成像需要试样保持干燥由于红外由水的吸光度。但是，FT-IR成像具有比其他技术显著的优点，在于它可以组织而导出丰富详尽生物化学信息非常迅速图像大的区域。该派生生物化学信息的无标记的方式中的类似技术包括拉曼光谱，但是数据采集的时间要慢得多，以获取图像。新拉曼成像方法正在出现，包括受激拉曼散射（SRS）和相干反斯托克斯拉曼散射（CARS）;但是，他们有机会获得有限的光谱范围，或单频成像。 在数据采集，空间分辨率，和计算方法的可用性的速度的进步已经在制造的FT-IR IMAG一直是巨大的价值荷兰国际集团翻译作为病理一种新的成像工具，比较可行的办法。在空间分辨率后的最新进展已经用于组织病理学尤其重要，因为关键细胞类型不被解析的使用常规的FT-IR成像系统。在最近的一份报告雷迪等人 。说明了如何一个理想系统获得的FT-IR成像系统5的最佳空间分辨率建模。在本文提出的肾组织的示例演示，以提取从肾小球结构（ 图3和图5）的生物化学信息的更高的空间分辨率的重要性。今后，在量子级联激光器非常明亮的红外光源的新进展54-57，三维光谱成像58，并在纳米技术，红外领域的突破52,53,59,60召开研究激动人心的新途径，可能有在组织成像的未来产生巨大影响。 <p cl屁股="jove_content"&gt;我们已经提出了在肝脏和肾脏疾病应用的例子，那里是一个需要额外的生化信息可以是诊断价值。光谱病理实验室病理学系在伊利诺伊大学芝加哥分校的重点是红外成像技术的翻译对改善疾病的诊断和改善患者预后预测。 FT-IR成像可以克服一些，其中定量和客观的信息，需要在实践中病理当前限制。尤其是，今后的工作重点是查明现有技术不能提供足够的诊断敏感性或提供的信息有限，目前病理学实践领域。显然需要存在于提高病理的现行做法，并朝着到大约患者的疾病状况的病理学家，采用高清FT-IR成像这可能是实现提供更多的信息。

IR光谱已经自1930年以某种形式提供的分析工具;然而，它仅是在过去的十年中组织成像用FT-IR的面积已分解。为组织成像FT-IR的进步已经驱动了很大一部分由三个关键的发展:数据采集1）提高速度，由于大型焦平面阵列的可用性（FPA）探测器通常有数以千计的红外敏感探测器1 ，2，2）开发先进的处理算法和计算能力来处理大量高光谱数据集3和FT-IR成像系统3）造型，以最大限度地提高空间分辨率4,5。已经有不少高品质和非常广泛的文章回顾了FT-IR光谱领域最近6-16，除了自然的协议文件，详细的步骤，以取得组织17点谱或地图 ​​。在本文中，我们将重点放在普罗特ocol得到使用在改良的FT-IR系统具有高清晰度能力的128×128焦平面探测器组织的图像。 的FT-IR成像早已被建议是一个潜在的理想的工具，细胞和组织成像，由于获得的图像，其中每一个像素具有丰富的生物化学信息的能力。的FT-IR成像是基于这样的原则，一个样本中不同的生物分子将定量吸收的中红外不同区域;这允许一个"生化指纹"的推导。该指纹已被证明在许多研究不同的细胞类型和疾病状态之间改变。不像在常规病理学的做法，即污点和免疫组化标记需要被用于可视化并确定用于指导诊断和治疗选择的细胞类型和组织的结构，从FT-IR的图像是基于组织的固有生物化学形成。目前techniq用于诊断染色组织的UE是费时的，破坏性的，费力，并且需要病理学家的主观专门知识，而FT-IR提供使这个过程的快速，非破坏性，自动化程度高，并且更加客观的潜力。此外，FT-IR提供了一种新途径，以获得额外的生物化学信息使用常规的染色技术可能不容易获得。 近年来的一个最令人振奋的进展一直是高分辨率成像方法现在，可以允许细胞类型和组织的结构，这对于全面的疾病诊断至关重要的可视化和表征的可用性。这些技术之一是衰减全反射（ATR）的FT-IR结合有高折射率其允许高分辨率成像18，具有许多非常令人兴奋的研究显示其应用19-25的固体浸没透镜（SIL）。此外，它瓦特最近证明，与ATR的成像相关联的增加的空间分辨率可允许内皮细胞和肌上皮细胞的乳腺组织中形成乳腺癌诊断26的一个关键组成部分的可视化和分类 ​​。而ATR成像是非常有用的，这种技术要求的SIL，使与组织形成的FT-IR图像接触;因此，它的使用受到一定限制的组织病理组织所在的大区域必须快速成像。 第二种方法所表明的耦合的高倍率物镜到使用同步加速器作为红外线的一个明亮的源的现有的FT-IR系统中，也能够充分照亮FPA和图像的0.54 X 0.54微米的有效像素尺寸。这允许我们能够想象关键结构在乳腺癌和前列腺组织了未解析使用传统的FT-IR系统4。虽然这些大幅增加红外图像空间地得到解决ñ是令人兴奋的，它的使用由于需要同步仍然有限。接着，一个最佳系统的设计也可能允许高清晰度成像能力与1.1×1.1微米的像素大小而不同步加速器源的要求，而是使用传统globar IR源5。在本文中，我们显示如何修改现有的商用的FT-IR成像系统，以允许组织的衍射极限红外成像与使用多个红外目标（15X，36X，和74X）可接受的信噪比。随着三个目标的有效像素尺寸为5.5×5.5微米（15X），2.2×2.2微米（36X）和1.1×1.1微米（74X）。然后，我们给出的收益空间分辨率在肝脏和肾脏活检27病检测的重要性的一些例子。

