Part of this work was supported by a Grant-in-Aid for Scientific Research (C) from the Japanese Society for the Promotion of Science (25350520, 22500401, 15K06105) and the US-ARMY ITC-PAC Research and Development Project (FA5209-15-P-0175).

动物保健，准备和实验方案是由东京农工大学的动物研究委员会的批准。对于这种方法，将大鼠圈养在受控环境中（24℃，12小时光照/黑暗周期），与食物和水可随意获得。 1.传统多光谱漫反射成像系统的构建<ol><li>摩9个的窄带光学干涉滤光器具有的中心波长500,520，540，560，570，580，600，730，和760纳米至机动滤光轮的过滤孔。 </li><li>构建使用宽带白色光源，机动滤光轮与上述组窄带干涉滤光片，光导，聚光透镜，视频变焦透镜，和一个单色CCD照相机的多光谱成像系统。光学元件，在图 2中所示的布局，可以被称为用于第是施工程序。 注:照明的角度是大约45°相对于样品表面。 </li><li>转动卤素灯光源经由一个干涉滤光器，光导，和聚光透镜来照射样品的表面。 </li><li>打开CCD相机的操作软件。 </li></ol> 2.动物的制备注:在这个协议中，并没有用于未来的实验大鼠和它多光谱图像的测量后，立即处死。 <ol><li>感应腔室的入口端口连接到麻醉机的与管出口端口。感应腔室的出口端口连接到麻醉机的进气口与第二管。 </li><li>放置老鼠进入吸气室和诱导具有5.0％异氟烷麻醉。在深度，使得老鼠并没有脚趾捏回应维持麻醉。大号奥尔〜2.0％异氟烷使用麻醉机上的旋钮。 </li><li>在立体定位框架固定所述大鼠头部。附上麻醉喉舌的立体框架。 </li><li>吸嘴的吸入口连接到麻醉机用管出口端口。吸嘴的出口端口连接到麻醉机用管入口端口。 </li><li>剃光头区域使用外推剪，直到出现在皮肤表面的预期切口部位。 </li><li>使形成纵向切口长大约20mm沿着所述头部的使用外科手术刀中线（ 图1（a））和暴露的皮下结缔组织（ 图1（b））。 </li><li>使用锋利刮匙或钳子去除皮下结缔组织并将其拉向头部两侧，以暴露颅骨（ 图1（c））。 </li><li>挖颅骨椭圆形沟颅sutur内es（冠状缝合，矢状缝合和羊角缝合）使用高速钻（ 图1（d） ）。 </li><li>使用高速钻机慢慢均匀地挖掘沟内的颅骨。 </li><li>用钳子的尖端轻轻地压在稀疏头骨的表面上，以估计脑血管出现后的骨厚度和强度。如果变薄的颅骨区域容易下压，则用高速钻机终止颅骨骨折。 </li><li>使用钳子或小手术剪刀切割瘦骨头碎片的椭圆形边界线。 </li><li>从脑表面缓慢地轻轻地使用钳子去除稀疏的头骨。 </li><li>用生理盐水轻轻沐浴颅窗，并用约0.1毫米厚的透明玻璃板覆盖。 </li></ol> 3.调节启发氧的分数注意:呼吸系统灰可以通过调节吸入氧的（ 吸入氧浓度）分数来改变。 <ol><li>使用管，一个Y形管连接器（连接器1）的第一端口连接到另一个Y形管连接器（连接器2）的第一端口。 </li><li>吸嘴的吸入口连接到管连接器1的第二端口。 </li><li>使用的管，管连接器1的第三端口连接到一个氧浓度监视器装置。 </li><li>用管，管连接器2的第二端口连接到麻醉机的出口。 </li><li>使用的管，管连接器2的第三端口连接到一个气体混合物装置的出口端口。 </li><li>气体混合物装置的一个进气口连接到一个高压95％O 2 - 5％使用管的CO 2气体圆柱体。 </li><li>气体混合物的设备的另一入口端口连接到一个高压95％N 2 -使用管5％CO 2气瓶。 </li><li>改变气体的流速Ø˚FO 2和N 2使用气体混合物的设备上的旋钮。 </li><li>检查并使用氧浓度监视器装置调节吸入氧浓度。 </li></ol> 4.多光谱漫反射图像采集<ol><li> 参考图像采集 注意:在这个实验中使用的光学部件，例如作为光源，光纤，和检测器有自己的光谱特性。因此，光的通过这些部件通过了强度应记录为参考图像。参考图像是用来自光源的光照亮的标准白色扩散器所拍摄的图像。 <ol><li>放标准白色扩散板在舞台上水平。 </li><li>通过在桶旋转变焦环聚焦白色扩散器的表面上的相机镜头。 </li><li>通过选择适当的值来调整摄像头的积分时间下拉的积分时间列表中相机的操作软件，使最大的光量产生的信号是最大计数的约75％。