这项工作是由加州大学理念授予的发现证明，由美国国立卫生研究院（R00 EB007241）。作者还要感谢杰奎琳·哈伯德对她的帮助，在这个实验中。

1。手术准备<ol><li>女CD 1岁之间的6-8周，在我们的实验中使用的小鼠。 </li><li>麻醉的小鼠腹膜内注射氯胺酮和甲苯噻嗪的组合（80毫克/公斤ketamine/10 mg / kg的甲苯噻嗪）。将鼠标在homeothermic垫，以确保最佳的体温〜37℃连续监测麻醉水平测试动物的反射（ 如 ，钝钳捏脚），并在必要时注入更多的麻醉剂。 </li><li>用人工泪液润滑双眼软膏。删除使用剃刀在头皮上的毛发，并用70％的酒精准备垫去除残留的头发。奈尔脱毛膏在头皮涂上薄薄的一层，等待2分钟生效。轻轻擦去Nair和剩余的头发用生理盐水浸湿棉签和酒精准备垫。的头皮现在应该是完全无毛。 </li><li>头皮使用优碘棉签棒的消毒和清洁70％的酒精准备垫。 </li><li>小心地包裹动物在手术单，以确保最佳的体温〜37°C，并安装到一个立体的框架，以固定的头骨的动物。轻轻敲击颅骨，以确保其稳定性。所使用的材料的列表被设置在表1中 。 </li></ol> 2。稀疏的头骨皮质窗口准备<ol><li>启动切口在中线点之间的眼睛。继续尾部的中线点之间的耳朵。用钳子部分的皮肤。 </li><li>在解剖显微镜下找到被摊薄和筋膜，用小镊子轻轻取出。头骨薄的皮质窗口前，用无菌棉签擦干。在我们的实验中，我们创建了一个4×4毫米薄的颅窗〜1毫米后方和侧面，前囟门。 </li><li>开始变薄的头骨采用圆硬质合金钻针，钻头尺寸0.75毫米手电钻在手术中使用淡扫运动ONLY。不要将头骨上的直接压力。停止钻，每隔20-30秒，去除骨灰尘，用无菌生理盐水和棉签，以避免过热的头骨。的盐水也将有助于在整个头骨的热量散发。 </li><li>一旦外层紧凑骨完全除去中间的海绵骨层现在应该可见。有可能会出现一些轻微的出血，如血管较明显的松质骨层。切换到一个绿色的石牙钻，继续钻探，使用格外小心，因为海绵层更加细腻。绿色的石牙钻去除骨材料少，同时创造均匀度在整个颅窗。停钻，偶尔去除骨的灰尘和冷却的头骨。 </li><li>最后，当头骨已变得更加透明和血管对大脑现在是可见的，开始抛光使用抛光牙钻的头骨。这将允许更精确的细化而平滑的头骨。检查薄的SKU会用钳子轻轻拍打就可以了。停止抛光时，的头骨变得稍微灵活。 </li><li>减薄的颅窗现在应该完全光滑的和反射的，并准备用于成像（ 图1）。由于高散射的大脑组织的性质，应减薄颅骨至少为55微米，最佳的穿透深度。所使用的材料的列表被设置在表1中 。 </li></ol> 3。光学相干断层成像<ol><li>手术完成后，检查动物的呼吸速率和反应能力，以确保适当的麻醉和管理额外的麻醉，如果必要的。删除动物脑立体定位框架，使动物在手术单包裹，运输动物的成像站。 </li><li>在成像检查对反射的迹象，如果需要申请额外的人工泪液。安装立体定位框架，以确保头骨的动物。 </li><li>广场动物下OCT相机和位置的TSCW下的光束（ 图2）。 A剖视图的头骨和大脑现在可以被可视化（ 图3）。 </li><li>一旦找到感兴趣的区域，就可以开始数据采集。对于成像的目的，我们使用检流计反射镜，具有4.0毫米的宽度来实现的摄像窗口。一种成像深度为2mm，得到6毫瓦的入射功率，以及焦点1毫米以下的薄的颅骨。每个横截面面积由2048与每幅图像的采集速率为0.14秒的轴向扫描。 </li><li>也可以通过以下方式获得的体积扫描的大脑收集一系列的二维截面图像通过使用检流计反射镜两套XY扫描与所述第一检流计镜扫描在弧矢方向上的光束和所述第二检流计镜扫描在冠状的方向发展。 </li></ol>

