P.林斯特龙博士是公认的ob / ob小鼠。 J.莱赫托宁是公认的援助，帮助与编辑视频制作和J.吉尔伯特。这项研究得到了糖尿病研究基金会（AP），青少年糖尿病研究基金会（AP和UA），欧盟委员会（FP-7，赠款协定。:CP-IP 228933-2）（JS和补助UA），肯普基础，于默奥大学和瑞典研究理事会UA

1。样品制备和扫描 1.1样品制备基本上如前所述7执行下面的过程。 <ol><li>收获的胰腺。用冰冷的PBS，以避免蛋白降解。 </li><li>在PBS中的4％PFA中修复的组织为2-3小时，在冰上。确保叶是"传播"期间固定。这将有利于确定后重建的解剖标志。 </li><li>过量的PBS洗涤30分钟。 </li><li>脱水胰腺逐步在甲醇（33，66％，100％），15分钟/步。这最大限度地减少漂白过程（见步骤5），并防止气泡的形成，细胞的破坏，在冷冻 - 解冻（见第7步）。 </li><li>孵育组织中新鲜制备的甲醇:H 2 O 2:DMSO一个= 2:1:3的比例在室温放置24小时，以便淬灭内源性组织荧光漂白缓冲器中。对于较大的样本，交流新bleachi纳克和另一个24小时的孵育缓冲。 </li><li>在过量的甲醇进行清洗，ON。 </li><li>冻结 - 解冻至少5个循环，在-80℃下 - RT以促进抗体渗透率。 </li><li>再水合逐步回TBST中（​​33，66％，100％），15分钟/步。 </li><li>座在TBST中含10％血清（优选来自同一物种的二次抗体生成），5％DMSO和0.01％NaAz为12-24小时在RT </li><li>与初级抗体孵育48小时的封闭缓冲液，在RT，延长至72小时，较大的样品（这里所用的抗体的试剂的表中列出）。 </li><li>超过TBST清洗，ON。 </li><li>与荧光共轭的次级抗体孵育48小时，在RT，延长至72小时，较大的样本。 </li><li>超过TBST清洗，ON。 </li></ol> 1.2 下面的过程介绍了如何安装在琼脂糖样品，将其附加到自定义的样品架（ 见图7）之前选择扫描。 <ol><li>分离脾，十二指肠和胃瓣图3A在Hörnblad 等[3]。波瓣之间的关系被进一步澄清在Hörnblad 等 11。 </li><li>准备1.5％（W / V）低熔点琼脂糖DH 2 O，过滤器，冷却至37°C和组织在卫生署2 O冲洗，以洗去清洁剂和消除气泡前琼脂糖上镶嵌冰。 </li><li>剪出的琼脂糖块包围您的样品，并留下一个〜1厘米的样品之间的间隔物和碱的琼脂糖。修剪琼脂糖块的尖锐的边缘（≤90°），以减少光散射。 </li><li>在MeOH（33，66％，100％）逐步脱水样本，允许每个步骤之间的时间以平衡。当试样下沉，它被认为是平衡。 </li><li>清除样品中苯甲醇1:2的解决方案:苄基奔驰烷基酯（巴布），直到它变得透明。交易所的巴布的解决方案和孵育12小时。 </li><li>定位清零的样品的样品架中，并通过插入2针通过琼脂糖的间隔件的保持架的凸缘通过钻孔，将它固定。 </li><li>将样品放置在扫描仪和淹没填充用巴布结算溶液到一个反应杯中。当比较胰腺的一系列相同的放大倍数应被用于所有扫描。应该优化的倍率系列中的最大的样本。 </li></ol> 1.3试样的定位在AR 以下协议描述一个示例使用的COM-AR算法的程序来精确定位。此程序仅适用时的投资回报率，包括整个标本。的算法的详细说明，请参阅Cheddad 等[2]。 <ol><li>获取图像的样本在两个位置的BOTh的解剖和信号通道。位置1，在0℃（用X-轴）相关联，显示最大的投影区域，在90℃（与Z-轴）和位置2。我们正在使用的的GFP通道可视化的解剖。 </li><li>应用期望最大化（EM）算法的解剖图像上的阈值的ROI。 </li><li>计算的COM点（x坐标），在步骤2中获得的在0°和90°突起的二进制图像。 </li><li>叠加的垂直线，通过在0°和90°的信号信道的图像的步骤3中所识别的COM计算点。 </li><li>使用在步骤4中获得的图像，作为参考样品，使视场的中心线穿过样品找到的COM点移动。 </li></ol> 1.4扫描<ol><li>调整曝光时间，以实现最高的信号噪声比成为可能，而不饱和婷的投影图像的任何区域。要扫描所有通道，重复上述步骤。 </li><li>选择要扫描的第一荧光通道的过滤器组。从更短的λ荧光基团的发射较长的λ激发荧光基团，从而导致光漂白。为了尽量减少这种可能性，扫描荧光激发λ最长的第一次。 </li><li>打开快门，以照亮样品和收集超过360°的荧光信号，为每个通道沿垂直轴转动样品。用于NIR-OPT设置的步距角为0.9°的Bioptonics 3001扫描仪0.45°。 </li><li>下一个通道，并选择适当的过滤器，请按以上。 </li></ol> 2。计算处理和重建 2.1收购后错位检测和纠正（A值调整） 在投影层析成像，它是在普通必要分配后的取向值微调的图像重建的前沿旋转轴线的位置的突起。然而，在相机的角度朝向光轴像差小可以导致非均匀的A值，沿样品的长度，从而诱导几何失真。为了避免这种扭曲，在整个样本的准确和统一后的调整值（A值）的计算方法可应用于2。 <ol><li>使用离散傅里叶变换的划分​​的投影的特定信号，在0°和180°成8像素高度块，并计算每个块之间沿x-轴的移位（A值）。 </li><li>应用最小二乘法的线性回归分析，以帮助计算的角度θ&#39;，它描述的x轴沿试样长度的移位的斜率，并找到一个旋转中心点。 </li><li>在扫描过程中，旋转所有幻灯纠正偏差ctionsθ&#39;/ 2左右的旋转中心点。 </li></ol> 2.2对比度受限自适应直方图均衡（CLAHE） 为了便于检测和分割对象（胰岛）表现出非常弱的信号，该信号是在风险的"阈值"在重建过程中和/或分割的定量评估，CLAHE算法可以被应用于投影图像。 CLAHE操作有两个主要的强度转换: <ol><li>估计的局部对比度的投影图像中的非重叠的块内均衡。 </li><li>标准化的强度，然后通过双线性内插块之间的边境地区。 </li></ol>对比度限制的名称是指到夹子的限制，它被设置为避免在图像中的饱和像素。在这个协议中，MATLAB内置函数"adapthisteq"使用默认的C应用<html>唇下限0.01和大小为256瓦。请注意，最优瓷砖大小需要凭经验进行测试，并且可以取决于分析的检体上。在Hörnblad 等[3]上的算法和实施例中可以找到更多细节。 注意上面列出的计算处理步骤（包括COM-AR，A值调整和CLAHE，见1.3-2.2）是建立在标准算法，并在MATLAB（MathWorks公司）执行。 2.3断层重建和异面绘制<ol><li>使用滤波反投影算法，现在可以被重建的校正和归一化的图像，用统一的不对中补偿和优化的动态范围的最低限度的要求。在该协议中，所有的重建进行滤波反投影法在NRecon软件（SKYSCAN），版本1.6.8（="_blank"&gt; http://www.skyscan.be/products/downloads.htm）。请注意，成像的对象的放大率取决于平行束几何结构中，除非它的镜头的焦点距离的摄像设置中实现。因此，导入的投影数据集的NRecon软件时，它是重要的，包括在随附的源间的距离（mm）和扫描仪的旋转方向（逆时针输入"cc"和顺时针输入"顺时针"）的正确的对象日志文件，以避免在重建过程中的锥束引起的工件。 </li><li>可视化和量化的堆栈获得的虚拟部分，产生三维等值面采用合适的图像处理软件，如Imaris或Volocity。 </li></ol>小鼠胰岛分离和移植程序进行糖尿病研究所的临床前细胞处理和转化模式的核心，根据协议，评论和批准MIAM大学我实验动物护理和使用委员会。动物研究的伦理委员会批准，瑞典北部，所有其他实验动物。