一边看像素值的直方图，调整积分时间，直到信号强度电平是最大计数的约75％。 </li><li>从文件菜单中的"保存"命令，将图像保存到文件中。 </li><li>通过旋转滤光轮改变滤波器位置。 </li><li>根据上述方法在其它波长保存的图像。文件名应识别样品和使用的波长（ 例如，W500，W520，W540 W760 ...）。 </li></ol></li><li> 样本图像采集 注意:在9个波长暴露的大鼠脑的漫反射光强度的图像被捕获并保存使用相同的采集条件的个人计算机的硬盘驱动器上。 <ol><li>轻轻地放在将R在舞台上，慢慢调整阶段水平，使相机可以对焦的大鼠脑的表面上。 </li><li>从文件菜单中的"保存"命令，将图像保存到文件中。 </li><li>通过旋转滤光轮改变滤波器位置。 </li><li>根据上述方法在其它波长保存的图像。文件名应识别样品和波长（ 如，R500，R520，R540 R760 ...）。 </li></ol></li><li> 深色图像采集 注:CCD照相机可以响应于电信号产生的光强度。然而，有一些小的输出由于在电气电路和检测器噪声，即使光不进入到检测器;这就是所谓的暗电流噪声。为了精确测量光的光谱强度时，暗电流成分应记录为暗图像，然后从所测量的信号中减去。黑暗的图像是取n，其中的光路阻断。 <ol><li>关闭卤素灯光源。 </li><li>阻挡光路，以利用屏蔽板的CCD摄像系统。 </li><li>从文件菜单中的"保存"命令，将图像保存到文件中。文件名应确定，样品（ 例如，暗）。 </li></ol></li></ol> 5.可视化血红蛋白含量和光散射参数注:一组多光谱漫反射的图像保存到个人电脑的硬盘驱动器和离线分析。通过在9个波长（500，520，540，560，570，580，600，730，和760纳米）的多光谱漫反射图像的蒙特卡罗模拟19辅助的多回归分析然后进行以可视化的二维氧化血红蛋白浓度，脱氧血红蛋白浓度，总血红蛋白浓度，局部脑氧饱和度，和SCAT的地图的TeringBay动力。具体算法已发表在文献17，18。 <ol><li>来自两个参考图像，并在每个波长处的样本图像中减去暗图像。 </li><li>在每个波长λ归一化由基准图像的样本图像。治疗的归一化的图像作为漫反射率图像R。 </li><li>通过取在每个波长λ的漫反射率图像R的倒数的对数计算的吸光度（或光密度）图像A: <img alt="式（1）" src="/files/ftp_upload/55399/55399eq1.jpg" /> （1） </li><li> y平面示出了用于在脑表面所获得的结构信息，而z -轴示出光谱信息-通过在它们的波长的顺序，其中x堆叠的吸光度图像生成的三维矩阵。 </li><li> PErform在每个xy坐标的吸收光谱A（λ）多元回归分析。 </li><li>使用的吸收光谱A（λ）作为因变量和氧合血红蛋白的摩尔消光系数光谱εHbO2的 （λ）和脱氧血红蛋白εHBR（λ）作为用于步骤5.5的独立变量（公布值εHbO2的 （λ）和εHBR（λ）在表1中提供）。 </li><li>检查三个多重回归的二维地图（图像）系数a HBO， 一个HBr和一个 0。 </li><li>通过堆叠多重回归系数的图像的次序的HBO， 一个HBr和一个 0，生成的三维矩阵，其中该<e米&gt; X - y平面示出了用于在脑表面，而z轴所获得的结构信息示出了多重回归系数。 </li><li>计算氧合血红蛋白浓度C HBO，脱氧血红蛋白浓度C HBr和从该组的多个回归系数a HBO， 一个HBr和 0在每个xy坐标使用以下经验式（他值β的散射电压B HBO，I，βHBR，i和β0，I（I = 0,1,2,3）在表2中提供）: <img alt="公式2" src="/files/ftp_upload/55399/55399eq2.jpg" /> （2） <img alt="公式3" src="/files/ftp_upload/55399/55399eq3.jpg" /> （3） <img alt="公式4" src="/files/ftp_upload/55399/55399eq4.jpg" /> （4） </li><li>检查氧化血红蛋白浓度C HBO，脱氧血红蛋白浓度C HBr和散射功率B的二维地图（图像）。 </li><li>计算由求和ÇHbO2的和C HBR在每个x总血红蛋白浓度C HBT的二维地图- y坐标。 </li><li> y坐标-通过在每个x除以氧化血红蛋白浓度C HbO2的由总血红蛋白浓度C HBT计算局部脑氧饱和度RSO 2的二维地图。 </li></ol>