<ol>
	<li>Bizheva, K., Unterhuber, A., Hermann, B., Povazay, B., Sattmann, H., Drexler, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+ex+vivo+and+in+vitro+brain+morphology+in+animal+models+with+ultrahigh+resolution+optical+coherence+tomography.">Imaging ex vivo and in vitro brain morphology in animal models with ultrahigh resolution optical coherence tomography.</a> Journal of Biomedical Optics. 9, 719-724 (2004).</li><li>Fujimoto, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+coherence+tomography+for+ultrahigh+resolution+in+vivo+imaging.">Optical coherence tomography for ultrahigh resolution in vivo imaging.</a> Nature Biotechnology. 21, 1361-1367 (2003).</li><li>Wantanabe, H., Rajagopalan, U. M., Nakamichi, Y., Igarashi, K. M., Kadono, H., Tanifuji, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Swept+source+optical+coherence+tomography+as+a+tool+for+real+time+visualization+and+localization+of+electrodes+used+in+electrophysiological+studies+of+brain+in+vivo.">Swept source optical coherence tomography as a tool for real time visualization and localization of electrodes used in electrophysiological studies of brain in vivo.</a> Biomedical Optics Express. 2, 3129-3134 (2011).</li><li>Huang, D., Swanson, E. A., Lin, C. P., Schuman, J. S., Stinson, W. G., Chang, W., Hee, M. R., Flottee, T., Gregory, K., Puliafito, C. A., Fujimoto, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+coherence+tomography.">Optical coherence tomography.</a> Science. 254, 1178-1181 (1991).</li><li>Mitsui, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+range+of+optical+reflectometry+with+spectral+interferometry.">Dynamic range of optical reflectometry with spectral interferometry.</a> Japanese Journal of Applied Physics. 38, 6133-6137 (1999).</li><li>de Boer, J. F., Cense, B., Park, B. H., Pierce, M. C., Tearney, G. J., Bouma, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+signal-to-noise+ratio+in+spectral-domain+compared+with+time-domain+optical+coherence+tomograhy.">Improved signal-to-noise ratio in spectral-domain compared with time-domain optical coherence tomograhy.</a> Optics Letters. 28, 2067-2069 (2003).</li><li>de Boer, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+5.">Ch. 5.</a> Optical Coherence Tomography: Technology and Applications. , (2008).</li><li>Choma, M. A., Sarunic, M. V., Yang, C., Izatt, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitivity+advantage+of+swept+source+and+fourier+domain+optical+coherence+tomography.">Sensitivity advantage of swept source and fourier domain optical coherence tomography.</a> Optics Express. 11, 2183-2189 (2003).</li><li>Leitgeb, R. A., Drexler, W., Unterhuber, A., Hermann, B., Bajraszewski, T., Le, T., Stingl, A., Fercher, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrahigh+resolution+fourier+domain+optical+coherence+tomography.">Ultrahigh resolution fourier domain optical coherence tomography.</a> Optics Express. 12, 2156-2165 (2004).</li><li>Drew, P. J., Shih, A. Y., Driscoll, J. D., Knutsen, P. M., Blinder, P., Davalos, D., Akassoglou, K., Tsai, P. S., Kleinfeld, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+optical+access+through+a+polished+and+reinforced+thinned+skull.">Chronic optical access through a polished and reinforced thinned skull.</a> Nature Methods. 7, 981-984 (2010).</li><li>Shih, A. Y., Mateo, C., Drew, P. J., Tsai, P. S., Kleinfeld, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Polished+and+Reinforced+Thinned-skull+Window+for+Long-term+Imaging+of+the+Mouse.">A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse.</a> J. Vis. Exp. 61, e3742 (2012).</li><li>Yang, G., Pan, F., Parkhurst, C. N., Grutzendler, J., Gan, W. <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 Protocols. 