我们感谢刘慧卿凯利头板设计。 我们感谢美国国立卫生研究院奖励支持HAG：RO1 MH56838 MH64570，PO1，RO1 MH078989，R21 MH03851，没有这项工作不可能有贺Waasdorp小儿神经基金的慷慨支持。 AKM的是由美国国立卫生研究院（EY019277），白厅基金，Alfred P. Sloan基金会和在生物医学科学研究所从宝来惠康基金事业奖。 MET是由一个全宗RECHERCHE EN SANTE魁北克（FRSQ）博士后奖。东风是一个在医学科学家培训项目由美国国立卫生研究院的T32 GM07356和T32 AI049815资助，学员。

1。准备成像动物<ol><li>在我们的实验中，我们使用成人CX 3 CR 1 GFP / +小鼠单核细胞谱系，包括小胶质细胞表达绿色荧光蛋白在4株小鼠不同，可用于满足特定项目的需要。 </li><li> 0.05毫克/公斤芬太尼，5.0毫克/公斤咪达唑仑和0.5毫克/公斤Medetomidine组成了三个药物鸡尾酒腹腔注射麻醉鼠标5 TSCW手术准备后开始的动物不再以疼痛刺激的反应，例如作为一个尾巴掐。整个手术和成像，我们一直在加热垫的动物，以防止麻醉后体温过低。如果有必要，原剂量的麻醉剂鸡尾酒助推器剂量的三分之一，可以考虑恢复原有的麻醉平面。 </li><li>盖与保护眼药膏的动物的眼睛保持在麻醉过程中的眼睛湿润了，这会抑制动物的眨眼反射。取下的头发从头皮的动物，用一把剃刀，剪刀，剪刀，或化学的方法。使用10％的碘伏溶液和70％的乙醇消毒头皮。所有手术器械需要进行高压灭菌，用玻璃珠的灭菌，或用70％乙醇消毒灭菌。 </li><li>制作的头皮，用小剪刀正中切口，开始4-5毫米，尾鳍的头骨和推进着眼睛的前面。重要的是，此切口足够长，皮肤不会干涉的双光子成像（图1），用于在稳定的小鼠的头部，头部板胶合。确定要根据解剖范围变薄的区域。避免直接位于颅缝线以上，头骨是不太稳定，在这些领域和潜在的大血管和脑膜会干扰成像感兴趣的领域。 </li><li>应用100％的乙醇，用无菌棉签干燥头骨顶部的膜，并用细镊子或刀片刮去他们走了10％三氯化铁溶液。未能消除不稳定的头板附着在膜的结果。 </li><li> PERMABOND 910胶头板下方的观景窗的边缘周围放置一个薄薄的一层。将头板用胶水涂在动物的头骨压光（图1）利息的面积。重要的是，头盘是只粘在颅骨骨。任何皮肤或陷入头板与颅骨之间的膜，将降低债券的稳定。周围观看的窗口使用一个注射器瞬间粘合胶，适用于少量的丙烯酸酯。最后，申请一个观景窗的边缘少量乐泰454，以防止泄漏。 </li><li>螺丝头盘动物的持有人（图1），并检查头板下一双镊子轻轻探清扫范围的稳定。不应该有骷髅头板相对运动。广场上的观景窗，以确保没有泄漏的生理盐水下降。 </li></ol> 2。准备减薄的头骨皮层窗口<ol><li>在准备颅骨变薄，干燥颅骨结合使用无菌棉签和压缩空气。最初，我们在Microtorque二钻无菌IRF 007钻在4000转位颅骨薄。轻轻地，开始变薄一个2-2.5毫米直径的圆形区域内的头骨。使用唯一的光线扫近于平行的头骨的议案;使用没有直接的下调压力。停钻，每20-30秒，去除骨灰尘，使用压缩空气。在钻井这些中断允许头骨凉爽，所以没有热诱导的基本脑组织的损害。 <ol class="lalpha"><li>由于钻井过程中，发现湿润的骨松质层（即“板障”）的过渡。一旦达到这一层与演练，锻炼时要格外小心。 </li></ol></li><li>偶尔检查放在盐水在薄区和观看下解剖范围内的头骨薄。萨林将进一步使散热。由于颅骨变薄，较小的血管变得可见。 </li><li>使用无菌牙科microblade实现下生理盐水的最后变薄，microblade提供触觉反馈的头骨稳定得多。这可以比单钻薄得多的筹备工作。从我们的经验，最佳的颅骨厚度为10-30微米。 <ol class="lalpha"><li>重要的是要检查epifluorescence多次下颅骨的薄，在最初的几次尝试使颅骨薄窗口。窗口时，成像的清晰度和可见的小胶质细胞和血管的深度给一个很好的迹象。 </li></ol></li><li>一旦窗口已准备就绪，胶水定做一块长方形玻璃罩无线0＃<html>第一个窗口每边尺寸小于2毫米以上。