<ol>
	<li>Sharpe, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+projection+tomography+as+a+tool+for+3D+microscopy+and+gene+expression+studies.">Optical projection tomography as a tool for 3D microscopy and gene expression studies.</a> Science. 296, 541-545 (2002).</li><li>Cheddad, A., Svensson, C., Sharpe, J., Georgsson, F., Ahlgren, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+Processing+Assisted+Algorithms+for+Optical+Projection+Tomography.">Image Processing Assisted Algorithms for Optical Projection Tomography.</a> IEEE Trans. Med. Imaging. , (2012).</li><li>Hornblad, A., Cheddad, A., Ahlgren, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+protocol+for+optical+projection+tomography+imaging+reveals+lobular+heterogeneities+in+pancreatic+islet+and+beta-cell+mass+distribution.">An improved protocol for optical projection tomography imaging reveals lobular heterogeneities in pancreatic islet and beta-cell mass distribution.</a> Islets. 3, 204-208 (2011).</li><li>Holmberg, D., Ahlgren, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+pancreas:+from+ex+vivo+to+non-invasive+technology.">Imaging the pancreas: from ex vivo to non-invasive technology.</a> Diabetologia. 51, 2148-2154 (2008).</li><li>Ahlgren, U., Gotthardt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Approaches+for+imaging+islets:+recent+advances+and+future+prospects.">Approaches for imaging islets: recent advances and future prospects.</a> Adv. Exp. Med. Biol. 654, 39-57 (2010).</li><li>Sharpe, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+projection+tomography.">Optical projection tomography.</a> Annu. Rev. Biomed. Eng. 6, 209-228 (2004).</li><li>Alanentalo, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tomographic+molecular+imaging+and+3D+quantification+within+adult+mouse+organs.">Tomographic molecular imaging and 3D quantification within adult mouse organs.</a> Nat. Methods. 4, 31-33 (2007).</li><li>Alanentalo, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+three-dimensional+imaging+of+islet-infiltrate+interactions+based+on+optical+projection+tomography+assessments+of+the+intact+adult+mouse+pancreas.">High-resolution three-dimensional imaging of islet-infiltrate interactions based on optical projection tomography assessments of the intact adult mouse pancreas.</a> J. Biomed. Opt. 13, 054070 (2008).</li><li>Alanentalo, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+and+Three-Dimensional+Imaging+of+the+Insulitis-Induced+Destruction+of+beta-Cells+in+Murine+Type+1+Diabetes.">Quantification and Three-Dimensional Imaging of the Insulitis-Induced Destruction of beta-Cells in Murine Type 1 Diabetes.</a> Diabetes. 59, 1756-1764 (2010).</li><li>Sun, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ablation+of+AMP-activated+protein+kinase+alpha1+and+alpha2+from+mouse+pancreatic+beta+cells+and+RIP2.Cre+neurons+suppresses+insulin+release+in+vivo.">Ablation of AMP-activated protein kinase alpha1 and alpha2 from mouse pancreatic beta cells and RIP2.Cre neurons suppresses insulin release in vivo.</a> Diabetologia. 53, 924-936 (2010).</li><li>Hornblad, A., Eriksson, A. U., Sock, E., Hill, R. E., Ahlgren, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+spleen+formation+perturbs+morphogenesis+of+the+gastric+lobe+of+the+pancreas.">Impaired spleen formation perturbs morphogenesis of the gastric lobe of the pancreas.</a> PLoS One. 6, e21753 (2011).</li><li>Lindstr&#246;m, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+physiology+of+the+Obese-Hyperglycemic+Mice+(ob/ob+Mice).">The physiology of the Obese-Hyperglycemic Mice (ob/ob Mice).</a> The Scientific World JOURNAL. 7, 665-685 (2007).</li><li>Bock, T., Pakkenberg, B., Buschard, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+background+determines+the+size+and+structure+of+the+endocrine+pancreas.">Genetic background determines the size and structure of the endocrine pancreas.</a> Diabetes. 54, 133-137 (2005).</li></ol>