5, (2010).</li><li>Lu, M., Majewska, S., K, A., Gelbard, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Thin-skull+Window+Technique+for+Chronic+Two-photon+In+vivo+Imaging+of+Murine+Microglia+in+Models+of+Neuroinflammation.">A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation.</a> J. Vis. Exp. (43), e2059 (2010).</li><li>Wang, Y., Oh, C. M., Oliveira, M. C., Islam, M. S., Ortega, A., Park, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GPU+accelerated+real-time+multi-functional+spectral-domain+optical+coherence+tomography+system+at+1300nm.">GPU accelerated real-time multi-functional spectral-domain optical coherence tomography system at 1300nm.</a> Optics Express. 20, 14797-14813 (2012).</li><li>Aguirre, A. D., Chen, Y., Fujimoto, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depth-resolved+imaging+of+functional+activation+in+the+rat+cerebral+cortex+using+optical+coherence+tomography.">Depth-resolved imaging of functional activation in the rat cerebral cortex using optical coherence tomography.</a> Opt. Lett. 31, 3459-3461 (2006).</li><li>Chen, Y., Aguirre, A. D., Ruvinskaya, L., Devor, A., Boas, D. A., Fujimoto, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+coherence+tomography+(OCT)+reveals+depth-resolved+dynamics+during+functional+brain+activation.">Optical coherence tomography (OCT) reveals depth-resolved dynamics during functional brain activation.</a> Journal of Neuroscience Methods. 178, 162-173 (2009).</li><li>Liang, C., Wierwille, J., Moreira, T., Schwartzbauer, G., Jafri, M. S., Tang, C., Chen, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+forward-imaging+needle-type+OCT+probe+for+image+guided+stereotactic+procedures.">A forward-imaging needle-type OCT probe for image guided stereotactic procedures.</a> Opt Express. 19, 26283-26294 (2011).</li><li>Srinivasan, V. J., Sakadzic, S., Gorczynska, I., Ruvinskaya, S., Wu, W., Fugimoto, J. G., Boas, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+cerebral+blood+flow+with+optical+coherence+tomography.">Quantitative cerebral blood flow with optical coherence tomography.</a> Optics Express. 18, 2477-2494 (2010).</li><li>Galetta, K. M., Calabresi, P. A., Frohman, E. M., Balcer, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+Coherence+Tomography+(OCT):+imaging+the+visual+pathway+as+a+model+for+neurodegeneration.">Optical Coherence Tomography (OCT): imaging the visual pathway as a model for neurodegeneration.</a> The Journal of the American Society for Experimental NeuroTherapeutics. 8, 117-132 (2011).</li><li>Seigo, M. A., Sotirchos, E. S., Newsome, S., Babiarz, A., Eckstein, C., Ford, E., Oakley, J. D., Syc, S. B., Frohman, T. C., Ratchford, J. N., Balcer, L. J., Frohman, E. M., Calabresi, P. A., Saidha, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+assessment+of+retinal+neuronal+layers+in+multiple+sclerosis+with+maual+and+automated+optical+coherence+tomography+segementation+techniques.">In vivo assessment of retinal neuronal layers in multiple sclerosis with maual and automated optical coherence tomography segementation techniques.</a> J. Neurol. , (2012).</li><li>Frohman, E. M., Fujimoto, J. G., Frohman, T. C., Calabresi, P. A., Cutter, G., Balcer, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+coherence+tomography:+a+window+into+the+mechanisms+of+multiple+sclerosis.">Optical coherence tomography: a window into the mechanisms of multiple sclerosis.</a> Nature Clinical Practice. 4, 664-675 (2008).</li><li>Gill, A. S., Rajneesh, K. F., Owen, C. M., Yeh, J., Hsu, M., Binder, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+optical+detection+of+cerebral+edema+in+vivo.">Early optical detection of cerebral edema in vivo.</a> J. Neurosurg. 114, 470-477 (2011).</li></ol>

