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 border="0" cellpadding="0" cellspacing="0" width="1102">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Cary 600 Series FT-IR system</td>
			<td style="width:308px;">Agilent</td>
			<td style="width:192px;">Multiple configurations&#160;</td>
			<td style="width:267px;">Alternate FT-IR imaging systems exist</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Adjustable ReflX Objective 74X/0.65NA IR</td>
			<td style="width:308px;">Edmund Optics</td>
			<td style="width:192px;">66-592</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adjustable ReflX Objective 36X/0.5NA IR</td>
			<td style="width:308px;">Edmund Optics</td>
			<td style="width:192px;">66-586</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">MirrIR slide</td>
			<td style="width:308px;">Kevley Technologies</td>
			<td style="width:192px;">CFR</td>
			<td>For FT-IR reflection-mode measurements</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Barium Fluoride slides</td>
			<td style="width:308px;">International Crystal Laboratories</td>
			<td style="width:192px;">Multiple sizes</td>
			<td>For FT-IR transmission-mode measurements</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Calcium Fluoride slides</td>
			<td style="width:308px;">International Crystal Laboratories</td>
			<td style="width:192px;">Multiple sizes</td>
			<td>For FT-IR transmission-mode measurements</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Dry Nitrogen/Dry Air gas</td>
			<td style="width:308px;">Multiple gas suppliers</td>
			<td style="width:192px;">Multiple sizes</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Hexane</td>
			<td style="width:308px;">Sigma Aldrich</td>
			<td style="width:192px;">Multiple sizes</td>
			<td>For deparafinizing tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Liquid Nitrogen</td>
			<td style="width:308px;">Multiple cryogenic liquid suppliers</td>
			<td style="width:192px;">Multiple sizes</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">ENVI-IDL software</td>
			<td style="width:308px;">Exelis-Vis</td>
			<td style="width:192px;"></td>
			<td>Other software packages available</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:336px;">Whole slide Imager</td>
			<td style="width:308px;">Scanscope (Aperio) or Nanozoomer (Hamamatsu)</td>
			<td style="width:192px;"></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>

Watch this Scientific Journal Video about High-definition Fourier Transform Infrared FT-IR Spectroscopic Imaging of Human Tissue Sections towards Improving Pathology at JoVE.com

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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33.0K 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

Technique and Considerations in the Use of 4x1 Ring High-definition Transcranial Direct Current Stimulation (HD-tDCS)

High-definition transcranial direct current stimulation (HD-tDCS) has recently been developed as a noninvasive brain stimulation approach that increases the accuracy of current delivery to the brain by using arrays of smaller "high-definition" electrodes, instead of the larger pad-electrodes of conventional tDCS. Targeting is achieved by energizing electrodes placed in predetermined configurations. One of these is the 4x1-ring configuration. In this approach, a center ring electrode (anode or cathode) overlying the target cortical region is surrounded by four return electrodes, which help circumscribe the area of stimulation. Delivery of 4x1-ring HD-tDCS is capable of inducing significant neurophysiological and clinical effects in both healthy subjects and patients. Furthermore, its tolerability is supported by studies using intensities as high as 2.0 milliamperes for up to twenty minutes.
Even though 4x1 HD-tDCS is simple to perform, correct electrode positioning is important in order to accurately stimulate target cortical regions and exert its neuromodulatory effects. The use of electrodes and hardware that have specifically been tested for HD-tDCS is critical for safety and tolerability. Given that most published studies on 4x1 HD-tDCS have targeted the primary motor cortex (M1), particularly for pain-related outcomes, the purpose of this article is to systematically describe its use for M1 stimulation, as well as the considerations to be taken for safe and effective stimulation. However, the methods outlined here can be adapted for other HD-tDCS configurations and cortical targets.

Technique and Considerations in the Use of 4x1 Ring High-definition Transcranial Direct Current Stimulation HD-tDCS

High-definition transcranial direct current stimulation (HD-tDCS), with its 4x1-ring montage, is a noninvasive brain stimulation technique that combines both the neuromodulatory effects of conventional tDCS with increased focality. This article provides a systematic demonstration of the use of 4x1 HD-tDCS, and the considerations needed for safe and effective stimulation.

技术和注意事项在使用4X1环高清晰度的经颅直流电刺激（HD-TDCS）

High-definition transcranial direct current stimulation (HD-tDCS) has recently been developed as a noninvasive brain stimulation approach that increases ...

