我们感谢少明路和哈里斯答：格尔巴德蠕动泵和vibratome的使用，以及在电子显微镜下研究的罗切斯特大学医学中心为技术援助的核心设施，克伦L. Bentley和盖尔施耐德。这项工作是由来自美国国立卫生研究院（EY019277），白厅基金，宝来惠康基金，和艾尔弗雷德斯​​隆基金会的AKM的资助。 M.-È.T.是由一个全宗RECHERCHE EN桑特魁北克（FRSQ）博士后奖。

1。动物灌注<ol><li>在灌注前一天，准备： - 2 L的磷酸盐缓冲液（PB，100毫米，pH值7.4）和1 L的磷酸钠缓冲液（PBS，0.9％NaCl的50 mM的PB，pH值7.4），双蒸水。存储PB的PBS和1L在4 ° C。这些将被用于灌注和预嵌入免疫细胞化学。 - 1L的4.0％多聚甲醛固定液（煤灰，pH值7.4），这将使灌注6只小鼠。为此，升温1 PB的大号下通风柜。称取40克颗粒多聚甲醛（PFA）的PB液倒入60 ° C，当它到达当该解决方案是明确的，与煤灰完全溶解，冷却至室温（RT）和储存于4 ° C。 </li><li>在灌注的日子，准备在PB 3.5％，丙烯醛的解决方案通风柜下500毫升。然后，过滤器的3.5％丙烯醛和4.0％煤灰的解决方案，用滤纸。 </li><li>对于鼠标麻醉，进入腹膜注入戊巴比妥钠（80毫克/公斤），与一个27号半“针。 </li><li>在灌注，50毫升的PBS，丙烯醛，75毫升和150毫升煤灰将依次传递到鼠标流通。要成立的蠕动泵，填补用PBS管，一端固定在了23号¾“蝴蝶针。油管的另一端浸入灌注液（PBS，丙烯醛或PFA）。蠕动的速度设置泵为20-25毫升/分少年和成年小鼠。整个灌注，小心地避免管路中的空气任何气泡形成。 </li><li>等待，直到不再响应痛苦的刺激，如一条尾巴捏，麻醉鼠标，然后再继续。在解剖盘莱动物和修复使用磁带的爪子。镊子和剪刀，打开皮肤和胸腔，暴露心脏。为了减少脑缺血再灌注的需求将迅速启动。小剪刀剖开右心房，并开始蠕动泵。用镊子按住心脏，左心室心尖的​​蝴蝶针插入。 </li><li>变化的解决方案时，停止蠕动泵，油管传输到另一个从一个解决方案，并立即重新启动泵。 </li><li>当150毫升煤灰的已经过去了，停止蠕动泵。使用小剪刀，切断头，开放的皮肤，并打破两眼之间的头骨。使用小镊子，小心关闭小块头骨芯片，直到大脑可以很容易地删除。后修复2个小时的大脑在4 °彗星在煤灰。在PBS洗3次，每次10分钟。 </li><li>立即切断在大脑中的横向冷却冰的PBS（50微米厚的部分）使用vibratome。用细刷，转成玻璃小瓶含有PBS的免疫细胞化学选定的部分。存放在冷冻保护剂-20 ° C（30％的乙二醇和30％甘油的PBS）在长达数年的其余部分。 </li></ol> 2。预嵌入免疫细胞化学<ol><li>免疫细胞化学，部分处理在玻璃小瓶自由浮动。整个过程中，需要小心避免让部分干出来的。 </li><li>首先，使用移液管取出PBS，并立即更换新鲜的溶液与0.1％PBS中的钠硼氢化物在室温下30分钟。 </li><li>冲洗用PBS 3次10分钟的部分，清除所有气泡，并在室温下2小时孵育的PBS阻断含有0.5％的明胶和5％在二级抗体生成的动物的正常血清（正常山羊血清中的解决方案在目前的Iba1免疫的例子）。 </li><li>在封闭液的主要抗体孵育48小时。 Iba1，免疫，用兔抗Iba1抗体（1:1000）。 </li><li>冲洗，用PBS 3次10分钟的部分，并以生物素标记的第二抗体（1:1000）孵育阻塞解决在室温下2小时。 Iba1，免疫，使用羊抗兔的IgG。 </li><li>冲洗干净，用PBS 3次10分钟的部分和链霉亲和素辣根过氧化物酶（1:1000）孵育阻断在室温下1小时的解决方案。 </li><li>冲洗用PBS 10分钟和部分用Tris /盐酸缓冲液（TBS电视台50毫米，pH值7.4），2次10分钟。为了揭示的标签，孵化，与0.05％民建联和0.01％，在TBS的过氧化氢（溶液新鲜配制）的部分。当染色黑褐色（见图1中的例子），停止反应的TBS冲洗。在任何情况下，最多5分钟后结束反应。冲洗用TBS 10分钟，用PB 2次，每次10分钟的部分。 </li></ol> 3。处理电子显微镜<ol><li>部分转移到PB中一个多孔培养板用细刷。