使用一个直径为1.0毫米的玻璃吸管，地方上薄的头骨面积氰基丙烯酸酯胶的小水滴和仔细下来的小块盖玻片，然后按对头骨轻轻挤压出过量的胶水。重要的是要避免泡沫形成之间的玻璃和胶水，因为残留的空气会导致该地区的下方，成为光学不透明。氰基丙烯酸酯胶使用，因为它保持透明，干燥时，还可以防止骨和膜的再生，维持颅骨薄，半透明的窗口下。如果有任何胶玻璃盖的顶部，使用microblade小心取出玻璃盖一次到位和胶水干。 </li><li>止痛药（例如，丁丙诺啡2毫克/公斤分cutaneously）管理后立即手术，重管理，在任何疼痛的迹象，包括不愿动，吃或喝，消瘦，流涎，立毛，呼吸音，等</li></ol> 3。双光子成像<ol><li>在准备双光子成像，盖用生理盐水TSCW和定位下epifluorescence利益的区域（图2）。为了使该地区的后续成像，使用照相机拍照。 </li><li>双光子成像，我们用一个特制的双光子显微镜与TI 6：蓝宝石激光调谐到920纳米。整个现场检测模式和一百八十零分之五百八十〇排放过滤器中使用光电倍增管检测荧光。 20倍水浸泡镜片（0.95 NA）的使用在整个成像会议。客观的激光功率的最大输出功率为50和65兆瓦之间。 </li><li>选择感兴趣的领域，在皮质层I或II（高达150微米以下的软脑膜表面）。为了便于重新成像，采取相同的视野图片1X，2X，3X数码变焦。血管可作为地标，很容易地找到在随后的成像会话相同的视野。根据80-90变焦的3倍，多的Z -栈在每个堆栈和一个0.69微米的一步的Z轴的步骤可以收购每5分钟至30分钟，这使小胶质细胞的形态和行为随着时间的推移，抽样（图3）。 Z -栈之间的收购，应核实在TSCW生理盐水水平，以及动物的麻醉深度。 </li></ol> 4。注射HIV - 1的神经毒素，达<ol><li>首先，silanize内衬的35号针和10μL微量注射器，防止达沉积。撤回的silanizing解决方案（Sigmacote），直到填满的针头和注射器。允许的解决方案，坐在在室温下5分钟。空注射器的解决方案，取出柱塞，并允许完全干燥的注射器和针头。最后，清洗彻底，用去离子水的注射器和针头。 </li><li>执行一个小开颅0.5毫米直径无论是3毫米延髓或横向的双光子图像已取得TSCW，使用较小的钻头，并仔细颅骨变薄，直到一双尖的镊子，可以去除薄层骨容易。我们用10μL微量注射器，装有35压力表控制针Ultramicropump三注射器泵安装一个3轴的显微和排队针尖开颅后，仔细先进的深度为700至900微米以下3μL达1-72（或其他亲消炎药）或生理盐水（或控制车辆），是在80 NL /分钟的软脑膜表面。 </li></ol> 5。畜舍<ol><li>如果动物是要在一天多次成像，盖用眼药膏和凡士林，以防止身体不适或损坏的头骨在会话之间的成像混合物的头骨的开放区域。断开小头板（见图1B）支持的桥梁，方便食品和水，并放置在一个温暖的笼子动物。所有动物都被安置在任何时候接受手术后单。一旦某一天的完整的动物成像会议，小心地取出整个头板和背部的皮肤在颅骨缝合＃6或更小的缝合。同样，单独房子的动物实验天之间的方便食品和水。每天检查的动物，任何压力的迹象，疼痛或感染。 </li></ol> 6。代表性的成果<img src="/files/ftp_upload/2059/2059fig1.jpg" alt="图1" /> 图1手术和双光子成像（A）和头板设计（二）动物的稳定。 <img src="/files/ftp_upload/2059/2059fig2.jpg" alt="图2" /> 图2 GFP标记的小胶质细胞的可视化与epifluorescence。 jove_content“&gt; <img src="/files/ftp_upload/2059/2059fig3.jpg" alt="图3" /> 图3。 GFP标记的小胶质细胞与双光子成像的可视化后植入TSCW，约50微米以下的软脑膜表面。单一双光子TSCW（0天）植入后15-30分钟的部分，以及7 17天后，展示的Windows保持清晰，而小胶质细胞在没有炎症侮辱的情况下仍然灭活。 Z - 6和预测，采取24小时后注射艾滋病毒的神经毒素达表现出令人印象深刻的激活小胶质细胞的形态学变化。比例尺：10微米。

