我们感谢头板设计博士，Nedergaard Maiken（罗切斯特大学医学中心）。这项工作已经由NIH奖项支持SD（R01DA026325和P30AI078498和基金会的资助KK（德纳基础脑和Immunoimaging计划，美国心脏协会0635595T和ALS协会[＃1112）]）。

1。准备成像动物注:我们用成人的C57BL小鼠。可以使用不同的转基因株系，根据所研究的问题。微血管手术和脉搏血氧饱和度，促进小鼠白色毛皮（例如的FVB株）。 <ol><li>准备手术部位在替补席上。所有的手术器械需要进行高压灭菌，或用70％乙醇消毒。 </li><li>插入直肠探头，整个过程持续测量动物的体温</li><li>使用最初5％的异氟醚麻醉鼠标。在15-30秒，当动物不动，将加热垫（37℃），适用于连续的空气含有1.3-1.5％的异氟醚）交付了口罩。确保该动物没有响应疼痛刺激（如尾捏）。 </li><li>使用人工泪液凝胶覆盖老鼠的眼睛，以防止暴露在干燥的空气。 </li><li>使用电动剃须刀，除毛fROM的头从两侧大腿。适用于头发卸妆（例如奈尔）2分钟的大腿，然后仔细擦拭以去除剩余的头发。这大腿，将用于血氧饱和度。 </li><li>使用10％碘伏溶液和70％乙醇消毒头皮。 </li></ol> 2。准备开放性颅骨颅窗<ol><li>从5毫米，尾端的头骨，使切口在头皮用剪刀，前进一厘米。双方将皮肤暴露颅骨。 </li><li>头骨的顶部，以消除膜适用10％的铁的头骨顶部的氯化物溶液。擦去多余的解决方案，并与45°角＃5镊子刮膜外。重要的是仔细准备网站，以确保头盘可以牢固地连接到头骨（步骤＃4至6）。 </li><li>使用解剖显微镜，找出感兴趣的领域。请注意，应避免颅缝，因为头骨ç打破难以预测沿着这些路线。 </li><li>应用窗口（图1）头板的边缘周围的快速胶水薄薄的一层（如Locktite 454）。在这样一个感兴趣的领域，在窗口公开的方式头板的位置。适用于光压头板。适用于少量的牙科水泥迅速聚合胶水。保持10秒，而胶聚合。 </li><li>快速胶水的少量应用到窗口封头板和头骨边缘。拧头板下的清扫范围内的动物持有人。 </li><li>使用微扭矩II在6000转和IRF的005钻头钻，从头骨和头板中删除所有多余的胶水。任何剩余的胶头板应去掉，因为它会以其他方式干扰玻璃盖的附件。 </li><li>演习将在1000 rpm，并开始头骨，头板"窗口内的一个圆圈，直径3〜5米钻米。使用轻抺，如果你是一个很软的铅笔画。停钻，每20-30秒，去除骨的灰尘，使用压缩空气。 注:在头板开幕的形状，使钻头（左手和右手的外科医生），这使得它更容易地创建一个圆形的颅骨窗口额外的访问。此外，两个奇形孔极谱或电额外测量颅窗口下的Clark电极或玻璃电极插入脑组织提供必要的空间。 </li><li>钻井过程中，洞穴是在中心周围形成完整的头骨。小心捅通过减薄头骨，但厚度均匀，使这个洞穴。要额外的小心周围的船只，他们不应该受到影响，如果可能的话，没有碰到大血。 </li><li>使用镊子＃3"腿"，解除骨（"轻弹关闭"）在中心的窗口。套用下降artificIAL脑脊液（ACSF）窗口。 </li><li>轻轻用软纸巾的角落（如Kimwipe）擦拭学联。一般硬脑膜被损坏，在一个或多个窗口的边缘周围的地方。在这些网站开始，轻轻抬起，然后取出脑表面硬脑膜，用45°角镊子＃5。当选择网站开始清除硬脑膜，避免大血管的毗邻地区。如果发生出血，应用学联下降，等待2-3分钟轻微出血停止。 </li><li>准备0.07％低熔点在学联溶解的琼脂糖）使体温融化的琼脂糖。从大脑的表面擦去学联过剩。琼脂糖倒在窗口，并盖上载玻片。轻轻按下玻璃，使玻璃和头板之间的接触，并等待10-20秒，直到琼脂糖是坚实的。 </li><li>使用镊子和软纸巾从玻璃盖，取出多余的琼脂糖。 </li><li>把一个小金额快速周围的玻璃胶头板玻璃胶。适用于少量的牙科水泥固化胶（图1）。 </li></ol> 3。静脉注射和血氧监测注:我们经常使用的得克萨斯红静脉应用，而不是尾静脉，股静脉。这是因为，股静脉注射提供安全和可再生丸注射染料。 <ol><li>转动部分过度暴露的左大腿内侧的动物。使用磁带，以确保动物在这个位置上。 </li><li>应用防腐剂擦洗，然后100％乙醇肌肤。 </li><li>做一个切口，沿大腿内侧从膝盖到耻骨。 ＃5使用镊子钝性分离的独立的软组织。 </li><li>填写1ml注射器130μL得克萨斯红（2.5毫克/毫升）。将新针（表30），并仔细弯曲30°，保持斜角。填补针无线网络日得克萨斯红的解决方案。 </li><li>解剖显微镜下，股静脉插入针头，并注入100μL德州红。取出针，并用纱布轻轻按下静脉止血。 </li><li>使用4-0缝合关闭皮肤。 </li><li>对于连续监测血SPO 2（以确保足够的血氧），血氧饱和度探头放在右大腿上（头发从以前删除）。 </li></ol> 4。双光子成像注:我们使用一个奥林巴斯Fluoview1000多光子成像系统（直立）1光谱物理茅台酒的惠普DeepSee飞秒钛:萨激光作为激发源。成像，我们用×10无0.45（蔡司C - 复消色差透镜），或×25无1.05（奥林巴斯XLPlan N）的水浸泡目标。我们需要在12位深度的图像分辨率为512×512像素一个像素停留时间为2μs。 NADH和得克萨斯州红-葡聚糖同时在740 nm的兴奋，和氟的escence使用分色镜/近红外阻塞过滤器组合（FF665-DI01从激发光分离; FF01-680/SP，Semrock，罗切斯特，纽约，美国）分为两个渠道使用分色镜（505DCXRU，色度罗金厄姆，VT，美国），带通滤波（NADH的Semrock FF460/80Texas-Red-dextran-Semrock FF607/36的）。后的客观测量激光功率为10-20毫瓦成像在Ⅰ层和第二层成像20-50兆瓦。 <ol><li>显微镜阶段转移的手术准备的鼠标。采取的颅窗网站的4倍放大倍率在使用亮场照明用高倍率的双光子造影作为登记的参考图的初始图片。 </li><li>放大面积×10倍率的利益。选择感兴趣的领域，在皮质层I或II（可达150微米以下的软膜表面）。如果需要更高的放大倍率，使用×25目标</li><li>开始记录的时间序列（Figu重2）。我们推荐使用平均3-5帧，以提高图像质量。 </li><li>加入50％的氮气，动物呼吸的空气，引起低氧血症。这将带来10％的氧气水平。通过监测血氧饱和度数据的验证低氧血症。 </li><li>继续记录的时间序列，并通过低氧血症（例如，30秒）后恢复正常氧气水平。 </li><li>收集区缺氧和周围血管的含氧组织气瓶检测，测量和量化计算的氧分布的数据。 </li></ol> 5。数据处理<ol><li>确定克罗组织缸半径为R，这是可以做到手动测量中心血管和高NADH的荧光检测的边界之间的距离（图3）。 </li><li>另外，使用的独立调查组织汽缸半径R计算半自动化的决心和中央BLOOD船只ŕ已先前所述补充形式，并在本议定书的讨论部分总结。 </li><li>确定使用开放获取的图像分析软件，如ImageJ的缺氧区。要做到这一点，使用NADH的信号，定义描绘NADH的高烈度区的阈值，然后升高NADH组织荧光测量面积。 </li></ol> 6。代表结果 图2显示了NADH /血管造影图像的视频，在短暂的低氧血症（此图的PDF版本，已选定静像）。吸入氧浓度从21％降低到10％，3分钟内。这个实验引起的低氧血症程度是足以引起严重缺氧，在大脑皮层。缺氧导致NADH的荧光增加，最初在动脉供血最远的地区。注意尖锐的NADH的组织边界，这代表观察组织的界限，从​​皮质微循环的氧气扩散。在图3中的图像显示脑血管周围的氧气扩散边界的几何形状。这是可能的推断克罗缸直径从这些界限，先前描述1。 <img alt="图1" src="/files/ftp_upload/3466/3466fig1.jpg" /> 图1（A）与成像准备颅窗口鼠标。 （二）头板的尺寸。 <img alt="图2" src="/files/ftp_upload/3466/3466fig2.jpg" /> 图2。内源性辅酶绿色荧光可视化双光子成像，通过颅窗脑膜表面，大约50微米以下。血管是与得克萨斯州红葡聚糖的可视化。吸入空气中的氧气减少到10％，为3分钟，然后恢复为21％。比例尺为50μm。 &lt;IMG ALT ="图3"的src ="/ files/ftp_upload/3466/3466fig3.jpg"/&gt; 图3。NADH的荧光边界几何。黄河大纲显示功能缺氧的边界;蓝色圆圈显示预计含氧（克罗）气瓶。蓝线显示汽缸半径;数字显示半径在微米。