福明引擎盖下一个准备1％锇在PB中的四氧化的解决方案。取出培养板井PB和<html>传播的部分，用细刷持平。立即浸入1％为30分钟，在室温下锇部分。 </li><li>转移到玻璃小瓶部分用PB 3次，每次10分钟，冲洗干净，并通过2分钟浸入脱水升序乙醇浓度35％的2倍，1次50％，70％，80％，90％，和95 ％，100％的3倍，3倍环氧丙烷。 </li><li>要准备的Durcupan树脂，结合20克的组件A，B组份为20克，0.6克组件C，0.4克的一次性烧杯ð组件到，拌匀，直到颜色变得均匀，并成为一个铝倒权衡菜。如果需要，可以制备树脂块涌入嵌入模具，并在55 ° C的烤箱48-72小时固化树脂。 </li><li>用细刷与环氧丙烷冲洗，到Durcupan浸渍树脂移除部分环氧丙烷和沉浸在一夜之间，在室温下。 </li><li>翌日，把铝的重量为55 ° C的烤箱10分钟，以软化树脂的菜。使用刷子用环氧丙烷，大衣ACLAR嵌入薄膜的树脂薄层冲洗，放下的部分，与另一嵌入薄膜覆盖，并均匀分布轻的重量（约2 g，例如玻璃瓶，塑料瓶盖）在上面，以帮助树脂蔓延。树脂聚合，培育在48-72小时（在55 ° C）的烤箱。 </li><li>聚合后，取出轻重量和嵌入电影节的顶部。在双目显微镜下，选择感兴趣的方形区域（约2 × 2毫米）和仔细的消费，从他们使用刀片的薄膜。 </li><li>粘在树脂使用的强力胶块尖端领域的兴趣和在55 ° C烘箱固化1小时。 </li><li>在准备为切片，修剪树脂块用刀片的等腰梯形的形状。 </li><li>使用ultramicrotome和玻璃刀，去除组织表面的胶和树脂。填充船的玻璃用双蒸水刀和切几半薄切片（0.5-1微米厚）。 </li><li>的部分转移到一个完美的循环SuperFrost幻灯片。干放置在加热板在80 ° C，1分钟的幻灯片部分。封面的0.1％甲苯胺蓝染色（2克硼酸钠和0.2克甲苯胺蓝在200毫升双蒸水）1分钟几滴。用双蒸水冲洗多余的污渍。与光学显微镜的部分考试将启用树脂（图2），以区别于染色组织。 </li><li>继续切割组织和树脂之间的边界，直到达到中间部分。注意：下面的过程，需要培训。更换玻璃用钻石刀的刀，并填写用双蒸水船。切断银银金超薄切片（60-80纳米厚），并认真收集铜网，用细倒镊子电网。干滤纸上的网格。 </li><li>为了提高对比度，超薄切片，可染色柠檬酸铅（0.03克柠檬酸铅和0.1毫升10毫升双蒸水1 10N氢氧化钠）。使用罚款倒镊子，转移到2分钟，柠檬酸铅下降一格。删除电网和微妙连续3双蒸水浴场冲洗。最后，干燥的滤纸上的电网，并存储在网格中，直到电子显微镜检查（图3-6）。 </li></ol> 4。代表性的成果<img src="/files/ftp_upload/2021/2021fig1.jpg" alt="图1" /> 图1。Iba1染色在光镜水平的部分。 Iba1蜂窝分布仅限于小胶质细胞，这表明，特异性的免疫。比例尺= 100微米。 <img src="/files/ftp_upload/2021/2021fig2.jpg" alt="图2" /> 图2，甲苯胺蓝染色半薄切片在光镜下水平。注意组织和树脂之间的边界将出现在电子显微镜的免疫最激烈的。 <img src="/files/ftp_upload/2021/2021fig3.jpg" alt="图3" /> 图3。超薄切片显示在电子显微镜水平Iba1免疫阳性胶质细胞perikaryon和流程。 Iba1阳性结构要素是公认的免疫过氧化物酶- DAB电子致密沉淀。比例尺= 2微米。 <img src="/files/ftp_upload/2021/2021fig4.jpg" alt="图4" /> 图4。超薄切片显示在电子显微镜水平Iba1小胶质细胞免疫反应过程的不同大小和形状。比例尺= 2微米。 E 5“/&gt; 图5。超薄切片显示在更高的放大倍率从图4中的一个Iba1阳性的小胶质细胞的过程，使细胞器的识别。比例尺= 1微米。 <img src="/files/ftp_upload/2021/2021fig6.jpg" alt="图6" /> 图6。超薄切片揭示在更高的放大倍率之间Iba1阳性小胶质细胞的过程中和附近的神经纤维元素，包括突触的超微结构的关系。比例尺=1μm的。