<table><tbody><tr><td>Cary 600 Series FT-IR system</td><td>Agilent</td><td>Multiple configurations&amp;nbsp;</td><td>Alternate FT-IR imaging systems exist</td></tr><tr><td>Adjustable ReflX Objective 74X/0.65 NA IR</td><td>Edmund Optics</td><td>66-592</td><td></td></tr><tr><td>Adjustable ReflX Objective 36X/0.5 NA IR</td><td>Edmund Optics</td><td>66-586</td><td></td></tr><tr><td>MirrIR slide</td><td>Kevley Technologies</td><td>CFR</td><td>For FT-IR reflection-mode measurements</td></tr><tr><td>Barium Fluoride slides</td><td>International Crystal Laboratories</td><td>Multiple sizes</td><td>For FT-IR transmission-mode measurements</td></tr><tr><td>Calcium Fluoride slides</td><td>International Crystal Laboratories</td><td>Multiple sizes</td><td>For FT-IR transmission-mode measurements</td></tr><tr><td>Dry Nitrogen/Dry Air gas</td><td>Multiple gas suppliers</td><td>Multiple sizes</td><td></td></tr><tr><td>Hexane</td><td>Sigma Aldrich</td><td>Multiple sizes</td><td>For deparafinizing tissue</td></tr><tr><td>Liquid Nitrogen</td><td>Multiple cryogenic liquid suppliers</td><td>Multiple sizes</td><td></td></tr><tr><td>ENVI-IDL software</td><td>Exelis-Vis</td><td></td><td>Other software packages available</td></tr><tr><td>Whole slide Imager</td><td>Scanscope (Aperio) or Nanozoomer (Hamamatsu)</td><td></td><td>To image stained slides</td></tr></tbody></table>

high-definition fourier transform infrared (ft-ir) spectroscopic imaging of human tissue sections towards improving pathology

高清晰度傅立叶变换红外（FT-IR）光谱成像是一个新兴的方法，得到具有关联生物化学信息的图像。组织的FT-IR成像是基于中红外的不同区域由不同的化学键（ 例如，C = O，CH，NH）内的细胞或组织，然后可与存在和组合物吸收的原理生物分子（ 例如，脂质，脱氧核糖核酸，糖原，蛋白质，胶原蛋白）。在的FT-IR图像，所述图像内的每个像素包括一个完整的红外光谱（IR），可以给信息上，然后可以利用对细胞类型或疾病的类型分类的细胞的生化状态。在本文中，我们将展示:如何从使用FT-IR系统的人体组织获得的红外图像，如何修改现有的仪器，使高清晰度成像能力，以及如何可视化FT-IR图像。然后，我们提出的FT-IR一些应用为病理使用肝肾作为例子。 FT-IR成像持有提供了一种新的途径获得的细胞和组织的生化信息完全无标记非扰动路线走向提供新的洞察生物分子的改变疾病进程的一部分，令人兴奋的应用。此外，该生物化学信息可以潜在地允许对疾病诊断的某些方面的目的和自动分析。