没有利益冲突的声明。

成像与OCT和减薄颅骨是一种新的神经成像技术，只有最近的调查15，16。在我们的实验中，我们证实SD-OCT成像的可行性通过一个TSCW在小鼠模型体内 。从我们的结果，颅骨被减薄到约55微米，和获得的穿透深度分别为8μm和20μm的在轴向和横向方向上的图像的分辨率，在约1毫米。在信号强度分布，通过TSCW OCT成像的信号强度和穿透深度增加，相比一个正常颅骨（ 图4）。相?...

在20世纪90年代初，自推出以来，华侨城已被广泛用于生物成像的组织结构和功能2。 OCT生成的横截面图像，通过测量通过实施低相干光源与光纤的Michelson干涉仪2,4回波时间延迟的反向散射光4。 SD-OCT，也被称为傅立叶域OCT（FD-OCT），在1995年5月首次推出，提供了出色的成像方式相比，传统的时域OCT（TD-OCT）。在SD-OCT中，参考臂保持静止导致在高速和超高分辨率图像采集6-9。 目前，TSCW模型已被主要用于代替传统的开颅双光子显微镜在体内脑成像应用。这些TSCW同时使用一个自定义的颅骨内板或玻璃盖片10-13提供额外的IMA晋稳定。在我们的研究中，我们已观察到，这些配件，如没有必要使用OCT成像时TSCW。因此，缺乏的颅骨外板或玻璃盖玻片允许摄像窗口的大小以便在更大的范围内，因为它们可能会干扰光束，并改变OCT图像。 阿稀疏颅骨制剂已被证明是有利的，在使用双光子显微镜10-13的大脑成像研究。在我们的实验中，我们使用了SD-OCT系统图像通过TSCW 体内的皮质。我们的自定义的SD-OCT成像设置包含一个宽带，低相干光源由两个超辐射发光二极管（SLD）的中心在1295纳米的带宽为97纳米，从而在轴向和横向分辨率为8μm和20μm的，分别为14组成的。我们的光学成像设备，我们设想，成像通过TSCW有很大的潜力，在识别和可视化的结构和功能的Optically密集的脑组织。

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thinned-skull cortical window technique for in vivo optical coherence tomography imaging

光学相干断层扫描（OCT）是生物医学成像技术，具有高时空分辨率。凭借其微创的方法，华侨城已被广泛用于眼科，皮肤科，消化内科1-3。使用稀疏的头骨皮层的窗口（TSCW），我们采用谱域OCT（SD-OCT）的方式作为一种工具， 体内的皮质图像。好评，打开颅骨已用于神经成像，因为它提供了更多的功能，但是，TSCW方法是微创的，是一种有效的长期平均成像在神经病理学研究。在这里，我们提出的方法在小鼠模型体内 OCT成像的大脑皮质创建一个TSCW的。

脉管系统创建一个减薄的窗口在大脑皮层后现在应该是视觉上更加突出（ 图1），并且将允许成像深度更深（可达1毫米）。右皮质减薄到约55微米，较到正常颅骨测定在140微米（ 图1），并提供了更大的光学清晰度。 11但没有必要的，因为使用的盖玻片和颅骨内板没有实现在我们的实验中（ 图1和图2），可以进一步细化到10-15微米。这个特定的方法，?...

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

我们提出一个方法创建一个稀疏的头骨皮质的窗口（TSCW）在小鼠模​​型<em在体内</emOCT成像的大脑皮质。

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 ...