<ol>
	<li>Venable, J. H., Coggeshall, 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+simplified+lead+citrate+stain+for+use+in+electron+microscopy.">A simplified lead citrate stain for use in electron microscopy.</a> J Cell Biol. 25, 407-407 (1965).</li><li>Riad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somatodendritic+localization+of+5-HT1A+and+preterminal+axonal+localization+of+5-HT1B+serotonin+receptors+in+adult+rat+brain.">Somatodendritic localization of 5-HT1A and preterminal axonal localization of 5-HT1B serotonin receptors in adult rat brain.</a> J Comp Neurol. 417 (2), 181-181 (2000).</li><li>Tremblay, 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=Localization+of+EphA4+in+axon+terminals+and+dendritic+spines+of+adult+rat+hippocampus.">Localization of EphA4 in axon terminals and dendritic spines of adult rat hippocampus.</a> J Comp Neurol. 501 (5), 691-691 (2007).</li><li>Tremblay, 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=Developmental+course+of+EphA4+cellular+and+subcellular+localization+in+the+postnatal+rat+hippocampus.">Developmental course of EphA4 cellular and subcellular localization in the postnatal rat hippocampus.</a> J Comp Neurol. 512 (6), 798-798 (2009).</li><li>Bouvier, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-synaptic+and+post-synaptic+localization+of+EphA4+and+EphB2+in+adult+mouse+forebrain.">Pre-synaptic and post-synaptic localization of EphA4 and EphB2 in adult mouse forebrain.</a> J Neurochem. 106 (2), 682-682 (2008).</li><li>Bouvier, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EphA4+is+localized+in+clathrin-coated+and+synaptic+vesicles+in+adult+mouse+brain.">EphA4 is localized in clathrin-coated and synaptic vesicles in adult mouse brain.</a> J Neurochem. , (2010).</li></ol>

在这里，我们描述了小鼠脑组织的准备，免疫电镜，最佳的超微结构的保存及免疫细胞化学检测时严格执行程序之间提供了一个很好的折衷协议。 用透射电子显微镜相结合，这种方法可以区分具有几个鲜明的特点和鉴定标准的蜂窝元素。特别是，Iba1免疫过氧化物酶- DAB染色，使确定的小胶质细胞perikarya和流程内的神经纤维，以及分析它们的形态，细胞器超微结构的关系，并?...

*应始终工作在通风橱和磨损丁腈手套时，丙烯醛，多聚甲醛，锇，也应妥善处理其废物。

preparation of mouse brain tissue for immunoelectron microscopy

透射电子显微镜（TEM）是非常有用的可视化的小胶质细胞，oligodendrocytic，星形胶质细胞和神经细胞的亚细胞（树突棘，枝蔓，轴突，轴突终端，perikaryon），以及他们的细胞器和细胞骨架在中枢神经系统，高空间分辨率。预嵌入免疫细胞化学与透射电镜相结合，允许具有几个鲜明的特点和鉴定标准（例如，小胶质细胞perikarya和流程，当使用一种抗体对小胶质细胞特异性标志物Iba1（钙离子结合适配器分子1的细胞成分的歧视;提出在这里）），识别细胞分子神经递质的内容（如五羟色胺）和可溶性或膜结合的蛋白质的超微结构定位（例如，5 HT1A和EphA4受体）。在这里，我们描述了协议transcardiac灌注小鼠丙烯醛固定液，拆除和大脑切片，以及免疫过氧化物酶 - 二氨基联苯胺（DAB）染色，树脂包埋，以及超薄的脑切片切片。这些程序完成后，免疫染色的材料是用透射电子显微镜检查的准备。如果严格执行，这种技术之间最佳的超微结构的保存及免疫细胞化学检测提供了一个很好的折衷。