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The authors have nothing to disclose.

在这个协议中最关键的步骤是去除减薄头骨区域，使颅窗口的;这应该小心进行，以避免意外出血。这个步骤是为了获得高品质的多光谱扩散高精度反射图像重要。建议手术过程如果可能的话，使用立体显微镜。明胶海绵小块止血有用。 这篇文章中描述的光学系统穿过位于所述光源的前面的干涉滤光器的单色的光。这可以通过将所述滤光轮在摄像机镜头或CCD摄像机的前面进行修改。在这种情况下，然而，焦平面可以是可变的，如果使用具有不同厚度的干涉滤光器，而这将造成图像质量的恶化。有必要从颅窗口取出玻璃板如果记录电极插入到皮质组织为电生理学测量，如电局部场电位的测量。在这种情况下，成像系统可以检测到来自皮质表面不期望的镜面反射。可以通过使用一组偏振板用正交尼科耳排列，可以避免此问题。 在这篇文章中展示了传统的多光谱成像设备是有点费时使用，由于在轮上的滤波器的位置被机械地改变。这意味着，所述成像系统捕获的每个漫反射图像依次以不同的波长点。由于此限制，该系统是不充分捕捉快速IOSS，如在反射光谱由于神经元的活动20的变化。虽然氧合血红蛋白和氧合血红蛋白是活脑组织的主要发色团，其他的发色团，如细胞色素C氧化酶，黄素腺嘌呤二核苷酸和烟酰胺腺嘌呤二核苷酸，还有助于在可见光波长区域中的吸收系数。因此，C HbO2的 ，C HBR，C HBT，RSO 2，和b的估计值可以由轻微的发色团所影响。此外，该方法综合了沿深度方向的所有信息，因为它依赖于漫反射。因此，成像系统不执行深度分辨的测量。 有利的是，用于本系统中的算法也可以应用到由其他快速光谱成像技术，例如声光可调滤光器21捕获的多光谱漫反射图像，多孔径小透镜阵列与干涉滤波器22，并从RGB图像17，2光谱图像重建3。将所提出的算法和快速光谱技术一起使用是评估快速IOS成像以及用于临床情况的有希望的方法。 目前大多数多光谱脑成像技术主要集中在皮质血液动力学和组织代谢，如脑血容量，局部脑氧饱和度以及氧气的代谢率10,11,12,13,14。假设散射功率恒定在15,16之间 ，现有的几种方法评估了散射振幅。然而，由于病理生理变化导致的组织形态学改变和生物皮层组织活力的降低可影响生物散射体4的尺寸，5，6，7，8，9。因此，为了估计B的散射参数定量评价大脑的组织形态是很重要的。相对于现有方法，本技术的意义是它能够同时测量在脑血流和皮层组织形态学的时空变化的能力。 在未来的应用而言，这种算法可以用于监测大脑功能，生命体征，和生存能力各种脑部疾病的动物模型，如脑外伤，癫痫发作，中风和缺血皮层组织。

多光谱漫反射成像是用于获得在皮质组织固有的光学信号（IOSS）的空间图的最常用的技术。 IOSS在体内脑电波中所见的主要归因于三个现象:在光吸收的变化和由于皮质血流动力学散射特性，这取决于降低或线粒体细胞色素氧化在吸收变化和变型中通过形态学改变诱导的光散射特性1。 在可见光（VIS）到近红外（NIR）的光谱范围内的光被有效地吸收，并通过生物组织散射。 体内脑的扩散反射光谱的特征在于，吸收和散射光谱。减小的散射系数μ在VIS到NIR波长范围内的结果的脑组织的S&#39;以单调散射光谱表现出在较长波长荷兰国际集团较小幅度。减小的散射系数μ频谱S"（λ）可以近似为在幂律函数2，3的形式为μS"（λ）=α×λ-b。散射能力b为在活组织2,3与生物散射体的大小。组织和减少活皮层组织的生存能力的形态改变可影响生物散射体4，5，6，7，8，9的大小。 多光谱漫反射成像光学系统，可以从白炽灯里容易地构建GHT源，简单的光学部件，和一个单色电荷耦合器件（CCD）。因此，各种算法和多光谱漫反射成像光学系统已被用来评估皮质血流动力学和/或组织形态10，11，12，13，14，15，16，17，18。 这篇文章中描述的方法，被用于可视化两者血液动力学和体内使用常规多光谱漫反射成像系统的大鼠脑组织的光散射性质。这种方法比其它技术的优点是，以评估在两个脑血流和皮层组织时空变化的能力形态，以及它适用于各种脑功能障碍的动物模型。因此，该方法将是适当的创伤性脑损伤，癫痫发作，中风和缺血调查。