没有利益冲突的声明。

所描述的技术为OPT摄像使空间和定量参数提取的整个体积的小鼠胰腺。由于在这种类型的介观成像它达到的分辨率的限制应该指出的是，对于大多数成像方式，较大的试样分辨率越低（虽然使用一个更高的分辨率的CCD应增加分辨率的OPT扫描） 。因此，完整无缺胰腺癌小鼠叶的评估，该技术目前不提供单细胞分辨率虽然接近（约15-20微米）7。尽管如此，BCM的分布在小鼠胰腺提取的协议所提供的数据，以上以及匹配得到的那些， 例如由点计数形态计量学3,13应该指出，虽然实施的CLAHE协议允许用于检测显着更多的胰岛这些小岛一般都比较小，不贡献特基本上整体的β细胞量。 免疫组化涉及的协议是相对较长（长达两个星期），但在实际操作样品准备时间很短，因此，该技术非常适合于大样本的研究，动物9。如果潜在的异种分布格局是调查的重点，应该强调的是，护理应采取的步骤，固定和安装，以避免胰腺组织成为一个不利的方式固定在平面（"传播"应努力促进这种评估）安装的组织。 执行OPT时的一个重要问题是，样品的COM被固定在旋转轴，它不会移动，垂直或水平方向，在扫描过程中。因此，必须有一个稳定的机械安装和运转良好的系统，附连在样本。我们解决了这个问题，通过构建一个新的安装（ 图7）。 并行几何NIR-3001 OPT或Bioptonics的扫描仪，其中检测作为周记录的投影图像中的对象的位置的正面和背面之间的垂直移位是不正确的。通过调整相应的扫描仪在日志文件中的对象源的距离（参见2.3.1节），我们可以显著提高我们的数据质量，并纠正在远边的投影图像的几何失真，这是特别重要时评估更大的标本。 在目前的方案，我们提供的过滤器设置，允许可视化三种不同的具体的渠道和完整的胰腺准备评估"解剖"通道的建议。显然，这些设置可调控的荧光染料用于一个给定的研究虽然以更好地适应，与各种形式的荧光百分之显微镜，应仔细评估潜在危险的信号渗出通过。与上述750nm的激发的荧光染料所标记的胰岛素胰岛的研究尚未能够由我们使用的金属卤化物灯，我们的设置利用。这是可能的替代光源（ 例如二极管激光器），结合在相关的波长具有更高的量子效率的照相机可以进一步增加NIR-OPT的电位，并允许在更高的波长成像。 OPT成像技术的生物医学样品的mm厘米尺度上的空间和定量评估是一个高度灵活的。虽然胰腺/糖尿病研究的主要目的，已经开发了用于此处介绍的协议，它们应该是可能的翻译对其他物种，标本类型和标记的研究。通过几个不同的渠道可视化的潜力在完整的胰腺准备，近红外光学成像f进一步的持有作为一种工具来评价用于非侵入性的评估的造影剂，由其他成像方式，只要这些造影剂可以设计也进行检测的OPT荧光团的吸收特异性的潜力。

A correction was made to <a href="/video/50238">Near Infrared Optical Projection Tomography for Assessments of &beta;-cell Mass Distribution in Diabetes Research</a>. In Protocol section 1.1, the order of the listed chemicals MeOH:H2O2:DMSO has accidentally been switched and should instead be MeOH:DMSO:H2O2.
Protocol section 1.1 was changed from:
Incubate the tissue in freshly prepared MeOH:H2O2:DMSO bleaching buffer in a 2:1:3 ratio at RT for 24 hr to quench endogenous tissue fluorescence. For larger samples, exchange to new bleaching buffer and incubate for another 24 hr.
to:
Incubate the tissue in freshly prepared MeOH:DMSO:H2O2 bleaching buffer in a 2:1:3 ratio at RT for 24 hr to quench endogenous tissue fluorescence. For larger samples, exchange to new bleaching buffer and incubate for another 24 hr.

A correction was made to <a href="/video/50238">Near Infrared Optical Projection Tomography for Assessments of &beta;-cell Mass Distribution in Diabetes Research</a>. In Protocol section 1.1, the order of the listed chemicals MeOH:H2O2:DMSO has accidentally been switched and should instead be MeOH:DMSO:H2O2.

Erratum: Near Infrared Optical Projection Tomography for Assessments of &beta;-cell Mass Distribution in Diabetes Research

产生胰岛素的β细胞是身体的能力，控制血糖平衡的关键。因此，胰腺癌的BCM分布的评估是必要的到糖尿病临床前研究的许多领域。例如在评估治疗制度，内分泌细胞分化在啮齿类动物模型的疾病或糖尿病的病因学的研究有针对性的基因消融的影响，往往依赖于这样的分析。传统上，这些类型的评估依赖于耗时体视学方法，是难以履行应尽的规模和复杂的解剖宪法的胰腺。目前高分辨率成像方法（通常是光纤），不提供足够的穿透深度，让整个胰腺成像在啮齿类动物。相反，其穿透深度（通常为核）成像方法，不仅限于提供，分辨率较低，解决BCM分布，并阻碍由于缺乏足够的造影剂4,5。 光学投影层析成像是3D成像方式，使生物医学样品的高分辨率评估厘米规模6毫米。据此，个别胰岛素表达胰岛的空间位置和体积上的信息可以被提取的整个体积的正常及糖尿病小鼠胰腺3,7-10。目前的研究的目的是为了进一步增进胰腺β-细胞的评估，这种技术的能力;其内源性的分布时，接枝到其他组织中，它们之间的关系的其他胰腺成分（如浸润细胞类型），并在较大的胰腺癌的准备工作比以前可能。 近红外光投影断层扫描（NIR-OPT）设置在下面的协议，根据原来的设置由夏普OPT扫描仪<e米&gt;等1，描述和使用在近红外范围内，适于成像。对于小鼠胰腺（ 如 BCM的）的单信道的评估，SKYSCAN 3001（Bioptonics）扫描仪可以被使用。 一种金属卤化物灯，可提供更高的激发态能量为650nm以上波长比汞弧灯，激发光的供给。被传输的光通过液体光导。在图3中示出的荧光染料和近红外荧光成像和信道分离的带通滤波器的一个有用的组合。所发射的光被检测到的背面照明的CCD照相机，在近红外光谱具有高量子效率。 OPT使用LabVIEW平台，控制摄像头和步进电机自动扫描。为了支持完整大鼠胰腺中，被保护的银包覆的镜子和大的比色皿的大小的样品中被使用。最后，样品架，消除不必要的垂直工运日ts的样品在扫描过程中的设计。