的FT-IR成像可以用于组织的红外图像，可以给根据所感兴趣的红外频率不同的对比度的推导。此外，在红外图像，每个像素包含了整个IR光谱，具有对应于不同的生物分子，可以得到大约的细胞类型或疾病状态（ 图1）的生化特性的信息不同的峰。在这里，我们已经展示了如何类之间比较光谱特征，但更先进的自动分类，可以使用额外的算法3,43-50，如贝叶斯分类，随机森林，人工神经网络和等级分明聚类分析可以在执行数据。监督分类方法将允许可以接受培训，以使细胞类型或疾病状态的自动识别分类器的建设。无监督分类方法可用于寻找天然存在的DIFferences在组织或由于生化方差细胞。 FT-IR仪器已经发展了数十年来，从一个单一的点/映射模式测量使用红外不透明孔以摄像模式中使用卡塞格伦目标，使用任一加上发射模式一收集目标或单一目标的照明目标这两个点亮，并收集在反射模式（ 图2）。据最近表明，在传输模式下，收集的目标可能被切换出了更高的放大倍率及数值孔径物镜，以允许有限的衍射红外成像，这导致了大量增加收集红外图像4,5的空间分辨率。对组织成像的空间分辨率的进步已经至关重要的，因为我们现在能够确定的细胞类型和组织结构，例如在肾，肾小球的功能单元，使用适于在内部的FT-IR系统（ 图3）。 高清晰度的FT-IR成像允许组织的详细图像，以进行检查，以确定异常区域并识别不同的细胞类型之间的生化差异。在肝组织核心，它是可能的可视化浸润纤维化的鸿沟发育不良和非发育异常的肝硬化（ 图4）的两个不同的区的肝细胞和地区。我们正在利用这一旨在使自动诊断工具，在肝脏疾病的疑难案件中使用。 重要的是，增加的空间分辨率，现在可以允许我们分离了可以由疾病进行化学修饰前的组织学变化是显而易见的具体的结构特征。例如，我们的重点是识别肾小球结构的生化改变，如肾小囊，系膜，肾小球基底膜和肾小管基底膜，前<html>确定的病理学家变化可以观察到（ 图5）。特别是，我们感兴趣的是确定与糖尿病肾病和慢性排斥在移植患者中，其中目前的技术不能识别在足够早的方式对成功的干预改变的进展相关的变化。 <img alt="图1" src="/files/ftp_upload/52332/52332fig1.jpg" /> （A） 的 H＆E染色的肝脏活组织检查和相同的核心序列部分的红外吸收芯的图像在（B）中 3286厘米-1和（C）2603图1的FT-IR图像和光谱从肝核心。图片厘米-1，强调不同的结构特点。 （ 四）组织的典型红外光谱，用标记重要的峰值。比例尺= 100微米。fig1large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/52332/52332fig2.jpg" /> 图2.光学示意图，详细的FT-IR显微镜操作模式:（A）在传输模式中，样品通过底部目标照亮，并且通过样品的光被顶客观收集。 （b）在反射模式中，最高目标供应照射样本，并收集反射光。底部的目标是不使用。 <a href="https://www.jove.com/files/ftp_upload/52332/52332fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52332/52332fig3.jpg" /> 图3的differen比较在肾脏肾小球FT-IR影像T显微镜物镜在2925厘米-1（A）15X收集与NA目标= 0.5（5.5×5.5微米像素大小）。 （B）36X采集与客观NA = 0.5（2.2×2.2微米像素大小）。 （C）74X收集目标与NA = 0.65（1.1×1.1微米像素大小）。比例尺= 50微米。 <a href="https://www.jove.com/files/ftp_upload/52332/52332fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52332/52332fig4.jpg" /> 图中的肝纤维化的核心和肝细胞之间4.光谱差异。（A）H＆E染色的核心，从肝活检。 （B）扫描FT-IR串行部分核心（36X目标设定）的图像。 （C）众议员肝细胞和纤维化余丹丹光谱，从（A）中由箭头指示组织区域采取和（B）。比例尺= 100微米。 <a href="https://www.jove.com/files/ftp_upload/52332/52332fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/52332/52332fig5.jpg" /> 要提取标记的图5.通过使用高清晰度的FT-IR成像分化肾脏组织活检的特征。（A）的高碘酸-希夫染色部分与特征。 （B）的相同组织的串联部分的CH 2不对称伸缩区域（36X物镜设置）的高清晰度的FT-IR图像。 （C）的特性标记在使用中的（B）的FT-IR图像，以便能够化学DIF（A）中提取ferentiate组织的四个特征。比例尺= 50微米。 <a href="https://www.jove.com/files/ftp_upload/52332/52332fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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High-definition Fourier Transform Infrared FT-IR Spectroscopic Imaging of Human Tissue Sections towards Improving Pathology