<table><tbody><tr><td>150-W halogen-lamp light source</td><td>Hayashi Watch Works Co., Ltd, Tokyo, Japan</td><td>LA-150SAE</td><td></td></tr><tr><td>Light guide</td><td>Hayashi Watch Works Co., Ltd, Tokyo, Japan</td><td>LGC1-5L1000</td><td></td></tr><tr><td>Collecting lens</td><td>Hayashi Watch Works Co., Ltd, Tokyo, Japan</td><td>SH-F16</td><td></td></tr><tr><td>Interference filters l@ 500 nm</td><td>Edmund Optics Japan Ltd, Tokyo, Japan</td><td>#65088</td><td></td></tr><tr><td>Interference filters l@ 520 nm</td><td>Edmund Optics Japan Ltd, Tokyo, Japan</td><td>#65093</td><td></td></tr><tr><td>Interference filters l@ 540 nm</td><td>Edmund Optics Japan Ltd, Tokyo, Japan</td><td>#65096</td><td></td></tr><tr><td>Interference filters l@ 560 nm</td><td>Edmund Optics Japan Ltd, Tokyo, Japan</td><td>#67766</td><td></td></tr><tr><td>Interference filters l@ 570 nm</td><td>Edmund Optics Japan Ltd, Tokyo, Japan</td><td>#67767</td><td></td></tr><tr><td>Interference filters l@ 580 nm</td><td>Edmund Optics Japan Ltd, Tokyo, Japan</td><td>#65646</td><td></td></tr><tr><td>Interference filters l@ 600 nm</td><td>Edmund Optics Japan Ltd, Tokyo, Japan</td><td>#65102</td><td></td></tr><tr><td>Interference filters l@ 730 nm</td><td>Edmund Optics Japan Ltd, Tokyo, Japan</td><td>#65115</td><td></td></tr><tr><td>Interference filters l@ 760 nm</td><td>Edmund Optics Japan Ltd, Tokyo, Japan</td><td>#67777</td><td></td></tr><tr><td>Motorized filter wheel&amp;nbsp;</td><td>Andover Corporation, NH, USA</td><td>FW-MOT-12.5</td><td></td></tr><tr><td>8-bit monochromatic CCD camera</td><td>THE IMAGINGSOURCE, Germany</td><td>DMK21BU618.H</td><td></td></tr><tr><td>Video zoom lens</td><td>Edmund Optics Japan Ltd, Tokyo, Japan</td><td>VZM&lt;sup&gt;TM&lt;/sup&gt;300i</td><td></td></tr><tr><td>Spectralon white standard with 99% diffuse reflectance</td><td>Labsphere Incorporated, North Sutton, NH, USA</td><td>SRS-99-020</td><td></td></tr></tbody></table>

simultaneous evaluation of cerebral hemodynamics and light scattering properties of the in vivo rat brain using multispectral diffuse reflectance imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system. This system is constructed from a broadband white light source, a motorized filter wheel with a set of narrowband interference filters, a light guide, a collecting lens, a video zoom lens, and a monochromatic charged-coupled device (CCD) camera. An ellipsoidal cranial window is made in the skull bone of a rat under isoflurane anesthesia to capture in vivo multispectral diffuse reflectance images of the cortical surface. Regulation of the fraction of inspired oxygen using a gas mixture device enables the induction of different respiratory states such as normoxia, hyperoxia, and anoxia. A Monte Carlo simulation-based multiple regression analysis for the measured multispectral diffuse reflectance images at nine wavelengths (500, 520, 540, 560, 570, 580, 600, 730, and 760 nm) is then performed to visualize the two-dimensional maps of hemodynamics and the light scattering properties of the in vivo rat brain.