<table border="1"><tbody><tr><td> 的试剂/材料名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>甲醇</td><td>沙尔劳</td><td> ME03162500 </td><td></td></tr><tr><td> 30％H 2 O 2 </td><td>沙尔劳</td><td> HI01362500 </td><td></td></tr><tr><td>苯甲醇</td><td>沙尔劳</td><td> AL01611000 </td><td></td></tr><tr><td>苯甲酸苄酯</td><td>沙尔劳</td><td> BE01851000 </td><td></td></tr><tr><td>低的熔点琼脂糖</td><td> LONZA </td><td> 50100 </td><td></td></tr><tr><td>多聚甲醛（PFA）的</td><td> Sigma-Aldrich公司</td><td> 158127 </td><td></td></tr><tr><td> DMSO </td><td> Sigma-Aldrich公司</td><td> D5879 </td><td></td></tr><tr><td>的Triton-X100 </td><td&gt; Sigma-Aldrich公司</td><td> T8787 </td><td></td></tr><tr><td>鼠抗ASMA-CY3 </td><td> Sigma-Aldrich公司</td><td> C6198 </td><td>一抗</td></tr><tr><td>兔抗-CD3 </td><td> Sigma-Aldrich公司</td><td> C7930 </td><td>一抗</td></tr><tr><td>豚鼠抗加载项</td><td> DAKO </td><td> A0564 </td><td>一抗</td></tr><tr><td>驴抗GP-IRDye680 </td><td> LI-COR Biosciences公司</td><td> 926-32421 </td><td>二抗</td></tr><tr><td>山羊抗RB-DyeLight750 </td><td> Thermo Scientific的</td><td> 35570 </td><td>二抗</td></tr><tr><td> ，山羊反GP-Alexa594 </td><td>分子探针</td><td> A-11076 </td><td>二抗</td></tr><tr><td>羊抗GP-ALEXA488 </td><td>分子探针</td><td> A-11008 </td><td>二抗</td></tr><tr><td>羊抗GP-Alexa594 </td><td>分子探针</td><td> A-11012 </td><td>二抗</td></tr><tr><td> ，山羊反GP-Alexa680 </td><td>分子探针</td><td> A-21076 </td><td>二抗</td></tr><tr><td> ，山羊反GP-Alexa750 </td><td>分子探针</td><td> A-21039 </td><td>二抗</td></tr><tr><td> OPT SKYSCAN 3001 </td><td> Bioptonics </td><td></td><td> OPT-扫描仪</td></tr><tr><td>徕卡MZ FLIII </td><td>徕卡显微系统</td><td></td><td>体视显微镜</td></tr><tr><td>徕卡物镜0.5X </td><td>徕卡显微系统</td><td> 10446157 </td><td></td></tr><tr><td>徕卡相机适配器1.0倍</td><td>徕卡显微系统</td><td> 10445930 </td><td></td></tr><tr><td> EL6000金属卤化物灯</td><td></td><td> 11504115 </td><td>光源</td></tr><tr><td>液体光导</TD&gt; <td></td><td> 11504116 </td><td></td></tr><tr><td>比色皿</td><td> HELLMA Analytics（分析） </td><td> 6030-OG </td><td> 55×55×52.5毫米</td></tr><tr><td>镜</td><td>爱特蒙特光学</td><td> F68-334 </td><td> 50 x 50毫米</td></tr><tr><td>安道​​尔依康-M </td><td>安道​​尔技术</td><td> DU934N-BV </td><td>背照式CCD </td></tr><tr><td> Filterset </td><td>色度技术</td><td> 41021-MZFLIII </td><td> TXR，Alexa的594，CY3 </td></tr><tr><td> Filterset </td><td>色度技术</td><td> 41022-MZFLIII </td><td> IRDye680灼-680 </td></tr><tr><td> Filterset </td><td>色度技术</td><td> 49037-MZFLIII </td><td> Dylight750灼-750 </td></tr><tr><td> ProteinG-Sepharose磁珠</td><td> GE医疗集团</td><td> 17-0618-01 </td><td>蛋白G Sepharose 4个快速流</td></tr><tr><td>叠氮化钠</td><td> Sigma-Aldrich公司</td><td> 08591 </td><td>叠氮化钠0.1 M溶液</td></tr></tbody></table>

near infrared optical projection tomography for assessments of &beta;-cell mass distribution in diabetes research

通过OPT来适应包括在近红外（NIR）光谱成像的能力，我们在这里示出的可能性的图像较大的机构胰腺组织，如大鼠胰腺，和增加的信道数目（细胞类型），可在一个单一的标本进行研究。我们进一步描述一个数的计算工具，提供:1 /准确定位的一个标本的（在我们的例子中胰腺）中心的质量（COM）的旋转轴（AR）2，2 /改进的算法进行后实施取向的断层重建期间2和3 /强度均衡OPT基于BCM测定3中，增加信号噪声比的一个协议，用于防止几何失真的调谐。此外，我们描述一个无意的标本变动的风险，最大限度地减少图像采集的样品架。总之，这些协议能够评价BCM分配和OTH的呃功能，要进行整个完好的胰腺或其它器官（ 例如，在胰岛移植的研究）的体积，其分辨率的电平下降到个别胰岛。