Fourier Transform Infrared (FT-IR) spectroscopic imaging is a fast and label-free approach to obtain biochemical data sets of cells and tissues. Here, we demonstrate how to obtain high-definition FT-IR images of tissue sections towards improving disease diagnosis.

高清傅立叶变换人体组织切片红外（FT-IR）光谱成像对提高病理

High-definition Fourier Transform Infrared (FT-IR) spectroscopic imaging is an emerging approach to obtain detailed images that have associated ...

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High-definition Fourier Transform Infrared (FT-IR) Spectroscopic Imaging of Human Tissue Sections towards Improving Pathology

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33.1K 次观看.  University of Illinois at Chicago. Fourier Transform Infrared (FT-IR) spectroscopic imaging is a fast and label-free approach to obtain biochemical data sets of cells and tissues. Here, we demonstrate how to obtain high-definition FT-IR images of tissue sections towards improving disease diagnosis. 

视频: High-definition Fourier Transform Infrared FT-IR Spectroscopic Imaging of Human Tissue Sections towards Improving Pathology

Optical Frequency Domain Imaging of Ex vivo Pulmonary Resection Specimens: Obtaining One to One Image to Histopathology Correlation

Lung cancer is the leading cause of cancer-related deaths1. Squamous cell and small cell cancers typically arise in association with the conducting airways, whereas adenocarcinomas are typically more peripheral in location. Lung malignancy detection early in the disease process may be difficult due to several limitations: radiological resolution, bronchoscopic limitations in evaluating tissue underlying the airway mucosa and identifying early pathologic changes, and small sample size and/or incomplete sampling in histology biopsies. High resolution imaging modalities, such as optical frequency domain imaging (OFDI), provide non-destructive, large area 3-dimensional views of tissue microstructure to depths approaching 2 mm in real time (Figure 1)2-6. OFDI has been utilized in a variety of applications, including evaluation of coronary artery atherosclerosis6,7 and esophageal intestinal metaplasia and dysplasia6,8-10.
Bronchoscopic OCT/OFDI has been demonstrated as a safe in vivo imaging tool for evaluating the pulmonary airways11-23 (Animation). OCT has been assessed in pulmonary airways16,23 and parenchyma17,22 of animal models and in vivo human airway14,15. OCT imaging of normal airway has demonstrated visualization of airway layering and alveolar attachments, and evaluation of dysplastic lesions has been found useful in distinguishing grades of dysplasia in the bronchial mucosa11,12,20,21. OFDI imaging of bronchial mucosa has been demonstrated in a short bronchial segment (0.8 cm)18. Additionally, volumetric OFDI spanning multiple airway generations in swine and human pulmonary airways in vivo has been described19. Endobronchial OCT/OFDI is typically performed using thin, flexible catheters, which are compatible with standard bronchoscopic access ports. Additionally, OCT and OFDI needle-based probes have recently been developed, which may be used to image regions of the lung beyond the airway wall or pleural surface17.
While OCT/OFDI has been utilized and demonstrated as feasible for in vivo pulmonary imaging, no studies with precisely matched one-to-one OFDI:histology have been performed. Therefore, specific imaging criteria for various pulmonary pathologies have yet to be developed. Histopathological counterparts obtained in vivo consist of only small biopsy fragments, which are difficult to correlate with large OFDI datasets. Additionally, they do not provide the comprehensive histology needed for registration with large volume OFDI. As a result, specific imaging features of pulmonary pathology cannot be developed in the in vivo setting. Precisely matched, one-to-one OFDI and histology correlation is vital to accurately evaluate features seen in OFDI against histology as a gold standard in order to derive specific image interpretation criteria for pulmonary neoplasms and other pulmonary pathologies. Once specific imaging criteria have been developed and validated ex vivo with matched one-to-one histology, the criteria may then be applied to in vivo imaging studies. Here, we present a method for precise, one to one correlation between high resolution optical imaging and histology in ex vivo lung resection specimens. Throughout this manuscript, we describe the techniques used to match OFDI images to histology. However, this method is not specific to OFDI and can be used to obtain histology-registered images for any optical imaging technique. We performed airway centered OFDI with a specialized custom built bronchoscopic 2.4 French (0.8 mm diameter) catheter. Tissue samples were marked with tissue dye, visible in both OFDI and histology. Careful orientation procedures were used to precisely correlate imaging and histological sampling locations. The techniques outlined in this manuscript were used to conduct the first demonstration of volumetric OFDI with precise correlation to tissue-based diagnosis for evaluating pulmonary pathology24. This straightforward, effective technique may be extended to other tissue types to provide precise imaging to histology correlation needed to determine fine imaging features of both normal and diseased tissues.