<ol>
	<li>Mostany, R., &amp;amp, P. o. r. t. e. r. a. -. C. a. i. l. l. i. a. u., C, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+craniotomy+surgery+procedure+for+chronic+brain+imaging.">A craniotomy surgery procedure for chronic brain imaging.</a> J Vis Exp. 12, (2008).</li><li>Holtmaat, A. <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+imaging+in+the+mouse+neocortex+through+a+chronic+cranial+window.">high-resolution imaging in the mouse neocortex through a chronic cranial window.</a> Nat Protoc. 4, (2009).</li><li>Yang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thinned-skull+cranial+window+technique+for+long-term+imaging+of+the+cortex+in+live+mice.">Thinned-skull cranial window technique for long-term imaging of the cortex in live mice.</a> Nature Protoc. 5, 213-21 (2010).</li><li>Jung, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+fractalkine+receptor+CX(3)CR1+function+by+targeted+deletion+and+green+fluorescent+protein+reporter+gene+insertion.">Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion.</a> Mol Cell Biol. 20, 4106-41 (2000).</li><li>Mrsic-Flogel, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homeostatic+regulation+of+eye-specific+responses+in+visual+cortex+during+ocular+dominance+plasticity.">Homeostatic regulation of eye-specific responses in visual cortex during ocular dominance plasticity.</a> Neuron. 54, 961-96 (2007).</li><li>Majewska, A., Yiu, G., &amp;amp, Y. u. s. t. e., R, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+custom-made+two-photon+microscope+and+deconvolution+system.">A custom-made two-photon microscope and deconvolution system.</a> Pflugers Arch. 441, 2-3 (2000).</li><li>Sharer, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathology+of+HIV-1+infection+of+the+central+nervous+system.">Pathology of HIV-1 infection of the central nervous system.</a> A review. J Neuropathol Exp Neurol. 51, (1992).</li><li>Everall, I. P., Aliberti, J., &amp;amp, B. a. l. c. e. r. z. y. k., M, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+density+in+the+superior+frontal+and+temporal+gyri+does+not+correlate+with+the+degree+of+human+immunodeficiency+virus-associated+dementia.">Neuronal density in the superior frontal and temporal gyri does not correlate with the degree of human immunodeficiency virus-associated dementia.</a> Acta Neuropathol (Berl. 88, 538-53 (1994).</li><li>Masliah, E., Banati, R. B., &amp;amp, Q. u. i. n. l. a. n., M, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+injury+is+a+pathological+substrate+for+human+immunodeficiency+virus-related+cognitive+disorders.">Dendritic injury is a pathological substrate for human immunodeficiency virus-related cognitive disorders.</a> Ann Neurol. 42, 963-96 (1997).</li><li>Everall, I. P., Banati, R. B., &amp;amp, U. p. e. n. d. e. r., B, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+synaptic+density+is+reduced+in+mild+to+moderate+human+immunodeficiency+virus+neurocognitive+disorder.">Cortical synaptic density is reduced in mild to moderate human immunodeficiency virus neurocognitive disorder.</a> Brain Pathol. 9, (1999).</li><li>Glass, J. D., Fedor, H., Wesselingh, S. L., &amp;amp, M. c. A. r. t. h. u. r., C, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunocytochemical+quantitation+of+human+immunodeficiency+virus+in+the+brain:+correlations+with+dementia.">Immunocytochemical quantitation of human immunodeficiency virus in the brain: correlations with dementia.</a> Ann Neurol. 38, (1995).</li><li>Feng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neuronal+subsets+in+transgenic+mice+expressing+multiple+spectral+variants+of+GFP.">Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP.</a> Neuron. 28, (2000).</li></ol>