<ol>
	<li>Kasischke, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+NADH+imaging+exposes+boundaries+of+oxygen+diffusion+in+cortical+vascular+supply+regions.">Two-photon NADH imaging exposes boundaries of oxygen diffusion in cortical vascular supply regions.</a> J. Cereb. Blood. Flow. Metab. 31, 68-81 (2011).</li><li>Ndubuizu, O., LaManna, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+tissue+oxygen+concentration+measurements.">Brain tissue oxygen concentration measurements.</a> Antioxid. Redox. Signal. 9, 1207-1219 (2007).</li><li>Sharan, M., Vovenko, E. P., Vadapalli, A., Popel, A. S., Pittman, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+and+theoretical+studies+of+oxygen+gradients+in+rat+pial+microvessels.">Experimental and theoretical studies of oxygen gradients in rat pial microvessels.</a> J. Cereb. Blood. Flow. Metab. 28, 1597-1604 (2008).</li><li>Sakadzic, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+high-resolution+measurement+of+partial+pressure+of+oxygen+in+cerebral+vasculature+and+tissue.">Two-photon high-resolution measurement of partial pressure of oxygen in cerebral vasculature and tissue.</a> Nat. Methods. 7, 755-759 (2010).</li><li>Vishwasrao, H. D., Heikal, A. A., Kasischke, K. A., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+dependence+of+intracellular+NADH+on+metabolic+state+revealed+by+associated+fluorescence+anisotropy.">Conformational dependence of intracellular NADH on metabolic state revealed by associated fluorescence anisotropy.</a> J. Biol. Chem. 280, 25119-25126 (2005).</li><li>Kleinfeld, D., Mitra, P. P., Helmchen, F., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluctuations+and+stimulus-induced+changes+in+blood+flow+observed+in+individual+capillaries+in+layers+2+through+4+of+rat+neocortex.">Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex.</a> Proc. Natl. Acad. Sci. U. S. A. 95, 15741-15746 (1998).</li><li>Brown, W. R., Thore, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review:+cerebral+microvascular+pathology+in+ageing+and+neurodegeneration.">Review: cerebral microvascular pathology in ageing and neurodegeneration.</a> Neuropathol. Appl. Neurobiol. 37, 56-74 (2011).</li><li>de la Torre, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+brain+microcirculation+may+trigger+Alzheimer's+disease.">Impaired brain microcirculation may trigger Alzheimer's disease.</a> Neurosci. Biobehav. Rev. 18, 397-401 (1994).</li><li>Brown, C. E., Boyd, J. D., Murphy, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+in+vivo+imaging+reveals+balanced+and+branch-specific+remodeling+of+mature+cortical+pyramidal+dendritic+arbors+after+stroke.">Longitudinal in vivo imaging reveals balanced and branch-specific remodeling of mature cortical pyramidal dendritic arbors after stroke.</a> J. Cereb. Blood. Flow. Metab. 30, 783-791 (2010).</li><li>Wilson, D. F., Erecinska, M., Drown, C., Silver, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+oxygen+dependence+of+cellular+energy+metabolism.">The oxygen dependence of cellular energy metabolism.</a> Arch. Biochem. Biophys. 195, 485-493 (1979).</li></ol>