Watch this Scientific Journal Video about Preparation of Mouse Brain Tissue for Immunoelectron Microscopy at JoVE.com

Preparation of Mouse Brain Tissue for Immunoelectron Microscopy

我们描述transcardiac灌注小鼠，拆除和切片的大脑，以及免疫过氧化物酶染色，树脂包埋，和脑切片切片超薄协议。这些程序完成后，免疫染色材料透射电子显微镜检查的准备。

制备小鼠脑组织中的免疫电镜

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments ...

preparation-of-mouse-brain-tissue-for-immunoelectron-microscopy

Research

JoVE Journal

Neuroscience

36.7K 次观看.  University of Rochester. 我们描述transcardiac灌注小鼠，拆除和切片的大脑，以及免疫过氧化物酶染色，树脂包埋，和脑切片切片超薄协议。这些程序完成后，免疫染色材料透射电子显微镜检查的准备。

视频: Preparation of Mouse Brain Tissue for Immunoelectron Microscopy

Focussed Ion Beam Milling and Scanning Electron Microscopy of Brain Tissue

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron microscope (FIB/SEM). The samples are fixed with aldehydes, heavy metal stained using osmium tetroxide and uranyl acetate. They are then dehydrated with alcohol and infiltrated with resin, which is then hardened. Using a light microscope and ultramicrotome with glass knives, a small block containing the region interest close to the surface is made. The block is then placed inside the FIB/SEM, and the ion beam used to roughly mill a vertical face along one side of the block, close to this region. Using backscattered electrons to image the underlying structures, a smaller face is then milled with a finer ion beam and the surface scrutinised more closely to determine the exact area of the face to be imaged and milled. The parameters of the microscope are then set so that the face is repeatedly milled and imaged so that serial images are collected through a volume of the block. The image stack will typically contain isotropic voxels with dimenions as small a 4 nm in each direction. This image quality in any imaging plane enables the user to analyse cell ultrastructure at any viewing angle within the image stack.

This protocol describes how resin embedded brain tissue can be prepared and imaged in the three dimensions in the focussed ion beam, scanning electron microscope.

聚焦离子束铣削和脑组织的扫描电子显微镜

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron ...

科研

神经科学

Multiphoton Microscopy of Cleared Mouse Brain Expressing YFP

Multiphoton microscopy of intrinsic fluorescence and second harmonic generation (SHG) of whole mouse organs is made possible by optically clearing the organ before imaging.1,2 However, for organs that contain fluorescent proteins such as GFP and YFP, optical clearing protocols that use methanol dehydration and clear using benzyl alcohol:benzyl benzoate (BABB) while unprotected from light3 do not preserve the fluorescent signal. The protocol presented here is a novel way in which to perform whole organ optical clearing on mouse brain while preserving the fluorescence signal of YFP expressed in neurons. Altering the optical clearing protocol such that the organ is dehydrated using an ethanol graded series has been found to reduce the damage to the fluorescent proteins and preserve their fluorescent signal for multiphoton imaging.4 Using an optimized method of optical clearing with ethanol-based dehydration and clearing by BABB while shielded from light, we show high-resolution multiphoton images of yellow fluorescent protein (YFP) expression in the neurons of a mouse brain more than 2 mm beneath the tissue surface.

Multiphoton microscopy of whole mouse organs is possible by optically clearing the organ before imaging, but not all protocols preserve the fluorescent signal of fluorescent proteins. Using an optical clearing method with ethanol-based dehydration and benzyl alcohol:benzyl benzoate clearing, we show high-resolution multiphoton images of whole mouse brain expressing YFP.