在当前的报告中，我们描述了一种用于提取和计算处理的BCM使用NIR-OPT（ 图1）在啮齿动物胰腺中的数据（和其他组织）协议。正如在图2中示出，如预期的那样组织autofluorescense胰腺标本NIR光谱中的显着降低。这导致到一个显着增加的平均信号对噪声比（S:N）为评估胰岛素标记胰岛。 OPT来在近红外部分的频谱，如本文所述的成像由适应特定信道，至少有三个可实现可视化与足够的S:N比，使抗体标记的细胞类型的评估，具有鲜明的整个体积的小鼠胰腺信道分离（参见图3和4）。致糖尿病的进程和/或BCM评估一般成像中的应用，因此，该技术允许的可视化和量化的胰岛素阳性的地区在围绕和/或相互作用的细胞类型（参见图4）的关系。这样的评估是由于增加中得到的近红外范围内可能执行标本比以前更大的，其中包括，这是其鼠标对应（参见图5）的3-5倍大于大鼠胰腺组织穿透深度。无论是否可见或近红外波长的光被利用，执行CLAHE可能显着方便OPT基于评估BCM的期间不同的遗传和生理条件下，通过增加的技术（参见图6）的检测灵敏度。在图7所示的样品架的蓝图。 <img src="/files/ftp_upload/50238/50238fig1.jpg" alt="图1" /> 图1。流程图的关键步骤OPT基础的分析BCM在MUR国家统计局胰腺所需要的时间来衡量一个典型的小鼠胰腺是13-14天。组织处理和免疫组织化学染色（10天）的时间过程中被消耗的大部分，组织结算需要约2天，而扫描的长度依赖于需要的曝光时间（通常约1小时）。随后的运算处理通常在一天之内进行。请注意，在相对较长的染色协议非常适合于大量标本批处理。 <img src="/files/ftp_upload/50238/50238fig2.jpg" alt="图2" fo:content-width="6in" fo:src="/files/ftp_upload/50238/50238fig2highres.jpg" /> 图2。信号噪声比为的BCM评估在不同的波长。鼠标十二指肠的胰腺叶，染色胰岛素的鸡尾酒荧光标记的二抗（Alexa的488，594，680和750），被用来确定在不同波长的S:N比。 A，图片显示出第投影为每个信道的帧。 B，曲线图的平均值S:N为每个信道。的比率被确定为平均胰岛除以背景强度（从外分泌腺组织）的内源性组织荧光的强度（根据215胰岛）。 C，图表显示小号的个别小岛Alexa的594通道每个通道归一化的S:N:N比。采用单因素方差分析进行统计分析。显着性水平对应于** P &lt;0.01。 （A）到1毫米的标尺。 <a href="https://www.jove.com/files/ftp_upload/50238/50238fig2large.jpg" target="_blank">点击此处查看大图</a> 。 <img src="/files/ftp_upload/50238/50238fig3.jpg" alt="图3" fo:content-width="4in" fo:src="/files/ftp_upload/50238/50238fig3highres.jpg"/> 图3。 A，信道分离。Alexafluor在表中列出的染料的共轭的第二抗体被固定上单独proteinG琼脂糖珠。 B，荧光微球，然后嵌入在不同的层次，在琼脂糖幻影成像使用指定的过滤器。 <img src="/files/ftp_upload/50238/50238fig4.jpg" alt="图4" fo:content-width="5.5in" fo:src="/files/ftp_upload/50238/50238fig4highres.jpg"/> 图4。 OPT基于多通道成像技术在糖尿病研究 A，OPT的等值面重建的胰，十二指肠球部叶（12周）1型糖尿病非肥胖型糖尿病（NOD）模型。试样染色胰岛素（胰岛β细胞，伪蓝色）;平滑肌α-肌动蛋白（血管，红色）和CD3（T淋巴细胞浸润，绿色）。相应的二次抗体，CY3，IRDye-680和DyeLight-750的分别。 "插图（A&#39;-A&#39;&#39;&#39;）显示的个别信号通道。 B，OPT打击了看法，与同基因胰岛移植和成像与NIR-OPT 2周后移植的小鼠肝的叶（lobus险恶外侧）。胰岛素表达的胰岛pseuodocolored在蓝色和平滑肌α-肌动蛋白阳性血管是红色的。这种方法有助于评估胰岛移植分配内的血管网。比例尺对应1毫米。 <img src="/files/ftp_upload/50238/50238fig5.jpg" alt="图5" fo:content-width="4in" fo:src="/files/ftp_upload/50238/50238fig5highres.jpg" /> 图5。 NIR-OPT利于较大的标本成像A，等值面渲染从只Zucker脂肪模型的2型糖尿病的（脾叶在9个月）在大鼠胰腺BCM的分布，例示对大鼠的可能性的图像试样的胰腺规模的NIR-OPT。由于确定通过这种技术，显示的叶〜6倍（体积/体积）比其鼠标和窝藏10139胰岛素表达胰岛的β-细胞的体积的1.32％的总小叶体积。乙，层析部分对应于（A）中示出，检测到胰岛从所有的组织深处的虚线。 C，等值面绘制的的BCM分布在一个小鼠胰腺（脾叶在8个星期）显示的大小引用。显示的叶窝藏2490胰岛素表达胰岛的β细胞的体积，使0.89％的总的小叶卷。被染成Alexa594标记的羊抗GP（鼠标）和IRDye 680标记的驴抗-GP抗体（鼠）分别与GP抗胰岛素的胰腺组织。标本在（AC）规模和比例尺描绘的（C）对应到2毫米。 <img src="/files/ftp_upload/50238/50238fig6.jpg" alt="图6" fo:content宽度="5英寸的FO:src ="/ files/ftp_upload/50238/50238fig6highres.jpg"/&gt; 图6。 CLAHE便于检测OPT成像在小鼠胰腺的胰岛。AC代表异面呈现OPT图像标记为胰岛素的C57B1 / 6老鼠胰腺（脾叶8周）。等值面重建的OPT图像的前（A，伪彩色绿色）CLAHE协议后的应用（B，伪红色）。 C，叠加的非规范化的数据（A）和（B）中的CLAHE处理的数据。 C&#39;-C"，代表的非标准化（A）和（B）CLAHE加工图像的高倍率覆盖。CLAHE脚本的"红只"小岛的存在，有利于小和低信号的检测在当前的示例中所描绘的试样（CLAHE加工后）的强度胰岛。窝藏2419胰岛的体积为1.74毫米3（相应的未处理的投影数据的基础上的编号为1057胰岛的Wi日:1.77毫米3）的体积。 D和E，对照组（D）和2型糖尿病12（E）在6个月实施CLAHE协议的ob / ob小鼠模型的示例数据。请注意胰岛大小的ob / ob胰腺（E）的大量增加。在（D）和（E）胰腺轮廓（灰色）的信号的基础上从组织的自发荧光。在C比例尺为500μm的AC。比例尺在C"相当于200μm的C&#39;和C"。比例尺在E对应到1毫米（D）和（E）在（AC）适于从Hörnblad 等 3图像，并使用生成的扫描仪Bioptonics 3001。 <img src="/files/ftp_upload/50238/50238fig7.jpg" alt="图7" /> 图7。用于附连的OPT标本的样本保持器，然后将试样固定通过插入针通过琼脂糖间隔件通过预先钻好的孔中的凸缘。保持架是铰接到步进马达通过强磁铁坐落在它的基础。该设置在扫描过程中忽略了不稳定的胶水的使用，防止不必要的运动的标本。