作者没有透露。

有关氧气扩散的高空间分辨率信息，重要的是了解如何在大脑的血流量调节脑细胞提供氧气，以满足代谢的需求。传统克拉克式玻璃电极的极谱氧测量高度侵袭性和空间分辨率低2-3和范围显着（第二）的响应时间。到目前为止，唯一的非侵入性方法测量脑组织中的PO 2燐光猝灭，激发探头的衰减率是成正比的氧气浓度4。这种方法提供了精确的氧气浓度，但需要一个专用的染料和技术上先进的磷光寿命成像系统。在这里，我们展示了一个简单的，简单的方法，可以在一个标准的双光子成像系统进行有两个flurescence通道。我们的方法需要一种内在的组织信号5的优势第二只需要对比的皮质微循环的可视化。由于非线性，基本上二进制增加NADH的荧光，在功能限制氧气浓度1，增加内在NADH的荧光观察，仅在显着，新陈代谢限制缺氧地区。一个重要的含义是，从皮质微血管的氧气扩散的组织界限，直接由圆柱形的内源性NADH的荧光强度的变化观察。我们是指这些结构作为克罗缸，因为定义周围血管组织的含氧体积的圆柱状结构的概念是由八月克罗最近已经利用双光子成像1 NADH的实验观察。克罗气瓶的图像可以被收集在3D一帧图像的Z-Stack。他们穿透动脉附近尤其突出，他们符合机智Ĥ毛细血管耗尽periarteriolar组织气瓶1,4。 提供克罗组织缸半径为R的客观测定（见5.2节），我们之间的气缸中心和外部边界使用MATLAB函数"improfile"定义段内测量其径向像素强度值。段外边界应选择延长与超越有形的边界的安全保证金。为了提高信号的噪音水平，我们averageed需要支付1°气缸可见段所有径向线。段内产生的平均径向强度分布呈现急剧增加，这相当于有形的组织边界 r。我们适合sigmoidal函数（如波兹曼函数）的平均径向强度分布，并用其拐点（又称X 0）为R 的定义。对应的两个-P华庭时尚商务microangiography（得克萨斯红）揭示了一个孤独的气缸中心的中央血管的横截面。中央血管的直径，可直接应用于确定ŕ。 并发高分辨率成像的皮质microangiography的双光子NADH的成像提供相同的空间分辨率。这种方法的定量化应用的一个重要特点是，磷的NADH荧光增加50已测得的3.4±0.6毫米汞柱和PO 2 microregional组织功能的NADH荧光强度可以用数学描述sigmoidal函数。 。我们表明，这种技术允许一个确定大脑缺氧（空气中的氧气含量降低10％）是最脆弱的地区。我们还表明，氧气扩散如下一个简单的几何血管周围模式。 一击ICAL此方法的步骤是颅窗口准备的质量。手术应产生最小的损伤，为了不惊动血流暴露面积。一个值得关注的是，在手术妥协的准备，窗下的皮层可能是缺氧开始，排除任何有意义的实验。一个精心准备的颅窗，应该有完整的主要和次要的血管，在所有船种生动的血流量和沿边缘没有明显的出血。常氧条件下（氧分压80-100毫米汞柱，SP氧气97-99％），脑实质表现不突出，明亮的组织补丁NADH的荧光升高均匀，均匀NADH的荧光。 我们的方法的一个基本的物理约束是有限的穿透深度。在脑的蓝绿色NADH的荧光是由血红蛋白在这些波长的吸收和散射组织迅速衰减。即使是用高数值孔径（如1.05）的水浸泡目标双光子NADH的成像目前仅限于皮质层I和II。这种限制是科学，因为能源或邻近白质的代谢有关，可能会不同于灰质。然而，如层IV-VI或皮层下结构，如白质束或纹状体深皮层结构的调查，将需要使用专门的微透镜在小鼠皮层体内6。 NADH基于氧气的扩散边界的测量与分析功能充血，毛细血管通量率检测7，如其他测量相结合时，特别有用。例如，这种技术可适应缺氧，在中风和阿尔茨海默氏病（AD）模型可视化。氧气扩散的简单的几何形状允许一个预测的情况下，毛细血管密度是氧气在微血管床梯度8折痕（例如，公元9），并检查是否与脑组织毛细血管密度降低地区微振由于缺氧损伤的风险增加。图像的能力microregionally还允许一个研究组织微振的几何形状和大小，确定缺氧发生在该组织的体积，以及之间的关系，组织缺氧和随后的神经元死亡或毛细管重塑10。 最后，由于增加内源性NADH的荧光急性线粒体功能障碍的直接后果，这种方法创造了机会，利用NADH的成像作为一个特定的神经能量代谢和线粒体功能障碍的代理记者。 总之，内源性NADH的荧光双光子成像是一种简单，要求不高的工具，可以用来了解在正常的大脑中的氧气输送和消费在病理状态。