多光子显微镜的清除小鼠脑组织表达YFP

Multiphoton microscopy of intrinsic fluorescence and second harmonic generation (SHG) of whole mouse organs is made possible by optically clearing the ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

小脑颗粒神经元的遗传操作<em&gt;体外</em&gt;和<em&gt;在体内</em&gt;为研究神经元形态和迁移

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

嗅觉系统作为模型来研究轴突生长的模式和形态<em&gt;在体内</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

Preparation of Non-human Primate Brain Tissue for Pre-embedding Immunohistochemistry and Electron Microscopy

Despite all the technological advances at the light microscopy&#160;level, electron microscopy remains the only tool in neuroscience to examine and characterize ultrastructural and morphological details of neurons, such as synaptic contacts. Good preservation of brain tissue for electron microscopy can be obtained by rigorous cryo-fixation methods, but these techniques are rather costly and limit the use of immunolabeling, which is crucial to understand the connectivity of identified neuronal systems. Freeze-substitution methods have been developed to allow the combination of cryo-fixation with immunolabeling. However, the reproducibility of these methodological approaches usually relies on costly freezing devices. Moreover, achieving reliable results with this technique is very time-consuming and skill-challenging. Hence, the traditional chemically fixed brain, particularly with acrolein fixative, remains a time-efficient and low-cost method to combine electron microscopy with immunohistochemistry. Here, we provide a reliable experimental protocol using chemical acrolein fixation that leads to the preservation of primate brain tissue and is compatible with pre-embedding immunohistochemistry and transmission electron microscopic examination.

Here, we provide an easy, low-cost, and time-efficient protocol to chemically fix primate brain tissue with acrolein fixative, allowing for long-term preservation that is compatible with pre-embedding immunohistochemistry for transmission electron microscopy.

非人类灵长类动物脑组织的制备预嵌入免疫组化和电子显微镜

Despite all the technological advances at the light microscopy&#160;level, electron microscopy remains the only tool in neuroscience to examine and ...

Combined Mechanical and Enzymatic Dissociation of Mouse Brain Hippocampal Tissue

This neural dissociation protocol (an adaptation of the protocol accompanying a commercial adult brain dissociation kit) optimizes tissue processing in preparation for detailed downstream analysis such as flow cytometry or single-cell sequencing. Neural dissociation can be conducted via mechanical dissociation (such as using filters, chopping techniques, or pipette trituration), enzymatic digestion, or a combination thereof. The delicate nature of neuronal cells can complicate efforts to obtain the highly viable, true single-cell suspension with minimal cellular debris that is required for single-cell analysis. The data demonstrate that this combination of automated mechanical dissociation and enzymatic digestion consistently yields a highly viable (&gt;90%) single-cell suspension, overcoming the aforementioned difficulties. While a few of the steps require manual dexterity, these steps lessen sample handling and potential cell loss. This manuscript details each step of the process to equip other laboratories to successfully dissociate small quantities of neural tissue in preparation for downstream analysis.

This neural cell dissociation protocol is intended for samples with a low amount of starting material and yields a highly viable single-cell suspension for downstream analysis, with optional fixation and staining steps.

小鼠脑海马组织机械和酶解离联合

This neural dissociation protocol (an adaptation of the protocol accompanying a commercial adult brain dissociation kit) optimizes tissue processing in ...

Deep-Tissue Three-Photon Fluorescence Microscopy in Intact Mouse and Zebrafish Brain

Multiphoton microscopy techniques, such as two-photon microscopy (2PM) and three-photon microscopy (3PM), are powerful tools for deep-tissue in vivo imaging with subcellular resolution. 3PM has two major advantages for deep-tissue imaging over 2PM that has been widely used in biology laboratories: (i) longer attenuation length in scattering tissues by employing ~1,300 nm or ~1,700 nm excitation laser; (ii) less background fluorescence generation due to higher-order nonlinear excitation. As a result, 3PM allows high-contrast structural and functional imaging deep within scattering tissues such as intact mouse brain from the cortical layers to the hippocampus and the entire forebrain of adult zebrafish.
Today, laser sources suitable for 3PM are commercially available, enabling the conversion of an existing two-photon (2P) imaging system to a three-photon (3P) system. Additionally, multiple commercial 3P microscopes are available, which makes this technique readily available to biology research laboratories. This paper shows the optimization of a typical 3PM setup, particularly targeting biology groups that already have a 2P setup, and demonstrates intravital 3D imaging in intact mouse and adult zebrafish brains. This protocol covers the full experimental procedure of 3P imaging, including microscope alignment, prechirping of ~1,300 and ~1,700 nm laser pulses, animal preparation, and intravital 3P fluorescence imaging deep in adult zebrafish and mouse brains.