科研

医学

Tissue-simulating Phantoms for Assessing Potential Near-infrared Fluorescence Imaging Applications in Breast Cancer Surgery

Inaccuracies in intraoperative tumor localization and evaluation of surgical margin status result in suboptimal outcome of breast-conserving surgery (BCS). Optical imaging, in particular near-infrared fluorescence (NIRF) imaging, might reduce the frequency of positive surgical margins following BCS by providing the surgeon with a tool for pre- and intraoperative tumor localization in real-time. In the current study, the potential of NIRF-guided BCS is evaluated using tissue-simulating breast phantoms for reasons of standardization and training purposes.
Breast phantoms with optical characteristics comparable to those of normal breast tissue were used to simulate breast conserving surgery. Tumor-simulating inclusions containing the fluorescent dye indocyanine green (ICG) were incorporated in the phantoms at predefined locations and imaged for pre- and intraoperative tumor localization, real-time NIRF-guided tumor resection, NIRF-guided evaluation on the extent of surgery, and postoperative assessment of surgical margins. A customized NIRF camera was used as a clinical prototype for imaging purposes.
Breast phantoms containing tumor-simulating inclusions offer a simple, inexpensive, and versatile tool to simulate and evaluate intraoperative tumor imaging. The gelatinous phantoms have elastic properties similar to human tissue and can be cut using conventional surgical instruments. Moreover, the phantoms contain hemoglobin and intralipid for mimicking absorption and scattering of photons, respectively, creating uniform optical properties similar to human breast tissue. The main drawback of NIRF imaging is the limited penetration depth of photons when propagating through tissue, which hinders (noninvasive) imaging of deep-seated tumors with epi-illumination strategies.

Near-infrared fluorescence (NIRF) imaging may improve therapeutic outcome of breast cancer surgery by enabling intraoperative tumor localization and evaluation of surgical margin status. Using tissue-simulating breast phantoms containing fluorescent tumor-simulating inclusions, potential clinical applications of NIRF imaging in breast cancer patients can be assessed for standardization and training purposes.

组织鬼怪模拟，以评估乳癌手术潜在的近红外荧光成像中的应用

Inaccuracies in intraoperative tumor localization and evaluation of surgical margin status result in suboptimal outcome of breast-conserving surgery ...

Cerenkov Luminescence Imaging of Interscapular Brown Adipose Tissue

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely related to metabolism and energy expenditure, and its dysfunction is one important contributor for obesity and diabetes. Contrary to previous belief, recent PET/CT imaging studies indicated the BAT depots are still present in human adults. PET imaging clearly shows that BAT has considerably high uptake of 18F-FDG under certain conditions. In this video report, we demonstrate that Cerenkov luminescence imaging (CLI) with 18F-FDG can be used to optically image BAT in small animals. BAT activation is observed after intraperitoneal injection of norepinephrine (NE) and cold treatment, and depression of BAT is induced by long anesthesia. Using multiple-filter Cerenkov luminescence imaging, spectral unmixing and 3D imaging reconstruction are demonstrated. Our results suggest that CLI with 18F-FDG is a practical technique for imaging BAT in small animals, and this technique can be used as a cheap, fast, and alternative imaging tool for BAT research.

In this video report, we show the application of Cerenkov Luminescence Imaging (CLI) for interscapular brown adipose tissue in mice under activated and depressed conditions.

肩胛间褐色脂肪组织的切伦科夫发光成像

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely ...

Working with Human Tissues for Translational Cancer Research

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human specimens, precious samples must be handled with the greatest care in order to ensure integrity of organs, tissues, and cells to the highest degree. Unfortunately, tissue processing by definition requires extraction of tissues from the host, a change which can cause great cellular stress and have major repercussions on subsequent analyses. These stresses could result in the specimen being no longer representative of the site from which it was retrieved. Therefore, a strict protocol must be adhered to while processing these specimens to ensure representativeness. The desired assay(s) must also be taken into consideration in order to ensure that an optimal technique is used for sample processing. Outlined here is a protocol for tissue retrieval, processing and various analyses which may be performed on processed tissue in order to maximize downstream production from limited tissue samples.

Translational cancer research is dependent on extraction of human tissues. Much work has gone into optimizing extraction methods for ex vivo analysis. Here, we describe tissue processing methods allowing for maximal data output from limited samples.

与人体组织转化为癌症研究工作

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human ...

Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of stroke than relying on indirect measurements, and will be a potent and robust vascular research tool. We use an optical imaging approach that labels thrombi with a molecular imaging thrombus marker&#160;&#8212; a Cy5.5 near-infrared fluorescent (NIRF) probe that is covalently linked to the fibrin strands of the thrombus by the fibrin-crosslinking enzymatic action of activated coagulation factor XIIIa during the process of clot maturation. A micro-computed tomography (microCT)-based approach uses thrombus-seeking gold nanoparticles (AuNPs) functionalized to target the major component of the clot: fibrin. This paper describes a detailed protocol for the combined in vivo microCT and ex vivo NIRF imaging of thromboemboli in a mouse model of embolic stroke. We show that in vivo microCT and fibrin-targeted glycol-chitosan AuNPs (fib-GC-AuNPs) can be used for visualizing both in situ thrombi and cerebral embolic thrombi. We also describe the use of in vivo microCT-based direct thrombus imaging to serially monitor the therapeutic effects of tissue plasminogen activator-mediated thrombolysis. After the last imaging session, we demonstrate by ex vivo NIRF imaging the extent and the distribution of residual thromboemboli in the brain. Finally, we describe quantitative image analyses of microCT and NIRF imaging data. The combined technique of direct thrombus imaging allows two independent methods of thrombus visualization to be compared: the area of thrombus-related fluorescent signal on ex vivo NIRF imaging vs. the volume of hyperdense microCT thrombi in vivo.

This protocol describes the application of combined near-infrared fluorescent (NIRF) imaging and micro-computed tomography (microCT) for visualizing cerebral thromboemboli. This technique allows the quantification of thrombus burden and evolution. The NIRF imaging technique visualizes fluorescently labeled thrombus in excised brain, while the microCT technique visualizes thrombus inside living animals using gold-nanoparticles.

结合近红外荧光成像和微计算机断层扫描直接可视化脑血栓

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of ...

3D Ultrasound Imaging: Fast and Cost-effective Morphometry of Musculoskeletal Tissue

The developmental goal of 3D ultrasound imaging (3DUS) is to engineer a modality to perform 3D morphological ultrasound analysis of human muscles. 3DUS images are constructed from calibrated freehand 2D B-mode ultrasound images, which are positioned into a voxel array. Ultrasound (US) imaging allows quantification of muscle size, fascicle length, and angle of pennation. These morphological variables are important determinants of muscle force and length range of force exertion. The presented protocol describes an approach to determine volume and fascicle length of m.&#160;vastus lateralis and m.&#160;gastrocnemius medialis. 3DUS facilitates standardization using 3D anatomical references. This approach provides a fast and cost-effective approach for quantifying 3D morphology in skeletal muscles. In healthcare and sports, information on the morphometry of muscles is very valuable in diagnostics and/or follow-up evaluations after treatment or training.

3D ultrasound imaging (3DUS) allows fast and cost-effective morphometry of musculoskeletal tissues. We present a protocol to measure muscle volume and fascicle length using 3DUS.

3D 超声成像: 快速、经济高效的肌骨骼组织形态分析

The developmental goal of 3D ultrasound imaging (3DUS) is to engineer a modality to perform 3D morphological ultrasound analysis of human muscles. 3DUS ...

Skeletal Muscle Neurovascular Coupling, Oxidative Capacity, and Microvascular Function with 'One Stop Shop' Near-infrared Spectroscopy

Exercise represents a major hemodynamic stress that demands a highly coordinated neurovascular response in order to match oxygen delivery to metabolic demand. Reactive hyperemia (in response to a brief period of tissue ischemia) is an independent predictor of cardiovascular events and provides important insight into vascular health and vasodilatory capacity. Skeletal muscle oxidative capacity is equally important in health and disease, as it determines the energy supply for myocellular processes. Here, we describe a simple, non-invasive approach using near-infrared spectroscopy to assess each of these major clinical endpoints (reactive hyperemia, neurovascular coupling, and muscle oxidative capacity) during a single clinic or laboratory visit. Unlike Doppler ultrasound, magnetic resonance images/spectroscopy, or invasive catheter-based flow measurements or muscle biopsies, our approach is less operator-dependent, low-cost, and completely non-invasive. Representative data from our lab taken together with summary data from previously published literature illustrate the utility of each of these end-points. Once this technique is mastered, application to clinical populations will provide important mechanistic insight into exercise intolerance and cardiovascular dysfunction.

Skeletal Muscle Neurovascular Coupling, Oxidative Capacity, and Microvascular Function with &#039;One Stop Shop&#039; Near-infrared Spectroscopy

Here, we describe a simple, non-invasive approach using near-infrared spectroscopy to assess reactive hyperemia, neurovascular coupling and skeletal muscle oxidative capacity in a single clinic or laboratory visit.

"一站式" 近红外光谱对骨骼肌神经血管的耦合、氧化能力及微血管功能的作用

Exercise represents a major hemodynamic stress that demands a highly coordinated neurovascular response in order to match oxygen delivery to metabolic ...