<table border="1"><tbody><tr valign="top"><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>暖气片</td><td>除了博迪热</td><td></td><td></td></tr><tr><td>眼药膏（人工泪液） </td><td>辉瑞公司</td><td></td><td></td></tr><tr><td>碘伏10％的解决方案</td><td>优碘</td><td></td><td></td></tr><tr><td>三氯化铁10％的解决方案</td><td></td><td></td><td></td></tr><tr><td>水泥</td><td> Stoelting公司</td><td> 51456 </td><td></td></tr><tr><td>瞬干胶454 </td><td>乐泰</td><td></td><td></td></tr><tr><td>学联</td><td>哈佛仪器</td><td> 597316 </td><td></td></tr><tr><td>微扭矩二机头套件</td><td>皮尔逊</td><td> R14的-0002 </td><td></td></tr><tr><td> IRF的007钻头</td><td>罚款科学工具</td><td> 19008-07 </td><td></td></tr><tr><td>镊子＃5 </td><td>罚款科学工具</td><td> 11295 </td><td></td></tr><tr><td> ＃5/45钳</td><td>罚款科学工具</td><td> 11251-35 </td><td></td></tr><tr><td> ＃0盖玻片</td><td>电子显微镜科学</td><td> 63750-01 </td><td></td></tr><tr><td>光电倍增管</td><td>滨松</td><td> HC125-02 </td><td></td></tr><tr><td lang="it-IT" xml:lang="it-IT">掺钛蓝宝石激光麦大</td><td>光谱物理</td><td> ＆NBSP; </td><td></td></tr></tbody></table>

detection of microregional hypoxia in mouse cerebral cortex by two-photon imaging of endogenous nadh fluorescence

大脑的正常代谢需求的高层次的能力，取决于血流量和组织氧扩散通过连续供氧。在这里，我们提出了一个可视化的实验和方法的协议，直接可视化microregional组织缺氧，并推断在小鼠大脑皮层血管周围的氧气梯度。它是基于非烟酰胺腺嘌呤二核苷酸（NADH）的内源性荧光强度和氧在组织中，在那里观 ​​察组织NADH的荧光突然增加组织的氧含量低于10毫米汞柱1分压之间的线性关系。在740 nm处NADH的内在组织与得克萨斯州红葡聚糖的荧光和对比血浆的同时激发允许我们使用双光子激发。这种方法比现有方法的优点包括以下内容:需要一种内在的组织信号的优势，可以在体内 IM使用标准的双光子设备老化，它允许在整场〜50微米的深度分辨率的连续监测。我们表明，脑组织，脑血管最远对应脆弱集水区，这是第一次成为功能缺氧后血管供氧下降。这种方法允许一个图像microregional皮质氧合，因此，用于检查神经血管疾病和中风的氧气供应不足或限制组织的作用。