从体内大鼠脑获取的漫反射的代表性光谱图像示于图3中的图像，在500，520，540，560，570和580nm处清楚地看到血管的密集网络在大脑皮层。的血管，在600，730中的图像中观察到的周围组织，和760纳米之间的对比度恶化反射光中的时间更长，NIR波长下吸收血红蛋白。 图4示出了暴露的大鼠脑的用于氧化血红蛋白浓度，脱氧血红蛋白浓度，总血红蛋白浓度，局部脑氧饱和度，和散射功率代表估计的图像。如从漫反射图像预期在较短波长在图3中，在血液中的VES的总血红蛋白浓度SEL区域比在周围组织区域更高。在另一方面，在小动脉氧合血红蛋白浓度比在静脉由于动脉血液中的血红蛋白比静脉血更多含氧较高。因此，小动脉和小静脉的分布可以清楚地区域的氧饱和度的所估计的图像中区别开来。 在500nm 的R（500），氧合血红蛋白C HBO，脱氧血红蛋白C HBR，总血红蛋白C HBT的浓度，局部脑氧饱和度的浓度的浓度在吸入氧浓度变化为漫反射过程中的暴露的大鼠脑的代表估计的图像RSO 2，和散射功率b的示于图5。 RSO 2的值高氧条件下增加的和缺氧条件诱导后显着降低。 b的值在从缺氧发作直到呼吸停止的期间稍微增加，而它连续缺氧发作后期间降低从5分钟至30分钟。这些变化在b的值分别表示的形态变化，如肿胀和细胞和亚细胞结构的收缩，通过组织的生存力在脑的损失引起的。 <img alt="图1" src="/files/ftp_upload/55399/55399fig1.jpg" /> 图1:在大鼠大脑皮质的手术暴露步骤。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55399/55399fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55399/55399fig2.jpg" /> 图2:实验装置用于施用麻醉和改变吸入氧的分数的示意图。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55399/55399fig2large.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/55399/55399fig3.jpg" /> 图3:在多光谱代表漫反射图像500，520，540，560，570，580，600，730和760nm处，从体内大鼠脑得到。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55399/55399fig3large.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/55399/55399fig4.jpg" /> 图4:暴露大鼠脑的代表推定图像。 &lt;强&gt;（一）含氧血红蛋白C 高压氧浓度，（b）中的脱氧血红蛋白C HBR的浓度，（c）中总血红蛋白C HBT的浓度，（d）中局部脑氧饱和度RSO 2，和（e）的散射功率湾 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55399/55399fig4large.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/55399/55399fig5.jpg" /> 图5:暴露的大鼠脑的代表性结果在在吸入氧浓度变化。在500nm 的R（500），氧合血红蛋白C 高压氧浓度，浓度在吸入氧浓度变化为漫反射期间在体内大鼠皮层组织的图像的脱氧血红蛋白C HBR，总血红蛋白C HBT，局部脑氧饱和度RSO 2的浓度，和散射功率湾 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55399/55399fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table fo:keep-together.within-page="1"><tbody><tr><td>波长λ处</td><td> εHbO2的 （λ） </td><td> εHBR（λ） </td></tr><tr><td> 500 </td><td> 113.03712 </td><td> 112.6548 </td></tr><tr><td> 520 </td><td> 130.69296 </td><td> 170.58384 </td></tr><tr><td> 540 </td><td> 287.4744 </td><td> 251.5968 </td></tr><tr><td> 560 </td><td> 176.11128 </td><td> 290.4552 </td></tr><tr><td> 570 </td><td> 240.2784 </td><td> 243.3888 </td></tr><tr><td> 580 </td><td> 270.5616 </td><td> 199.908 </td></tr> &lt;TR&gt; <td> 600 </td><td> 17.28 </td><td> 79.25688 </td></tr><tr><td> 730 </td><td> 2.106 </td><td> 5.95188 </td></tr><tr><td> 760 </td><td> 3.1644 </td><td> 8.36201 </td></tr></tbody></table> 表1:ε 高压氧的价值观和εHbO2的用于多元回归分析。在每个波长λ氧合血红蛋白εHBO和脱氧血红蛋白εHBR的摩尔消光系数。 <table fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 一世 </td><td> βHBO，我 </td><td> βHBR，我 </td><td> βB，I </td></tr><tr><td> 0 </td><td> -8.3302 </td><td> -5.85271 </td><td> -0.76587 </td></tr><tr><td> 1 </td><td> 4405.877</td><td> -143.23 </td><td> 53.34134 </td></tr><tr><td> 2 </td><td> 2740.622 </td><td> 3798.067 </td><td> 124.4656 </td></tr><tr><td> 3 </td><td> -4.40454 </td><td> -2.81699 </td><td> -1.36919 </td></tr></tbody></table> 表2: C HbO ， C HbR和b的经验公式中使用的βHbO ， i ， βHbR ， i和β0 ， i （ i = 0,1,2,3）的值。注意，从这些经验公式得出的C HbO和C HbR的单位是体积浓度，其中血细胞比容读数为44％的全血的血红蛋白浓度被认为是血红蛋白的100体积浓度。血红蛋白浓缩的经验公式ations可以从通过光传输19的蒙特卡罗模拟中计算出的漫反射光谱来导出。对于经验公式的推导的详细过程已经在文献17,18进行了描述。

Watch this Scientific Journal Video about Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging at J

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

脑血流和体内大鼠脑组织的光散射特性的同时评估用常规多光谱漫反射成像系统证明。

脑血流动力学和的光散射特性的同步评估<em&gt;体内</em&gt;大鼠脑使用多光谱漫反射成像

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional ...

simultaneous-evaluation-cerebral-hemodynamics-light-scattering

Nanyang Technological University

Research

JoVE Journal

Neuroscience

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

7.6K 次观看.  Tokyo University of Agriculture & Technology. 脑血流和体内大鼠脑组织的光散射特性的同时评估用常规多光谱漫反射成像系统证明。 

视频: Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

How to Build a Laser Speckle Contrast Imaging (LSCI) System to Monitor Blood Flow

Laser Speckle Contrast Imaging (LSCI) is a simple yet powerful technique that is used for full-field imaging of blood flow. The technique analyzes fluctuations in a dynamic speckle pattern to detect the movement of particles similar to how laser Doppler analyzes frequency shifts to determine particle speed. Because it can be used to monitor the movement of red blood cells, LSCI has become a popular tool for measuring blood flow in tissues such as the retina, skin, and brain. It has become especially useful in neuroscience where blood flow changes during physiological events like functional activation, stroke, and spreading depolarization can be quantified. LSCI is also attractive because it provides excellent spatial and temporal resolution while using inexpensive instrumentation that can easily be combined with other imaging modalities. Here we show how to build a LSCI setup and demonstrate its ability to monitor blood flow changes in the brain during an animal experiment.