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

15.0K 次观看.  University of California, Riverside. 我们提出一个方法创建一个稀疏的头骨皮质的窗口（TSCW）在小鼠模​​型

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

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

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or thinned-skull 3 preparations. While the open-skull technique is very versatile, it is not optimal for studying microglia because it is invasive and can cause microglial activation. Even though the thinned-skull approach is minimally invasive, the repeated re-thinning of skull required for chronic imaging increases the risks of tissue injury and microglial activation and allows for a limited number of imaging sessions. Here we present a chronic thin-skull window method for monitoring murine microglia in vivo over an extended period of time using two-photon microscopy. We demonstrate how to prepare a stable, accessible, thinned-skull cortical window (TSCW) with an apposed glass coverslip that remains translucent over the course of three weeks of intermittent observation. This TSCW preparation is far more immunologically inert with respect to microglial activation than open craniotomy or repeated skull thinning and allows an arbitrary number of imaging sessions during a time period of weeks. We prepare TSCW in CX3CR1 GFP/+ mice 4 to visualize microglia with enhanced green fluorescent protein to &le;150 &mu;m beneath the pial surface. We also show that this preparation can be used in conjunction with stereotactic brain injections of the HIV-1 neurotoxic protein Tat, adjacent to the TSCW, which is capable of inducing durable microgliosis. Therefore, this method is extremely useful for examining changes in microglial morphology and motility over time in the living brain in models of HIV Associated Neurocognitive Disorder (HAND) and other neurodegenerative diseases with a neuroinflammatory component.

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

We describe a method for repeatedly visualizing murine microglia and circulating monocytes in vivo over hours, days or weeks using transcranial two-photon microscopy. We demonstrate how to prepare a thinned-skull window that allows intermittent observation of quiescent microglia that can be activated by adjacent stereotactic injection of the HIV-1 regulatory protein Tat.

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 ...

科研

神经科学

Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The technique presented here allows the imaging of the same area of the brain at several time points (chronic imaging) with microscopic resolution allowing the tracking of dendritic spines which are the small structures that represent the majority of postsynaptic excitatory sites in the CNS. The ability to clearly resolve fine cortical structures over several time points has many advantages, specifically in the study of brain plasticity in which morphological changes at synapses and circuit remodeling may help explain underlying mechanisms. In this video and supplementary material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation. The thinned-skull preparation is a minimally invasive approach, which avoids potential damage to the dura and/or cortex, thus reducing the onset of an inflammatory response. When this protocol is performed correctly, it is possible to clearly monitor changes in dendritic spine characteristics in the intact brain over a prolonged period of time.

In this video and supplemental material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation.

慢性成像小鼠的视觉皮层使用减薄，颅骨的制备

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The ...

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form a polished and reinforced thinned skull (PoRTS) window in the mouse skull that spans several millimeters in diameter and is stable for months. The skull is thinned to 10 to 15 &mu;m in thickness with a hand held drill to achieve optical clarity, and is then overlaid with cyanoacrylate glue and a cover glass to: 1) provide rigidity, 2) inhibit bone regrowth and 3) reduce light scattering from irregularities on the bone surface. Since the skull is not breached, any inflammation that could affect the process being studied is greatly reduced. Imaging depths of up to 250 &mu;m below the cortical surface can be achieved using two-photon laser scanning microscopy. This window is well suited to study cerebral blood flow and cellular function in both anesthetized and awake preparations. It further offers the opportunity to manipulate cell activity using optogenetics or to disrupt blood flow in targeted vessels by irradiation of circulating photosensitizers.

We present a method to form an imaging window in the mouse skull that spans millimeters and is stable for months without inflammation of the brain. This method is well suited for longitudinal studies of blood flow, cellular dynamics, and cell/vascular structure using two-photon microscopy.

抛光和钢筋，颅骨变薄窗口，为长期的小鼠脑成像

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form ...

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 ...

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 ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

光学相干断层扫描: 成像小鼠视网膜神经节细胞在体内

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

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 ...