Optical Frequency Domain Imaging of Ex vivo Pulmonary Resection Specimens: Obtaining One to One Image to Histopathology Correlation

A method to image ex vivo pulmonary resection specimens with optical frequency domain imaging (OFDI) and obtain precise correlation to histology is described, which is essential to developing specific OFDI interpretation criteria for pulmonary pathology. This method is applicable to other tissue types and imaging techniques to obtain precise imaging to histology correlation for accurate image interpretation and assessment. Imaging criteria established with this technique would then be applicable to image assessment in future in vivo studies.

光频域成像<em&gt;体外</em肺切除标本：获得一对一的图像病理组织学的相关性

Lung cancer is the leading cause of cancer-related deaths1. Squamous cell and small cell cancers typically arise in association with the conducting ...

科研

生物工程

Three-dimensional Imaging of Nociceptive Intraepidermal Nerve Fibers in Human Skin Biopsies

A punch biopsy of the skin is commonly used to quantify intraepidermal nerve fiber densities (IENFD) for the diagnosis of peripheral polyneuropathy 1,2. At present, it is common practice to collect 3 mm skin biopsies from the distal leg (DL) and the proximal thigh (PT) for the evaluation of length-dependent polyneuropathies 3. However, due to the multidirectional nature of IENFs, it is challenging to examine overlapping nerve structures through the analysis of two-dimensional (2D) imaging. Alternatively, three-dimensional (3D) imaging could provide a better solution for this dilemma.
In the current report, we present methods for applying 3D imaging to study painful neuropathy (PN). In order to identify IENFs, skin samples are processed for immunofluorescent analysis of protein gene product 9.5 (PGP), a pan neuronal marker. At present, it is standard practice to diagnose small fiber neuropathies using IENFD determined by PGP immunohistochemistry using brightfield microscopy 4. In the current study, we applied double immunofluorescent analysis to identify total IENFD, using PGP, and nociceptive IENF, through the use of antibodies that recognize tropomyosin-receptor-kinase A (Trk A), the high affinity receptor for nerve growth factor 5. The advantages of co-staining IENF with PGP and Trk A antibodies benefits the study of PN by clearly staining PGP-positive, nociceptive fibers. These fluorescent signals can be quantified to determine nociceptive IENFD and morphological changes of IENF associated with PN. The fluorescent images are acquired by confocal microscopy and processed for 3D analysis. 3D-imaging provides rotational abilities to further analyze morphological changes associated with PN. Taken together, fluorescent co-staining, confocal imaging, and 3D analysis clearly benefit the study of PN.

In order to study the changes of nociceptive intraepidermal nerve fibers (IENFs) in painful neuropathies (PN), we developed protocols that could directly examine three-dimensional morphological changes observed in nociceptive IENFs. Three-dimensional analysis of IENFs has the potential to evaluate the morphological changes of IENF in PN.

三维成像伤害表皮内神经纤维在人体皮肤活检

A punch biopsy of the skin is commonly used to quantify intraepidermal nerve fiber densities (IENFD) for the diagnosis of peripheral polyneuropathy 1,2. ...

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 ...