有越来越多的共识，为HIV - 1相关的神经认知赤字（手）的实际基板是突触结构的破坏，大概是从HIV - 1感染单核吞噬细胞释放炎症介质，引起。的小胶质细胞激活，多核巨细胞，胶质细胞增生由于星形胶质细胞肥大，被认为是在中枢神经系统7 HIV - 1感染和炎症的非特异性标志。相比之下，生前神经系统疾病的严重程度已与损失突触的复杂性，以及在中枢神经系统的巨噬细胞8,9,10,11负担。但是，小胶质细胞，周边浸润的巨噬细胞，患者用手的神经元之间的动态交互根本无法使用传统的静态的处理从传统的免疫细胞化学研究取得建模。从我们的实验室最近的研究表明，至少有一些外周和中枢的单核细胞和神经元之间的这些交互可以模拟立体定向注射的HIV - 1蛋白达1-72部分脑实质（路，标记，特朗布莱益气，和格尔巴德，未发表数据）。因此，我们决定申请在体内的双光子成像检查单核细胞衍生的巨噬细胞，脑居民小胶质细胞和神经元之间的相互作用，neuroAIDS一个听话的小动物模型。虽然开放的颅骨或变薄，颅骨准备买得起一个一个时间短暂的时期（小时）皮质架构单一的检查，调查的亚急性事件的时间当然可以不使用不artifactually激活小胶质细胞/巨噬细胞因手术操纵的这些准备工作感兴趣颅骨窗口。在这里，我们展示了一个简单的方法绕过这些限制胶合薄的头骨面积小片，防止再生的颅骨在TSCW以及为≥3个星期保持半透明的玻璃盖玻片。我们进一步证明了这种技术，使我们能够监测在应对艾滋病毒达 1-72立体注射皮质CX 3 CR 1 / GFP的杂合子小鼠外周血单核细胞进入大脑微血管，以及大脑居民的小胶质细胞的行为（确定的单核血统细胞）。这种技术可以很容易地适应使用上具有不同的遗传背景的小鼠，例如，我们经常使用杂合子CX 3 CR 1 / GFP的X THY - 1 YFP的 12只小鼠，以探讨单核细胞衍生的巨噬细胞，脑驻地小胶质细胞和神经元之间的相互作用在体内模型的neuroinflammation有关手。我们TSCW准备允许调查研究之间的周边和中枢神经系统，可能发生在周期间，其中的动物，可反复实验性治疗前后成像的细胞间的相互作用。因此，每个动物可以作为自己的控制。这个TSCW模型的一个需要注意的是，受调查的靶细胞是基因工程表达增强荧光蛋白（eXFP），或与荧光染料没有违反颅骨的机械标记; eXFP表达必须强大到足以使细胞结构可视化后，通过一个星期内重复成像会议。

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		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>Fentanyl Citrate</td>
<td>&nbsp;</td>
<td>Hospira</td>
<td>&nbsp;</td>
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<td>Midazolam hydrochloride</td>
<td>&nbsp;</td>
<td>Baxter</td>
<td>&nbsp;</td>
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<td>Medetomidine hydrocholoride (Domitor)</td>
<td>&nbsp;</td>
<td>Pfizer</td>
<td>&nbsp;</td>
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<td>Heating pads</td>
<td>&nbsp;</td>
<td>Beyond Bodi Heat</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Tobramycin and dexamethasone ophthalmic ointment (TobraDex)</td>
<td>&nbsp;</td>
<td>Alcon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Povidone-iodine 10% solution</td>
<td>&nbsp;</td>
<td>Betadine</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Ferric chloride 10% solution</td>
<td>&nbsp;</td>
<td>Ricca Chemical Company</td>
<td>3120-32</td>
<td>&nbsp;</td>
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<td>Methyl cyanoacrylate 910</td>
<td>&nbsp;</td>
<td>Permabond</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Cyanoacrylate 454</td>
<td>&nbsp;</td>
<td>Loctite</td>
<td>&nbsp;</td>
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<td>TEETS denture material crosslinking methyl methacrylate</td>
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<td>Co-Oral-lte Dental Mfg CO</td>
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<td>Microtorque II handpiece kit</td>
<td>&nbsp;</td>
<td>Pearson</td>
<td>R14-0002</td>
<td>&nbsp;</td>
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<td>IRF 007 drill bits</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
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		</div>

a thin-skull window technique for chronic two-photon in vivo imaging of murine microglia in models of neuroinflammation