Using Optical Coherence Tomography and Optokinetic Response As Structural and Functional Visual System Readouts in Mice and Rats

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for the investigation of processes of neurodegeneration, neuroprotection and neuro-repair involving the visual system, as these often correlate well with retinal changes. As a functional readout, visually evoked compensatory eye and head movements are commonly used in experimental models involving the visual function. Combining both techniques allows a quantitative in vivo investigation of structure and function, which can be used to investigate the pathological conditions or to evaluate the potential of novel therapeutics. A great benefit of the presented techniques is the possibility to perform longitudinal analyses allowing the investigation of dynamic processes, reducing variability and cuts down the number of animals needed for the experiments. The protocol described aims to provide a manual for acquisition and analysis of high quality retinal scans of mice and rats using a low cost customized holder with an option to deliver inhalational anesthesia. Additionally, the proposed guide is intended as an instructional manual for researchers using optokinetic response (OKR) analysis in rodents, which can be adapted to their specific needs and interests.

A detailed protocol for the assessment of structural and visual readouts in rodents by optical coherence tomography and optokinetic response is presented. The results provide valuable insights for ophthalmologic as well as neurologic research.

光学相干层析成像和光运动响应在小鼠和大鼠中的结构和功能视觉系统读数

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for ...

Retinal Vascular Reactivity as Assessed by Optical Coherence Tomography Angiography

The vascular supply to the retina has been shown to dynamically adapt through vasoconstriction and vasodilation to accommodate the metabolic demands of the retina. This process, referred to as retinal vascular reactivity (RVR), is mediated by neurovascular coupling, which is impaired very early in retinal vascular diseases such as diabetic retinopathy. Therefore, a clinically feasible method of assessing vascular function may be of significant interest in both research and clinical settings. Recently, in vivo imaging of the retinal vasculature at the capillary level has been made possible by the FDA approval of optical coherence tomography angiography (OCTA), a noninvasive, minimal risk and dyeless angiography method with capillary level resolution. Concurrently, physiological and pathological changes in RVR have been shown by several investigators. The method shown in this manuscript is designed to investigate RVR using OCTA with no need for alterations to the clinical imaging procedures or device. It demonstrates real time imaging of the retina and retinal vasculature during exposure to hypercapnic or hyperoxic conditions. The exam is easily performed with two personnel in under 30 min with minimal subject discomfort or risk. This method is adaptable to other ophthalmic imaging devices and the applications may vary based on the composition of the gas mixture and patient population. A strength of this method is that it allows for an investigation of retinal vascular function at the capillary level in human subjects in vivo. Limitations of this method are largely those of OCTA and other retinal imaging methods including imaging artifacts and a restricted dynamic range. The results obtained from the method are OCT and OCTA images of the retina. These images are amenable to any analysis that is possible on commercially available OCT or OCTA devices. The general method, however, can be adapted to any form of ophthalmic imaging.

This article describes a method for measuring retinal vasculature reactivity in vivo with human subjects using a gas breathing provocation technique to deliver vasoactive stimuli while acquiring retinal images.

光相一致性断层扫描血管造影评估的视网膜血管反应

The vascular supply to the retina has been shown to dynamically adapt through vasoconstriction and vasodilation to accommodate the metabolic demands of ...

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their activity in vivo in behaving mice. This paper describes a method for the implantation of a chronic cranial window to allow for the longitudinal imaging of brain cells in awake, head-restrained mice. In combination with genetic strategies and viral injections, it is possible to label specific cells and regions of interest with structural or physiological markers. This protocol demonstrates how to combine viral injections to label neurons in the vicinity of GCaMP6-expressing astrocytes in the cortex for simultaneous imaging of both cells through a cranial window. Multiphoton imaging of the same cells can be performed for days, weeks, or months in awake, behaving animals. This approach provides researchers with a method for viewing cellular dynamics in real time and can be applied to answer a number of questions in neuroscience.

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

Presented here is a protocol for the implantation of a chronic cranial window for the longitudinal imaging of brain cells in awake, head-restrained mice.

植入颅窗以在清醒小鼠 中重复体内 成像

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their ...