Expanding the Comprehension of the Tumor Microenvironment using Mass Spectrometry Imaging of Formalin-Fixed and Paraffin-Embedded Tissue Samples

Advances in immune-based therapies have revolutionized cancer treatment and research. This has triggered growing demand for the characterization of the tumor immune landscape. Although standard immunohistochemistry is suitable for studying tissue architecture, it is limited to the analysis of a small number of markers. Conversely, techniques such as flow cytometry can evaluate multiple markers simultaneously, although information about tissue morphology is lost. In recent years, multiplexed strategies that integrate phenotypic and spatial analysis have emerged as comprehensive approaches to the characterization of the tumor immune landscape. Herein, we discuss an innovative technology combining metal-labeled antibodies and secondary ion mass spectrometry focusing on the technical steps in assay development and optimization, tissue preparation, and image acquisition and processing. Before staining, a metal-labeled antibody panel must be developed and optimized. This hi-plex image system supports up to 40 metal-tagged antibodies in a single tissue section. Of note, the risk of signal interference increases with the number of markers included in the panel. After panel design, particular attention should be given to the metal isotope assignment to the antibody to minimize this interference. Preliminary panel testing is performed using a small subset of antibodies and subsequent testing of the entire panel in control tissues. Formalin-fixed, paraffin-embedded tissue sections are obtained and mounted on gold-coated slides and further stained. The staining takes 2 days and closely resembles standard immunohistochemical staining. Once samples are stained, they are placed in the image acquisition instrument. Fields of view are selected, and images are acquired, uploaded, and stored. The final stage is image preparation for the filtering and removal of interference using the system&#39;s image processing software. A disadvantage of this platform is the lack of analytical software. However, the images generated are supported by different computational pathology software.

In the era of cancer immunotherapy, interest in elucidating tumor microenvironment dynamics has increased strikingly. This protocol details a mass spectrometry imaging technique with respect to its staining and imaging steps, which allow for highly multiplexed spatial analysis.

使用福尔马林固定和石蜡包埋组织样品的质谱成像扩展肿瘤微环境的理解

Advances in immune-based therapies have revolutionized cancer treatment and research. This has triggered growing demand for the characterization of the ...

癌症研究

Terahertz Imaging and Characterization Protocol for Freshly Excised Breast Cancer Tumors

This manuscript presents a protocol to handle, characterize, and image freshly excised human breast tumors using pulsed terahertz imaging and spectroscopy techniques. The protocol involves terahertz transmission mode at normal incidence and terahertz reflection mode at an oblique angle of 30&#176;. The collected experimental data represent time domain pulses of the electric field. The terahertz electric field signal transmitted through a fixed point on the excised tissue is processed, through an analytical model, to extract the refractive index and absorption coefficient of the tissue. Utilizing a stepper motor scanner, the terahertz emitted pulse is reflected from each pixel on the tumor providing a planar image of different tissue regions. The image can be presented in time or frequency domain. Furthermore, the extracted data of the refractive index and absorption coefficient at each pixel are utilized to provide a tomographic terahertz image of the tumor. The protocol demonstrates clear differentiation between cancerous and healthy tissues. On the other hand, not adhering to the protocol can result in noisy or inaccurate images due to the presence of air bubbles and fluid remains on the tumor surface. The protocol provides a method for surgical margins assessment of breast tumors.

Freshly excised human breast cancer tumors are characterized with terahertz spectroscopy and imaging following fresh tissue handling protocols. Tissue positioning is taken into consideration to enable effective characterization while providing analysis in a timely manner for future intraoperative applications.

新切除乳腺癌肿瘤的Terahertz成像和表征协议

This manuscript presents a protocol to handle, characterize, and image freshly excised human breast tumors using pulsed terahertz imaging and spectroscopy ...