Watch this Scientific Journal Video about Near Infrared Optical Projection Tomography for Assessments of Î²-cell Mass Distribution in Diabetes Research at JoVE.com

Near Infrared Optical Projection Tomography for Assessments of &#946;-cell Mass Distribution in Diabetes Research

我们描述了适应光学投影层析成像（OPT）<sup&gt; 1</sup&gt;成像技术在近红外光谱，并实施了一些计算工具。这些协议使胰岛β-细胞团（BCM）的评估，在更大的标本，增加多渠道的技术能力，并增加OPT数据的质量。

近红外光学投影层析成像技术在糖尿病研究β细胞质量分布的评估

By adapting OPT to include the capability of imaging in the near infrared (NIR) spectrum, we here illustrate the possibility to image larger bodies of ...

near-infrared-optical-projection-tomography-for-assessments-cell-mass

Research

JoVE Journal

Medicine

Near Infrared Optical Projection Tomography for Assessments of &beta;-cell Mass Distribution in Diabetes Research

16.3K 次观看.  Umeå University. 我们描述了适应光学投影层析成像（OPT） 1成像技术在近红外光谱，并实施了一些计算工具。这些协议使胰岛β-细胞团（BCM）的评估，在更大的标本，增加多渠道的技术能力，并增加OPT数据的质量。

视频: Near Infrared Optical Projection Tomography for Assessments of β-cell Mass Distribution in Diabetes Research

In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading to an accumulation of lipid-laden intra-luminal plaque, smooth muscle cell proliferation and progressive narrowing of the vessel lumen. The formation of such vulnerable plaques prone to rupture underlies the majority of cases of acute myocardial infarction. The complex molecular and cellular inflammatory cascade is orchestrated by the recruitment of T lymphocytes and macrophages and their paracrine effects on endothelial and smooth muscle cells.1
Molecular imaging in atherosclerosis has evolved into an important clinical and research tool that allows in vivo visualization of inflammation and other biological processes. Several recent examples demonstrate the ability to detect high-risk plaques in patients, and assess the effects of pharmacotherapeutics in atherosclerosis.4 While a number of molecular imaging approaches (in particular MRI and PET) can image biological aspects of large vessels such as the carotid arteries, scant options exist for imaging of coronary arteries.2 The advent of high-resolution optical imaging strategies, in particular near-infrared fluorescence (NIRF), coupled with activatable fluorescent probes, have enhanced sensitivity and led to the development of new intravascular strategies to improve biological imaging of human coronary atherosclerosis.
Near infrared fluorescence (NIRF) molecular imaging utilizes excitation light with a defined band width (650-900 nm) as a source of photons that, when delivered to an optical contrast agent or fluorescent probe, emits fluorescence in the NIR window that can be detected using an appropriate emission filter and a high sensitivity charge-coupled camera. As opposed to visible light, NIR light penetrates deeply into tissue, is markedly less attenuated by endogenous photon absorbers such as hemoglobin, lipid and water, and enables high target-to-background ratios due to reduced autofluorescence in the NIR window. Imaging within the NIR 'window' can substantially improve the potential for in vivo imaging.2,5
Inflammatory cysteine proteases have been well studied using activatable NIRF probes10, and play important roles in atherogenesis. Via degradation of the extracellular matrix, cysteine proteases contribute importantly to the progression and complications of atherosclerosis8. In particular, the cysteine protease, cathepsin B, is highly expressed and colocalizes with macrophages in experimental murine, rabbit, and human atheromata.3,6,7 In addition, cathepsin B activity in plaques can be sensed in vivo utilizing a previously described 1-D intravascular near-infrared fluorescence technology6, in conjunction with an injectable nanosensor agent that consists of a poly-lysine polymer backbone derivatized with multiple NIR fluorochromes (VM110/Prosense750, ex/em 750/780nm, VisEn Medical, Woburn, MA) that results in strong intramolecular quenching at baseline.10 Following targeted enzymatic cleavage by cysteine proteases such as cathepsin B (known to colocalize with plaque macrophages), the fluorochromes separate, resulting in substantial amplification of the NIRF signal. Intravascular detection of NIR fluorescence signal by the utilized novel 2D intravascular NIRF catheter now enables high-resolution, geometrically accurate in vivo detection of cathepsin B activity in inflamed plaque.
In vivo molecular imaging of atherosclerosis using catheter-based 2D NIRF imaging, as opposed to a prior 1-D spectroscopic approach,6 is a novel and promising tool that utilizes augmented protease activity in macrophage-rich plaque to detect vascular inflammation.11,12 The following research protocol describes the use of an intravascular 2-dimensional NIRF catheter to image and characterize plaque structure utilizing key aspects of plaque biology. It is a translatable platform that when integrated with existing clinical imaging technologies including angiography and intravascular ultrasound (IVUS), offers a unique and novel integrated multimodal molecular imaging technique that distinguishes inflammatory atheromata, and allows detection of intravascular NIRF signals in human-sized coronary arteries.

In vivo Near Infrared Fluorescence NIRF Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

We detail a new near-infrared fluorescence (NIRF) catheter for 2-dimensional intravascular molecular imaging of plaque biology in vivo. The NIRF catheter can visualize key biological processes such as inflammation by reporting on the presence of plaque-avid activatable and targeted NIR fluorochromes. The catheter utilizes clinical engineering and power requirements and is targeted for application in human coronary arteries. The following research study describes a multimodal imaging strategy that utilizes a novel in vivo intravascular NIRF catheter to image and quantify inflammatory plaque in proteolytically active inflamed rabbit atheromata.

体内近红外荧光（NIRF）血管内分子成像炎性斑块，多式联运方针成像动脉粥样硬化

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading ...