Watch this Scientific Journal Video about Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence at JoVE.com

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

在这里，我们描述了一个方法，直接可视化在小鼠皮层microregional组织缺氧<em&gt;在体内</em&gt;。它是基于并发双光子成像烟酰胺腺嘌呤二核苷酸（NADH）和皮质微循环。这种方法是有用的组织供氧的高分辨率分析。

检测内源NADH的荧光双光子成像的小鼠大脑皮层Microregional缺氧

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

detection-microregional-hypoxia-mouse-cerebral-cortex-two-photon

Nanyang Technological University

Research

JoVE Journal

Neuroscience

16.0K 次观看.  University of Rochester Medical Center. 在这里，我们描述了一个方法，直接可视化在小鼠皮层microregional组织缺氧在体内。它是基于并发双光子成像烟酰胺腺嘌呤二核苷酸（NADH）和皮质微循环。这种方法是有用的组织供氧的高分辨率分析。

视频: Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

在体内脊髓的鼠标线使用双光子显微镜成像

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

科研

神经科学

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

在体内双光子成像的经验依赖的分子在大脑皮质神经元的变化

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal models, including monkeys, ferrets, bats, rodents, and cats. In order to draw suitable parallels between human and animal models of auditory function, it is important to establish a bridge between human functional imaging studies and animal electrophysiological studies. Functional magnetic resonance imaging (fMRI) is an established, minimally invasive method of measuring broad patterns of hemodynamic activity across different regions of the cerebral cortex. This technique is widely used to probe sensory function in the human brain, is a useful tool in linking studies of auditory processing in both humans and animals and has been successfully used to investigate auditory function in monkeys and rodents. The following protocol describes an experimental procedure for investigating auditory function in anesthetized adult cats by measuring stimulus-evoked hemodynamic changes in auditory cortex using fMRI. This method facilitates comparison of the hemodynamic responses across different models of auditory function thus leading to a better understanding of species-independent features of the mammalian auditory cortex.

Functional studies of the auditory system in mammals have traditionally been conducted using spatially-focused techniques such as electrophysiological recordings. The following protocol describes a method of visualizing large-scale patterns of evoked hemodynamic activity in the cat auditory cortex using functional magnetic resonance imaging.

听觉皮层在成年猫用高场磁共振成像功能成像

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

双光子<em&gt;在体内</em&gt;树突棘中的鼠标皮质用稀疏的头颅成像准备

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

细胞动力学在小鼠脊髓双光子成像

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

同时双光子<em&gt;在体内</em&gt;突触输入的影像和突触后目标中的鼠标Retrosplenial的Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced Neuroinflammation

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic neuroinflammatory state within the central nervous system (CNS), thought to be driven principally by virally-mediated activation of microglia and brain resident macrophages. HIV-1 infection is also accompanied by changes in cerebrovascular blood flow (CBF), raising the possibility that HIV-associated chronic neuroinflammation may lead to changes in CBF and/or in cerebral vascular architecture. To address this question, we have used a mouse model for HIV-induced neuroinflammation, and we have tested whether long-term exposure to this inflammatory environment may damage brain vasculature and result in rarefaction of capillary networks. In this paper we describe a method to quantify changes in cortical capillary density in a mouse model of neuroinflammatory disease (HIV-1 Tat transgenic mice). This generalizable approach employs in vivo two-photon imaging of cortical capillaries through a thin-skull cortical window, as well as ex vivo two-photon imaging of cortical capillaries in mouse brain sections. These procedures produce images and z-stack files of capillary networks, respectively, which can be then subjected to quantitative analysis in order to assess changes in cerebral vascular architecture.

This paper describes a method by which the vascular architecture in the brain can be quantified using in vivo and ex vivo two-photon microscopy.

利用双光子显微镜的艾滋病毒引起的神经炎症的小鼠模型脑血管结构的量化

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

脑切片神经元树突的双光子钙成像

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

体内新生小鼠皮层神经元的双光子成像

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...