Characterization of Immune Cells in Human Adipose Tissue by Using Flow Cytometry

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the development of obesity-associated complications such as type 2 diabetes. To quantitatively and qualitatively investigate the immune cell subsets in human AT deposits, we have developed a flow cytometry approach. The stromal vascular fraction (SVF), containing the immune cells, is isolated from subcutaneous and visceral AT biopsies by collagenase digestion. Adipocytes are removed after centrifugation. The SVF cells are stained for multiple membrane-bound markers selected to differentiate between immune cell subsets and analyzed using flow cytometry. As a result of this approach, pro- and anti-inflammatory macrophage subsets, dendritic cells (DCs), B-cells, CD4+ and CD8+ T-cells, and NK cells can be detected and quantified. This method gives detailed information about immune cells in AT and the amount of each specific subset. Since there are numerous fluorescent antibodies available, our flow cytometry approach can be adjusted to measure various other cellular and intracellular markers of interest.

This article describes a method to analyze immune cell content of adipose tissue by isolation of immune cells from adipose tissue and subsequent analysis using flow cytometry.

应用流式细胞仪对人体脂肪组织免疫细胞的表征

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the ...

Nanoscopic Imaging of Human Tissue Sections via Physical and Isotropic Expansion

In modern pathology, optical microscopy plays an important role in disease diagnosis by revealing microscopic structures of clinical specimens. However, the fundamental physical diffraction limit prevents interrogation of nanoscale anatomy and subtle pathological changes when using conventional optical imaging approaches. Here, we describe a simple and inexpensive protocol, called expansion pathology (ExPath), for nanoscale optical imaging of common types of clinical primary tissue specimens, including both fixed-frozen or formalin-fixed paraffin embedded (FFPE) tissue sections. This method circumvents the optical diffraction limit by chemically transforming the tissue samples into tissue-hydrogel hybrid and physically expanding them isotropically across multiple scales in pure water. Due to expansion, previously unresolvable molecules are separated and thus can be observed using a conventional optical microscope.

Nanoscale imaging of clinical tissue samples can improve understanding of disease pathogenesis. Expansion pathology (ExPath) is a version of expansion microscopy (ExM), modified for compatibility with standard clinical tissue samples, to explore the nanoscale configuration of biomolecules using conventional diffraction limited microscopes.

通过物理和各向异性扩张对人体组织部分的纳米成像

In modern pathology, optical microscopy plays an important role in disease diagnosis by revealing microscopic structures of clinical specimens. However, ...

DIPLOMA Approach for Standardized Pathology Assessment of Distal Pancreatectomy Specimens

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignant cancers. A minority (20%) of PDACs are found in the pancreatic body and tail. Accurate pathology assessment of the pancreatic specimen is essential for providing prognostic information and it may guide further treatment strategies. The recent 8th edition of the American Joint Committee on Cancer/Union for International Cancer Control (AJCC/UICC) staging system for pancreatic tumors has incorporated significant changes to tumor (pT) stage, which is predominantly based on tumor size. This change emphasizes the importance of careful block selection. Owing to the greater prevalence of tumors in the head of the pancreas, efforts are made to standardize the assessment of pancreatoduodenectomy specimens. However, consensus regarding the macroscopic assessment of distal (i.e., left) pancreatectomy specimens is lacking. The DIPLOMA approach includes the standardized measurement of pancreas and other resected organs, inking of relevant surgical margins and anatomical surfaces without removing covering layers of fat, measurement of tumor size (for T-stage), together with assessment of splenic vessel involvement (and other organs if present). All relevant margins are assessed, and relevant blocks are selected to confirm these parameters microscopically. The current protocol describes a standardized approach to the macroscopic assessment of distal pancreatectomy specimens. This approach was developed during several meetings with pathologists and surgeons during the preparation phase for an international multicenter trial (DIPLOMA, ISRCTN44897265), which focuses on radicality of distal pancreatectomy for pancreatic ductal adenocarcinoma. This standardized approach can be instrumental in the design of studies and will uniform reporting on the outcomes of distal pancreatectomy. The described technique is used in the DIPLOMA trial for pancreatic ductal adenocarcinoma but may also be useful for other indications.

The current study highlights a standardized approach to the macroscopic assessment of distal pancreatectomy specimens for pancreatic ductal adenocarcinoma, with special emphasis on the measurement of pancreatic dimensions and those of other organs, inking of margins, measurement of tumor size and proximity to margins, lymph node sampling and block selection.

分离性造形切除术标本标准化病理学评估的DIPLOMA方法

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignant cancers. A minority (20%) of PDACs are found in the pancreatic body and tail. ...