How to Build a Laser Speckle Contrast Imaging LSCI System to Monitor Blood Flow

This video demonstrates how to build a Laser Speckle Contrast Imaging (LSCI) system that can easily be used to monitor blood flow.

如何建立一个激光散斑对比度成像（LSCI）系统，以监测血流

Laser Speckle Contrast Imaging (LSCI) is a simple yet powerful technique that is used for full-field imaging of blood flow.  The technique analyzes ...

科研

神经科学

Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants

Perinatal brain injury remains a significant cause of infant mortality and morbidity, but there is not yet an effective bedside tool that can accurately screen for brain injury, monitor injury evolution, or assess response to therapy. The energy used by neurons is derived largely from tissue oxidative metabolism, and neural hyperactivity and cell death are reflected by corresponding changes in cerebral oxygen metabolism (CMRO2). Thus, measures of CMRO2 are reflective of neuronal viability and provide critical diagnostic information, making CMRO2 an ideal target for bedside measurement of brain health.
Brain-imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) yield measures of cerebral glucose and oxygen metabolism, but these techniques require the administration of radionucleotides, so they are used in only the most acute cases.
Continuous-wave near-infrared spectroscopy (CWNIRS) provides non-invasive and non-ionizing radiation measures of hemoglobin oxygen saturation (SO2) as a surrogate for cerebral oxygen consumption. However, SO2 is less than ideal as a surrogate for cerebral oxygen metabolism as it is influenced by both oxygen delivery and consumption. Furthermore, measurements of SO2 are not sensitive enough to detect brain injury hours after the insult 1,2, because oxygen consumption and delivery reach equilibrium after acute transients 3. We investigated the possibility of using more sophisticated NIRS optical methods to quantify cerebral oxygen metabolism at the bedside in healthy and brain-injured newborns. More specifically, we combined the frequency-domain NIRS (FDNIRS) measure of SO2 with the diffuse correlation spectroscopy (DCS) measure of blood flow index (CBFi) to yield an index of CMRO2 (CMRO2i) 4,5.
With the combined FDNIRS/DCS system we are able to quantify cerebral metabolism and hemodynamics. This represents an improvement over CWNIRS for detecting brain health, brain development, and response to therapy in neonates. Moreover, this method adheres to all neonatal intensive care unit (NICU) policies on infection control and institutional policies on laser safety. Future work will seek to integrate the two instruments to reduce acquisition time at the bedside and to implement real-time feedback on data quality to reduce the rate of data rejection.

We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain.

婴幼儿的脑代谢和血流动力学的非侵入式光学测量

Perinatal brain injury remains a significant cause of infant mortality  and morbidity, but there is not yet an effective bedside tool that can  accurately ...

医学

Simultaneous PET/MRI Imaging During Mouse Cerebral Hypoxia-ischemia

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics disturbance in affected cells. Diffusion weighted magnetic resonance imaging (MRI) identifies regions that are damaged, potentially irreversibly, by hypoxia-ischemia. Alterations in glucose utilization in the affected tissue may be detectable by positron emission tomography (PET) imaging of 2-deoxy-2-(18F)fluoro-&#7429;-glucose ([18F]FDG) uptake. Due to the rapid and variable nature of injury in this animal model, acquisition of both modes of data must be performed simultaneously in order to meaningfully correlate PET and MRI data. In addition, inter-animal variability in the hypoxic-ischemic injury due to vascular differences limits the ability to analyze multi-modal data and observe changes to a group-wise approach if data is not acquired simultaneously in individual subjects. The method presented here allows one to acquire both diffusion-weighted MRI and [18F]FDG uptake data in the same animal before, during, and after the hypoxic challenge in order to interrogate immediate physiological changes.

The method presented here uses simultaneous positron emission tomography and magnetic resonance imaging. In the cerebral hypoxia-ischemia model, dynamic changes in diffusion and glucose metabolism occur during and after injury. The evolving and irreproducible damage in this model necessitates simultaneous acquisition if meaningful multi-modal imaging data are to be acquired.

同时PET / MRI成像在小鼠脑缺氧缺血

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics ...

Cerebral Blood Flow-Based Resting State Functional Connectivity of the Human Brain using Optical Diffuse Correlation Spectroscopy

To obtain a comprehensive understanding of the human brain, utilization of cerebral blood flow (CBF) as a source of contrast is desired because it is a key hemodynamic parameter related to cerebral oxygen supply. Resting state low frequency fluctuations based on oxygenation contrast have been shown to provide correlations between functionally connected regions. The presented protocol uses optical diffuse correlation spectroscopy (DCS) to assess blood flow-based resting state functional connectivity (RSFC) in the human brain. Results of CBF-based RSFC in human frontal cortex indicate that intra-regional RSFC is significantly higher in the left and right cortices compared to inter-regional RSFC in both cortices. This protocol should be of interest to researchers who employ multi-modal imaging techniques to study human brain function, especially in the pediatric population.