科研

医学

Doppler Optical Coherence Tomography of Retinal Circulation

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double circular scan (CDCS) that scans around the optic nerve head at 3.40 mm and 3.75 mm diameters. The double concentric circles are performed 6 times consecutively over 2 sec. The CDCS scan is saved with Doppler shift information from which flow can be calculated. The standard clinical protocol calls for 3 CDCS scans made with the OCT beam passing through the superonasal edge of the pupil and 3 CDCS scan through the inferonal pupil. This double-angle protocol ensures that acceptable Doppler angle is obtained on each retinal branch vessel in at least 1 scan. The CDCS scan data, a 3-dimensional volumetric OCT scan of the optic disc scan, and a color photograph of the optic disc are used together to obtain retinal blood flow measurement on an eye. We have developed a blood flow measurement software called "Doppler optical coherence tomography of retinal circulation" (DOCTORC). This semi-automated software is used to measure total retinal blood flow, vessel cross section area, and average blood velocity. The flow of each vessel is calculated from the Doppler shift in the vessel cross-sectional area and the Doppler angle between the vessel and the OCT beam. Total retinal blood flow measurement is summed from the veins around the optic disc. The results obtained at our Doppler OCT reading center showed good reproducibility between graders and methods (&lt;10%). Total retinal blood flow could be useful in the management of glaucoma, other retinal diseases, and retinal diseases. In glaucoma patients, OCT retinal blood flow measurement was highly correlated with visual field loss (R2&gt;0.57 with visual field pattern deviation). Doppler OCT is a new method to perform rapid, noncontact, and repeatable measurement of total retinal blood flow using widely available Fourier-domain OCT instrumentation. This new technology may improve the practicality of making these measurements in clinical studies and routine clinical practice.

Total retinal blood flow is measured by Doppler optical coherence tomography and semi-automated grading software.

多普勒光学相干断层扫描视网膜循环

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double ...

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating important information on the pathogenesis of lesion formation and the action of novel therapies. Use of atherosclerosis-prone mice, surgical methods of lesion induction, and dietary modification has dramatically improved understanding of the mechanisms that contribute to disease development and the potential of new treatments.
Classically, analysis of lesions is performed ex vivo using 2-dimensional histological techniques. This article describes application of optical projection tomography (OPT) to 3-dimensional quantitation of arterial lesions. As this technique is non-destructive, it can be used as an adjunct to standard histological and immunohistochemical analyses.
Neointimal lesions were induced by wire-insertion or ligation of the mouse femoral artery whilst atherosclerotic lesions were generated by administration of an atherogenic diet to apoE-deficient mice.
Lesions were examined using OPT imaging of autofluorescent emission followed by complementary histological and immunohistochemical analysis. OPT clearly distinguished lesions from the underlying vascular wall. Lesion size was calculated in 2-dimensional sections using planimetry, enabling calculation of lesion volume and maximal cross-sectional area. Data generated using OPT were consistent with measurements obtained using histology, confirming the accuracy of the technique and its potential as a complement (rather than alternative) to traditional methods of analysis.
This work demonstrates the potential of OPT for imaging atherosclerotic and neointimal lesions. It provides a rapid, much needed ex vivo technique for the routine 3-dimensional quantification of vascular remodelling.

Ex vivo analysis of arterial lesions from animal models of cardiovascular disease classically relies on histological and immunohistochemical techniques. These provide 2-dimensional measurements in 3-dimensional lesions. This manuscript describes the generation of arterial lesions for quantitative analysis in 3-dimensions using optical projection tomography.

生成和动脉病变的小鼠的三维定量使用光学投影层析成像

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

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 ...

In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

Optical coherence tomography (OCT) is the optical equivalent of ultrasound imaging, based on the backscattering of near infrared light. OCT provides real time images with a 15 &#181;m axial resolution at an effective tissue penetration of 2-3 mm. Within the OCT images the loss of signal intensity per millimeter of tissue penetration, the attenuation coefficient, is calculated. The attenuation coefficient is a tissue specific property, providing a quantitative parameter for tissue differentiation. 
Until now, renal mass treatment decisions have been made primarily on the basis of MRI and CT imaging characteristics, age and comorbidity. However these parameters and diagnostic methods lack the finesse to truly detect the malignant potential of a renal mass. A successful core biopsy or fine needle aspiration provides objective tumor differentiation with both sensitivity and specificity in the range of 95-100%. However, a non-diagnostic rate of 10-20% overall, and even up to 30% in SRMs, is to be expected, delaying the diagnostic process due to the frequent necessity for additional biopsy procedures. 
We aim to develop OCT into an optical biopsy, providing real-time imaging combined with on-the-spot tumor differentiation. This publication provides a detailed step-by-step approach for percutaneous, needle based, OCT of renal masses.

In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

Optical coherence tomography (OCT) is a high resolution imaging technique that allows analysis of tissue specific optical properties providing the means for tissue differentiation. We developed needle based OCT, providing real-time imaging combined with on-the-spot tumor differentiation. This publication describes a method for percutaneous, needle based OCT of renal masses.

在体内 ，经皮，基于针，肾群众的光学相干断层扫描

Optical coherence tomography (OCT) is the optical equivalent of ultrasound imaging, based on the backscattering of near infrared light. OCT provides real ...

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 ...

Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the subretinal space, and to evaluate toxicity and efficacy in vivo. HR-SD-OCT uses near infrared light (800-880 nm) and has optics specifically designed for the unique optics of the mouse eye with sub-2-micron axial resolution. Transgenic mouse models of outer retinal (photoreceptor) degeneration and controls were imaged to assess the disease progression. Pulled glass microneedles were used to deliver sub retinal injections of adeno-associated virus (AAV) or nanoparticles (NP) via a trans-scleral and trans-choroidal approach. Careful positioning of the needle into the subretinal space was required prior to a calibrated pressure injection, which delivers fluid into the sub retinal space. Real time subretinal surgery was conducted on our retinal imaging system (RIS). HR-SD-OCT demonstrated progressive uniform retinal degeneration due to expression of a toxic mutant human mutant rhodopsin (P347S) (RHOP347S) transgene in mice. HR-SD-OCT allows rigorous quantification of all the retinal layers. Outer nuclear layer (ONL) thickness and photoreceptor outer segment length (OSL) measurements correlate with photoreceptor vitality, degeneration, or rescue. The RIS delivery system allows real-time visualization of subretinal injections in neonatal (~P10-14) or adult mice, and HR-SD-OCT immediately determines success of delivery and maps areal extent. HR-SD-OCT is a powerful tool that can evaluate the success of subretinal surgery in mice, in addition to measuring vitality of photoreceptors in vivo. HR-SD-OCT can also be used to identify uniform animal cohorts to evaluate the extent of retinal degeneration, toxicity, and therapeutic rescue in preclinical gene therapy research studies.

Here we demonstrate a novel approach to using high resolution spectral-domain optical coherence tomography (HR-SD-OCT) to assist delivery of gene therapy agents into the subretinal space, assess its areal coverage, and characterize photoreceptor vitality.