神经科学的传统， 在体内小鼠中枢神经系统的两个光子成像，无论是涉及使用开放性颅骨 1,2或变薄， 颅骨 3准备工作。虽然开颅骨技术是非常灵活，它是研究小胶质细胞最优的，因为它是侵入性，并可能导致小胶质细胞激活。即使颅骨变薄，方法是微创，反复慢性成像所需的头骨重新变薄，增加组织损伤和小胶质细胞激活的风险，并允许数量有限的成像会议。在这里，我们提出一个用于监测小鼠小胶质细胞在体内长时间使用双光子显微镜的时间的慢性颅骨薄窗口的方法。我们演示了如何准备一个稳定，方便，皮质变薄，颅骨窗口（TSCW）apposed仍然超过三个星期的间歇性观察过程中的半透明的玻璃盖玻片。这TSCW准备是远远方面比开放开颅手术或反复颅骨变薄的小胶质细胞激活免疫惰性，并允许在一个星期的时间内任意数量的成像会议。我们准备在CX 3 CR 1 GFP / +小鼠4 TSCW可视化与增强型绿色荧光蛋白≤150下方的软脑膜表面微米的小胶质细胞。我们还表明，该制剂可与HIV - 1蛋白的神经毒性达，毗邻TSCW，这是能够诱导持久microgliosis的立体定向脑注射液配合使用。因此，这种方法是非常有用的研究生活在艾滋病毒有关的神经认知障碍（手）和其他神经退行性疾病与neuroinflammatory组件模型的脑小胶质细胞形态和活力随着时间的推移变化的。

Watch this Scientific Journal Video about A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation at JoVE.com

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

我们描述了一个反复可视化小鼠小胶质细胞和单核细胞循环的方法<em&gt;在体内</em&gt;超过几小时，几天或几周内，采用经颅双光子显微镜。我们将演示如何编写一个薄的头骨的窗口，让静态的小胶质细胞，激活，可以通过相邻的立体定向注射HIV - 1调控蛋白达间歇观察。

一个慢性双光子薄颅骨窗口技术在体内 Neuroinflammation模型小鼠小胶质细胞成像

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or ...

a-thin-skull-window-technique-for-chronic-two-photon-vivo-imaging

Research

JoVE Journal

Neuroscience

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

23.4K 次观看.  University of Rochester. 我们描述了一个反复可视化小鼠小胶质细胞和单核细胞循环的方法在体内超过几小时，几天或几周内，采用经颅双光子显微镜。我们将演示如何编写一个薄的头骨的窗口，让静态的小胶质细胞，激活，可以通过相邻的立体定向注射HIV - 1调控蛋白达间歇观察。

视频: A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

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

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

Optical coherence tomography (OCT) is a biomedical imaging technique with high spatial-temporal resolution. With its minimally invasive approach OCT has been used extensively in ophthalmology, dermatology, and gastroenterology1-3. Using a thinned-skull cortical window (TSCW), we employ spectral-domain OCT (SD-OCT) modality as a tool to image the cortex in vivo. Commonly, an opened-skull has been used for neuro-imaging as it provides more versatility, however, a TSCW approach is less invasive and is an effective mean for long term imaging in neuropathology studies. Here, we present a method of creating a TSCW in a mouse model for in vivo OCT imaging of the cerebral cortex.

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

We present a method of creating a thinned-skull cortical window (TSCW) in a mouse model for in vivo OCT imaging of the cerebral cortex.

稀疏的头骨皮层的窗口技术<em在体内</em光学相干断层成像

Optical coherence tomography (OCT) is a biomedical imaging technique  with high spatial-temporal resolution. With its minimally invasive approach OCT  has ...

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 ...

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their close-to-surface positioning facilitates microscopic imaging of skin nerve endings in living intact animal. Using multiphoton microscopy, it is possible to obtain fine images overcoming the problem of strong light scattering of the skin tissue. Reporter transgenic mice that express EYFP under the control of Thy-1 promoter in neurons (including periphery sensory neurons) are well suited for the longitudinal studies of individual nerve endings over extended periods of time up to several months or even life-long. Furthermore, using the same femtosecond laser as for the imaging, it is possible to produce highly selective lesions of nerve fibers for the studies of the nerve fiber restructuring. Here, we present a simple and reliable protocol for longitudinal multiphoton in vivo imaging and laser-based microsurgery on mouse skin nerve endings.

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

The protocol for dynamic longitudinal imaging and selective laser lesion of nerve endings in reporter transgenic mice is presented.&#160;

在皮肤中的单神经末梢体内双光子显微镜

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their ...

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 ...

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 ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

小鼠视网膜的经瞳双光子体内成像

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...