This protocol demonstrates how to measure resting state functional connectivity in the human prefrontal cortex using a custom-made diffuse correlation spectroscopy instrument. The report also discuss practical aspects of the experiment as well as detailed steps for analyzing the data.

使用光学漫反射相关光谱，基于脑血流的休息状态功能连接

To obtain a comprehensive understanding of the human brain, utilization of cerebral blood flow (CBF) as a source of contrast is desired because it is a ...

生物工程

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 ...

Advanced Diffusion Imaging in The Hippocampus of Rats with Mild Traumatic Brain Injury

Mild traumatic brain injury (mTBI) is the most common type of acquired brain injury. Since patients with traumatic brain injury show a tremendous variability and heterogeneity (age, gender, type of trauma, other possible pathologies, etc.), animal models play a key role in unraveling factors that are limitations in clinical research. They provide a standardized and controlled setting to investigate the biological mechanisms of injury and repair following TBI. However, not all animal models mimic the diffuse and subtle nature of mTBI effectively. For example, the commonly used controlled cortical impact (CCI) and lateral fluid percussion injury (LFPI) models make use of a craniotomy to expose the brain and induce widespread focal trauma, which are not commonly seen in mTBI. Therefore, these experimental models are not valid to mimic mTBI. Thus, an appropriate model should be used to investigate mTBI. The Marmarou weight drop model for rats induces similar microstructural alterations and cognitive impairments as seen in patients who sustain mild trauma; therefore, this model was selected for this protocol. Conventional computed tomography and magnetic resonance imaging (MRI) scans commonly show no damage following a mild injury, because mTBI induces often only subtle and diffuse injuries. With diffusion weighted MRI, it is possible to investigate microstructural properties of brain tissue, which can provide more insight into the microscopic alterations following mild trauma. Therefore, the goal of this study is to obtain quantitative information of a selected region-of-interest (i.e., hippocampus) to follow up disease progression after obtaining a mild and diffuse brain injury.

The overall goal of this procedure is to obtain quantitative microstructural information of the hippocampus in a rat with mild traumatic brain injury. This is done using an advanced diffusion-weighted magnetic resonance imaging protocol and region-of-interest based analysis of parametric diffusion maps.

轻度创伤性脑损伤大鼠海马学的高级扩散成像

Mild traumatic brain injury (mTBI) is the most common type of acquired brain injury. Since patients with traumatic brain injury show a tremendous ...

Cerebrovascular Reactivity Measurement with Functional Near Infrared Spectroscopy

Cerebrovascular reactivity (CVR) is the capacity of blood vessels in the brain to alter cerebral blood flow (either with dilation or constriction) in response to chemical or physical stimuli. The amount of reactivity in the cerebral microvasculature depends on the integrity of the capacitance vasculature and is the primary function of endothelial cells. CVR is, therefore, an indicator of the microvasculature&#8217;s physiology and overall health. Imaging methods that can measure CVR are available but can be costly, and require magnetic resonance imaging centers and technical expertise. In this study, we used fNIRS technology to monitor changes of oxyhemoglobin (HbO) and deoxyhemoglobin (HbR) in the cerebral microvasculature to assess the CVR of 15 healthy controls (HC) in response to a vasoactive stimulus (inhaled 5% carbon dioxide or CO2). Our results suggest that this is a promising imaging technology that offers a non-invasive, accurate, portable, and cost-effective method of mapping cortical CVR and associated microvasculature function, resulting from a traumatic brain injury or other conditions associated with cerebral microvasculopathy.

Presented here is a protocol for imaging and measurement of cerebrovascular reactivity in humans with functional Near Infrared Spectroscopy (fNIRS). fNIRS is a novel imaging modality that captures the concentration changes of hemoglobin species in the brain&#8217;s outermost cortex under specific stimuli.

Cerebrovascular reactivity (CVR) is the capacity of blood vessels in the brain to alter cerebral blood flow (either with dilation or constriction) in ...

Systems Analysis of the Neuroinflammatory and Hemodynamic Response to Traumatic Brain Injury

Mild traumatic brain injuries (mTBIs) are a significant public health problem. Repeated exposure to mTBI can lead to cumulative, long-lasting functional deficits. Numerous studies by our group and others have shown that mTBI stimulates cytokine expression and activates microglia, decreases cerebral blood flow and metabolism, and impairs cerebrovascular reactivity. Moreover, several works have reported an association between derangements in these neuroinflammatory and hemodynamic markers and cognitive impairments. Herein we detail methods to characterize the neuroinflammatory and hemodynamic tissue response to mTBI in mice. Specifically, we describe how to perform a weight-drop model of mTBI, how to longitudinally measure cerebral blood flow using a non-invasive optical technique called diffuse correlation spectroscopy, and how to perform a Luminex multiplexed immunoassay on brain tissue samples to quantify cytokines and immunomodulatory phospho-proteins (e.g., within the MAPK and NF&#954;B pathways) that respond to and regulate activity of microglia and other neural immune cells. Finally, we detail how to integrate these data using a multivariate systems analysis approach to understand the relationships between all of these variables. Understanding the relationships between these physiologic and molecular variables will ultimately enable us to identify mechanisms responsible for mTBI.