超高分辨率小鼠光学相干层析成像辅助眼内注射在视网膜基因治疗中的应用

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the ...

A New Best Practice for Validating Tail Vein Injections in Rat with Near-infrared-Labeled Agents

Intravenous (IV) administration of agents into the tail vein of rats can be both difficult and inconsistent. Optimizing tail vein injections is a key part of many experimental procedures where reagents need to be introduced directly into the bloodstream. Unwittingly, the injection can be subcutaneous, possibly altering the scientific outcomes. Utilizing a nanoemulsion-based biological probe with an incorporated near-infrared fluorescent (NIRF) dye, this method offers the capability of imaging a successful tail vein injection in vivo. With the use of a NIRF imager, images are taken before and after the injection of the agent. An acceptable IV injection is then qualitatively or quantitatively determined based on the intensity of the NIRF signal at the site of injection.

Here we present a method to validate tail vein injections in rats by utilizing near-infrared fluorescence imaging data from dyes incorporated into agents or biological probes. The tail is imaged before and after the injection, the fluorescent signal is quantified, and an assessment of the injection quality is made.

近红外标记剂对大鼠尾静脉注射的新的最佳验证

Intravenous (IV) administration of agents into the tail vein of rats can be both difficult and inconsistent. Optimizing tail vein injections is a key part ...

Semi-Minimal Invasive Method to Induce Myocardial Infarction in Rats and the Assessment of Cardiac Function by an Isolated Working Heart System

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily and highly reproducible MI induction procedure is required to obtain further insight and better understanding of the underlying pathological changes. This procedure can also be used to evaluate the effects or potency of new and promising treatments (as drugs or interventions) in acute MI, subsequent remodeling and heart failure (HF). After intubation and pre-operative preparation of the animal, an anesthetic protocol with isoflurane was performed, and the surgical procedure was conducted&#160;quickly. Using a minimally invasive approach, the left anterior descending artery (LAD) was located and occluded by a ligature. The occlusion can be performed acutely for subsequent reperfusion (ischemia/reperfusion injury). Alternatively, the vessel can be ligated permanently to investigate the development of chronic MI, remodeling or HF. Despite common pitfalls, the drop-out rates are minimal. Various treatments such as remote ischemic conditioning can be examined for their cardioprotective potential pre-, peri- and post-operatively. The post-operative recovery was quick as the anesthesia was precisely controlled and the duration of the operation was short. Post-operative analgesia was administered for three days. The minimally invasive procedure reduces the risk of infection and inflammation. Furthermore, it facilitates rapid recovery. The &#8220;working heart&#8221; measurements were performed ex vivo and enabled precise control of preload, afterload and flow. This procedure requires specific equipment and training for adequate performance. This manuscript provides a detailed step-by-step introduction for conducting these measurements.

This article presents an efficient method to perform myocardial ischemia and subsequent chronic reperfusion in rats using a minimally invasive approach. In addition, left ventricular hemodynamic function of rats is assessed by echocardiography and isolated working heart methods.&#160;

诱导大鼠心肌梗死的半微创方法及独立工作心脏系统对心脏功能的评估

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily ...

How to Administer Near-Infrared Spectroscopy in Critically ill Neonates, Infants, and Children

Near infrared spectroscopy (NIRS) calculates regional tissue oxygenation (rSO2) using the different absorption spectra of oxygenated and deoxygenated hemoglobin molecules. A probe placed on the skin emits light that is absorbed, scattered, and reflected by the underlying tissue. Detectors in the probe sense the amount of reflected light: this reflects the organ-specific ratio of oxygen supply and consumption - independent of pulsatile flow. Modern devices enable the simultaneous monitoring at different body sites. A rise or dip in the rSO2 curve visualizes changes in oxygen supply or demand before vital signs indicate them. The evolution of rSO2 values in relation to the starting point is more important for interpretation than are absolute values.
A routine clinical application of NIRS is the surveillance of somatic and cerebral oxygenation during and after cardiac surgery. It is also administered in preterm infants at risk for necrotizing enterocolitis, newborns with hypoxic ischemic encephalopathy and a potential risk of impaired tissue oxygenation. In the future, NIRS could be increasingly used in multimodal neuromonitoring, or applied to monitor patients with other conditions (e.g., after resuscitation or traumatic brain injury).

This protocol is designed to assist clinicians to measure regional tissue oxygenation at different body sites in infants and children. It can be used in situations where tissue oxygenation is potentially compromised, particularly during cardiopulmonary bypass, when using non-pulsatile cardiac-assist devices, and in critically ill neonates, infants and children.

如何管理危重新生儿、婴儿和儿童近红外光谱

Near infrared spectroscopy (NIRS) calculates regional tissue oxygenation (rSO2) using the different absorption spectra of oxygenated and deoxygenated ...

近红外光学投影层析成像技术在糖尿病研究β细胞质量分布的评估

摘要

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此视频中的章节

体内近红外荧光（NIRF）血管内分子成像炎性斑块，多式联运方针成像动脉粥样硬化

多普勒光学相干断层扫描视网膜循环

生成和动脉病变的小鼠的三维定量使用光学投影层析成像

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

在体内，经皮，基于针，肾群众的光学相干断层扫描

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

超高分辨率小鼠光学相干层析成像辅助眼内注射在视网膜基因治疗中的应用

近红外标记剂对大鼠尾静脉注射的新的最佳验证

诱导大鼠心肌梗死的半微创方法及独立工作心脏系统对心脏功能的评估

如何管理危重新生儿、婴儿和儿童近红外光谱

近红外光学投影层析成像技术在糖尿病研究β细胞质量分布的评估

摘要

探索更多视频

此视频中的章节

体内近红外荧光（NIRF）血管内分子成像炎性斑块，多式联运方针成像动脉粥样硬化

多普勒光学相干断层扫描视网膜循环

生成和动脉病变的小鼠的三维定量使用光学投影层析成像

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

在体内 ，经皮，基于针，肾群众的光学相干断层扫描

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

超高分辨率小鼠光学相干层析成像辅助眼内注射在视网膜基因治疗中的应用

近红外标记剂对大鼠尾静脉注射的新的最佳验证

诱导大鼠心肌梗死的半微创方法及独立工作心脏系统对心脏功能的评估

如何管理危重新生儿、婴儿和儿童近红外光谱

在体内，经皮，基于针，肾群众的光学相干断层扫描