Three-photon microscopy enables high-contrast fluorescence imaging deep in living biological tissues, such as mouse and zebrafish brains, with high spatiotemporal resolution.

完整小鼠和斑马鱼大脑的深组织三光子荧光显微镜

Multiphoton microscopy techniques, such as two-photon microscopy (2PM) and three-photon microscopy (3PM), are powerful tools for deep-tissue in vivo ...

Whole-Brain Single-Cell Imaging and Analysis of Intact Neonatal Mouse Brains Using MRI, Tissue Clearing, and Light-Sheet Microscopy

Tissue clearing followed by light-sheet microscopy (LSFM) enables cellular-resolution imaging of intact brain structure, allowing quantitative analysis of structural changes caused by genetic or environmental perturbations. Whole-brain imaging results in more accurate quantification of cells and the study of region-specific differences that may be missed with commonly used microscopy of physically sectioned tissue. Using light-sheet microscopy to image cleared brains greatly increases acquisition speed as compared to confocal microscopy. Although these images produce very large amounts of brain structural data, most computational tools that perform feature quantification in images of cleared tissue are limited to counting sparse cell populations, rather than all nuclei.
Here, we demonstrate NuMorph (Nuclear-Based Morphometry), a group of analysis tools, to quantify all nuclei and nuclear markers within annotated regions of a postnatal day 4 (P4) mouse brain after clearing and imaging on a light-sheet microscope. We describe magnetic resonance imaging (MRI) to measure brain volume prior to shrinkage caused by tissue clearing dehydration steps, tissue clearing using the iDISCO+ method, including immunolabeling, followed by light-sheet microscopy using a commercially available platform to image mouse brains at cellular resolution. We then demonstrate this image analysis pipeline using NuMorph, which is used to correct intensity differences, stitch image tiles, align multiple channels, count nuclei, and annotate brain regions through registration to publicly available atlases.
We designed this approach using publicly available protocols and software, allowing any researcher with the necessary microscope and computational resources to perform these techniques. These tissue clearing, imaging, and computational tools allow measurement and quantification of the three-dimensional (3D) organization of cell-types in the cortex and should be widely applicable to any wild-type/knockout mouse study design.

This protocol describes methods for conducting magnetic resonance imaging, clearing, and immunolabeling of intact mouse brains using iDISCO+, followed by a detailed description of imaging using light-sheet microscopy, and downstream analyses using NuMorph.

使用MRI，组织清除和光片显微镜对完整的新生小鼠大脑进行全脑单细胞成像和分析

Tissue clearing followed by light-sheet microscopy (LSFM) enables cellular-resolution imaging of intact brain structure, allowing quantitative analysis of ...

上丘的双光子和宽场钙成像

Calcium Imaging in Mouse Superior Colliculus

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the emergence of the cerebral cortex. It receives direct inputs from ~30 types of retinal ganglion cells (RGCs), with each encoding a specific visual feature. It remains elusive whether the SC simply inherits retinal features or if additional and potentially de novo processing occurs in the SC. To reveal the neural coding of visual information in the SC, we provide here a detailed protocol to optically record visual responses with two complementary methods in awake mice. One method uses two-photon microscopy to image calcium activity at single-cell resolution without ablating the overlaying cortex, while the other uses wide-field microscopy to image the whole SC of a mutant mouse whose cortex is largely undeveloped. This protocol details these two methods, including animal preparation, viral injection, headplate implantation, plug implantation, data acquisition, and data analysis. The representative results show that the two-photon calcium imaging reveals visually evoked neuronal responses at single-cell resolution, and the wide-field calcium imaging reveals&#160;neural activity across the entire SC. By combining these two methods, one can reveal the neural coding in the SC at different scales, and such combination can also be applied to other brain regions.

作者聚焦：揭示上丘的神经编码和视觉处理机制 

This protocol details the procedure for imaging calcium responses in the superior colliculus (SC) of awake mice, including imaging single-neuron activity with two-photon microscopy while leaving the cortex intact in wild-type mice, and imaging the entire SC with wide-field microscopy in partial-cortex mutant mice.

小鼠上丘的钙成像

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the ...