This protocol presents methods to characterize the neuroinflammatory and hemodynamic response to mild traumatic brain injury and to integrate these data as part of a multivariate systems analysis using partial least squares regression.

创伤性脑损伤的神经炎症和血流动力学反应的系统分析

Mild traumatic brain injuries (mTBIs) are a significant public health problem. Repeated exposure to mTBI can lead to cumulative, long-lasting functional ...

Quantification of Cerebral Perfusion using Laser Speckle Imaging and Infarct Volume using MRI in a Pre-clinical Model of Posterior Circulation Stroke

Background: Basilar artery occlusion (BAO) is a subset of posterior circulation stroke that carries a mortality as high as 90%.&#160; The current clinical standard to diagnose ischemic stroke include computerized tomography (CT), CT angiography and perfusion and magnetic resonance imaging (MRI). Large animal pre-clinical models to accurately reflect the clinical disease as well as methods to assess stroke burden and evaluate treatments are lacking.
Methods: We describe a canine model of large vessel occlusion (LVO) stroke in the posterior circulation, and developed a laser speckle imaging (LSI) protocol to monitor perfusion changes in real time.&#160; We then utilized high b-value DWI (b=1800s/mm2) MRI to increase detection sensitivity. We also evaluated the ability of magnetic resonance angiography (MRA) to assess arterial occlusion and correlate with DSA. Finally, we verified infarct size from apparent diffusion coefficient (ADC) mapping with histology.&#160;
Results:&#160; Administration of thromboembolism occluded the basilar artery as tracked by DSA (n=7).&#160; &#160;LSI correlated with DSA, demonstrating a reduction in perfusion after stroke onset that persisted throughout the experiment, allowing us to monitor perfusion in real time.&#160; DWI with an optimized b-value for dogs illustrated the stroke volume and allowed us to derive ADC and magnetic resonance angiography (MRA) images. The MRA performed at the end of the experiment correlated with DSA performed after occlusion. Finally, stroke burden on MRI correlated with histology.
Conclusions: Our studies demonstrate real time perfusion imaging using LSI of a canine thromboembolic LVO model of posterior circulation stroke, which utilizes multimodal imaging important in the diagnosis and treatment of ischemic stroke.

A canine model of LVO stroke was utilized to develop laser speckle imaging to monitor cerebral perfusion in real-time.&#160; Diffusion-weighted MRI was optimized to image infarct volume utilizing a high b-value, enabling ADC and MRA, correlated with DSA at the time of stroke.&#160; Finally, ADC reconstructions correlated with histological findings.

Background: Basilar artery occlusion (BAO) is a subset of posterior circulation stroke that carries a mortality as high as 90%.&#160; The current clinical ...

Real-Time Monitoring of Neurocritical Patients with Diffuse Optical Spectroscopies

Neurophysiological monitoring is an important goal in the treatment of neurocritical patients, as it may prevent secondary damage and directly impact morbidity and mortality rates. However, there is currently a lack of suitable non-invasive, real-time technologies for continuous monitoring of cerebral physiology at the bedside. Diffuse optical techniques have been proposed as a potential tool for bedside measurements of cerebral blood flow and cerebral oxygenation in case of neurocritical patients. Diffuse optical spectroscopies have been previously explored to monitor patients in several clinical scenarios ranging from neonatal monitoring to cerebrovascular interventions in adults. However, the feasibility of the technique to aid clinicians by providing real-time information at the bedside remains largely unaddressed. Here, we report the translation of a diffuse optical system for continuous real-time monitoring of cerebral blood flow, cerebral oxygenation, and cerebral oxygen metabolism during intensive care. The real-time feature of the instrument could enable treatment strategies based on patient-specific cerebral physiology rather than relying on surrogate metrics, such as arterial blood pressure. By providing real-time information on the cerebral circulation at different time scales with relatively cheap and portable instrumentation, this approach may be especially useful in low-budget hospitals, in remote areas and for&#160;monitoring in open fields (e.g., defense and sports).

Presented here is a protocol for non-invasively monitoring cerebral hemodynamics of neurocritical patients in real-time and at the bedside using diffuse optics. Specifically, the proposed protocol uses a hybrid diffuse optical systems to detect and display real-time information on cerebral oxygenation, cerebral blood flow and cerebral metabolism.

使用漫反射光谱实时监测神经危重症患者

Neurophysiological monitoring is an important goal in the treatment of neurocritical patients, as it may prevent secondary damage and directly impact ...

脑血流动力学和的光散射特性的同步评估

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