工程学

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

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of spectroscopy techniques. Combining imaging and spectroscopy techniques into a single optical probe may provide a more complete analysis of tissue health. In this article, two dissimilar modalities are combined, high-resolution fluorescence microendoscopy imaging and diffuse reflectance spectroscopy, into a single optical probe. High-resolution fluorescence microendoscopy imaging is a technique used to visualize apical tissue micro-architecture and, although mostly a qualitative technique, has demonstrated effective real-time differentiation between neoplastic and non-neoplastic tissue. Diffuse reflectance spectroscopy is a technique which can extract tissue physiological parameters including local hemoglobin concentration, melanin concentration, and oxygen saturation. This article describes the specifications required to construct the fiber-optic probe, how to build the instrumentation, and then demonstrates the technique on in vivo human skin. This work revealed that tissue micro-architecture, specifically apical skin keratinocytes, can be co-registered with its associated physiological parameters. The instrumentation and fiber-bundle probe presented here can be optimized as either a handheld or endoscopically-compatible device for use in a variety of organ systems. Additional clinical research is needed to test the viability of this technique for different epithelial disease states.

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 ...

Three-Dimensional Imaging of Tumor-Bearing Tissue Using the Iterative Bleaching Extends Multiplexity Approach

The spatial heterogeneity of the tumor microenvironment (TME) is a critical determinant of therapeutic response, particularly for immune-oncology agents, where success depends on the distribution of specific immune cell subpopulations. Over the past decade, multiple sophisticated technologies have been introduced to achieve detailed resolution of the TME using two-dimensional sections from either formalin-fixed, paraffin-embedded (FFPE) or fixed-frozen tissues. While these thin sections are easier to procure and analyze, they lack the three-dimensional architecture needed to reliably and comprehensively characterize a tumor. To address this limitation, a tissue mounting and imaging technique was developed to enable the three-dimensional analysis of tumor lesions in their native in vivo state. This protocol outlines the procurement of human tumor tissue, the mounting of samples on custom-printed platforms, and staining procedures for post-fixation samples. The multiplexed immunofluorescence technique, IBEX (Iterative Bleaching Extends Multiplexity), was adapted to characterize the three-dimensional TME with up to 15 markers for tumor, immune, and stromal cells using commercially available antibodies. Imaging depths of up to 100 &#181;m were achieved using an inverted white-light laser confocal microscope with a custom-printed imaging adaptor and commercial glass-bottom dishes to ensure optimal tissue orientation. This protocol highlights the potential of the IBEX method to expand multiplexed immunofluorescence studies, providing a more comprehensive understanding of TME composition.

This article describes a protocol for multiplex immunofluorescence optimized to characterize the three-dimensional architecture of peritoneal metastases.

使用迭代漂白对荷瘤组织进行三维成像扩展了多重方法

The spatial heterogeneity of the tumor microenvironment (TME) is a critical determinant of therapeutic response, particularly for immune-oncology agents, ...

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 ...

<meta charset="utf8"></head><body>使用 OPTIR-FISH 进行细胞鉴定和代谢分析的综合方案

Optical Photothermal Infrared-Fluorescence In Situ Hybridization (OPTIR-FISH)

Understanding the metabolic activities of individual cells within complex communities is critical for unraveling their role in human disease. Here, we present a comprehensive protocol for simultaneous cell identification and metabolic analysis with the OPTIR-FISH platform by combining rRNA-tagged FISH probes and isotope-labeled substrates. Fluorescence imaging provides cell identification by the specific binding of rRNA-tagged FISH probes, while OPTIR imaging provides metabolic activities within single cells by isotope-induced red shift on OPTIR spectra. Using bacteria cultured with 13C-glucose as a test bed, the protocol outlines microbial culture with isotopic labeling, fluorescence in situ hybridization (FISH), sample preparation, optimization of the OPTIR-FISH imaging setup, and data acquisition. We also demonstrate how to perform image analysis and interpret spectral data at the single-cell level with high throughput. This protocol&#39;s standardized and detailed nature will greatly facilitate its adoption by researchers from diverse backgrounds and disciplines within the broad single-cell metabolism research community.

<meta charset="utf8"></head><body>作者聚焦:用于复杂环境中单细胞代谢和身份分析的集成 OPTIR-FISH

Here, we present a protocol using optical photothermal infrared-fluorescence in situ hybridization (OPTIR-FISH), also known as mid-infrared photothermal-FISH (MIP-FISH), to identify individual cells and understand their metabolism. This methodology can be applied broadly for diverse applications, including mapping cellular metabolism with single-cell resolution.

光学光热红外荧光原位杂交 （OPTIR-FISH）

Understanding the metabolic activities of individual cells within complex communities is critical for unraveling their role in human disease. Here, we ...

高清傅立叶变换人体组织切片红外（FT-IR）光谱成像对提高病理

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