这项工作是支持由美国国立卫生研究院授予2 R15 GM061539-02（以BAS），2 R15 NS40425-03（对JEL），MH 66179（俄勒冈健康与科学大学/ OHSU的银行家加里博士），和P30 NS061800（以OHSU的苏艾舍博士）。我们感谢芭芭拉Smoody与海马神经元的文化的广泛支持，和Drs。布赖恩长和詹姆斯阿布尼这个手稿的批判性阅读。

1，样品制备<ol><li>制备DNA编码靶向DCV值，使用标准分子生物学技术的photoconvertible或光活化的嵌合体。 注意:一种可能性是DNA编码一个组织型纤溶酶原活化剂 - Dendra2嵌合体（TPA-Dendra2）。 Dendra2是一个photoconvertible蛋白质切换由绿暴露于紫外线12红光发射。 </li><li>高性能＃1.5封面文化的海马神经元的滑落〜5-10天，下面的标准协议13。 </li><li>转染开发的海马神经元使用阳离子脂质体试剂。 注:病毒介导的感染是更有效的成熟神经元。这种替代方法已被详细描述在别处14。 <ol><li>制备1.5ml试管含有〜1微克DNA与250μl的最低必需培养基（MEM）和第二1.5毫升管含有4微升阳离子脂质试剂和250微升的MEM。 </li><li>孵育5分钟的解决方案。 </li><li>结合上述两种溶液并孵育另外的30分钟，所得混合物。 </li><li>在孵育，制备含几毫升培养基的培养皿加热至37℃。 </li><li>含有神经元的盖玻片轻轻地转移到含中等温暖的菜，并加入转染混合物。 </li><li>倾斜的菜轻轻地，然后将其放置在孵化器在37℃90分钟。 </li><li>回到神经元的"家菜"，等待〜12-18小时。 </li></ol></li><li>固定细胞45分钟，在磷酸盐缓冲盐水中的4％低聚甲醛/ 4％蔗糖（PBS，pH 7.4）中加热到37℃。 注意:在PALM实验，以达到良好的保存超微结构的是重要的，并用于免疫荧光染色标准甲醛固定协议通常是不足够15。对贫困的结构性保存的重要原因之一为f不足ixation时间，因为甲醛固定涉及加合物的形成和蛋白质的交联，而后者的过程是相当慢16。一个45分钟的固定有助于与荧光检测不到overfixation引起的损失减轻这个问题。 </li><li>洗盖玻片〜10倍带过滤的PBS。 注意:这降低了荧光污染物。图像样本相对快速地固定后。在一个不透光的容器中期，商店细胞在4℃过滤的PBS。 </li><li>摩盖玻片含在过滤PBS一腔神经元。 注:样品必须安装在水性介质中，如PBS中，用折射率比玻璃低，以实现全内反射荧光（TIRF）为基础的照明。 TIRF为基础的照明是非常有用的，因为只有盖玻片近端荧光团被激发，并且在背景荧光17大相关的下降。 </li></ol> 2，图像采集&lt;/ P&gt; <ol><li>把超分辨率成像系统上。 <ol><li>开启电弧灯。 </li><li>翻转"系统/ PC"和"组件"开关（电源遥控开关）为"on"。 </li><li>打开计算机（显微镜控制平板电脑/扩展坞后亮起）。 注意:该基座可被用于控制和监测的光学和光路的许多属性，尤其是所选择的目标和焦点。 </li><li>双击软件图标，并选择在登录对话框中的"启动系统"选项。 </li></ol></li><li>检查样本。 <ol><li>打开对激光安全柜正面和顶部检修面板。 </li><li>倾斜透射光的手臂访问阶段和目标。 </li><li>放油的阿尔法计划复消色差透镜100X数值孔径/ NA = 1.46（TIRF）的目标。 注:63X高数值孔径的物镜可以代替100X的使用。 </li><li>安装在舞台上的样品，并提高第Ë目标朝向样品。 </li><li>监视使用对接站的XYZ功能的透镜位置。停止当镜头处于焦点的球场（ 如 Z〜2.50毫米）。 </li><li>完全关闭检修面板。 注意:要从事激光安全。 </li><li>微调使用的定位选项卡中的目镜和按钮的焦点。 注:定位标签可以访问产生弧光灯的照明度，便于直接观察样品与目镜按钮。同样，收购选项卡提供了对生产基于激光的照明和相机有利于图像捕捉工具。 <ol><li>转到查找选项卡，选择"跨开。" </li><li>点击眼部扩张的象征。 注:这带来了光路的示意图。光路的属性可以通过左击在原理图中相应的图标或通过使用触摸屏基座上的改变。例如，一个过滤器适合于从Dendra2观看发射可以使用反射左轮手枪图标来选择。 </li><li>将眼睛对准目镜，以及使用该基座为指导使细胞成为关注焦点。 注:样本会很人烟稀少的荧光细胞，因为海马神经元都很难转染，并因此它可以是一个有点棘手使用反射照明集中。如果是这种情况，可使用透射照明和对接站的z读数作为一个指南。 </li><li>对焦后切换光"关"。 </li></ol></li></ol></li><li>识别电池，它是相当明亮的，具有点状荧光，并拥有经典的形态是反映身体健康。 <ol><li>选择反射光和过滤器适用于观看Dendra2绿色发光。 </li><li>扫描盖玻片的细胞使用低强度的激发光表达Dendra2嵌合体。 </li></ol></li><li>切换到ACQuisition标签。 <ol><li>使用"实验管理器"来加载适当设计或简单地修改了收购Palm的配置。 注意:如果没有这样的实验存在，使用以下设置作为指导设计开始实验配置:激光功率滑块:405纳米（〜为50毫瓦激光0.01％）; 488纳米（〜为100毫瓦的激光3％）; 561纳米（〜40％为100毫瓦激光）;曝光时间:约50毫秒;相机增益:〜200;多维采集:时间序列（周期= 30,000，间隔= 0）;专辑曲目:488 nm处落射照明，和561 nm的全内反射荧光为基础的照明（见步骤2.5.1）;目的:100X（NA = 1.46）; TIRF角度滑块:入射角〜67-72°;像素尺寸:100纳米;视场角:全内反射荧光（替代选择产生更高的激光强度和用于荧光团更难以漂白）。 </li><li>点击"是"时，弹出菜单会出现一个询问关于激光器的开关。 </li></ol></li><li>把图像相机平面细胞成焦点。 <ol><li>选择488 nm的落射照明激光跟踪。 注:曲目是在成像过程中使用的光束配置。这里，轨迹名称是由能产生被传递给相机的发射激发波长测定。例如，561纳米的轨道使用405和561 nm的激光线两者，但滤波器立方体通过从光转换Dendra2通过561纳米的光的相机激发仅发射。 </li><li>专注于细胞的图像使用"连续"采集按钮在相机上。 </li></ol></li><li>收集传统的宽视场图像。 <ol><li>使用"快照"按钮。 </li><li>通过转到"文件"菜单，选择保存图像"保存/另存为"。 </li></ol></li><li>成立了全内反射荧光为基础的照明。 <ol><li>选择488轨道，切换到使用EPI / TIRF按钮"全内反射荧光"。 </li><li>选择"连续"采集。 </li><li>调节吨他TIRF为基础的照明滑块。 注:当存在的背景突然变暗和试样可以被聚焦在一个平面18 TIRF的实现。如果新的功能通过向上聚焦到样品变得明显，入射角是太低了，因为在TIRF只有一个平面上聚焦。 </li><li>使用561轨道与405 nm激光仔细检查TIRF角度"关闭"。 </li><li>打开405 nm激光背"上"推动PALM实验前。 </li></ol></li><li> PALM收集数据。 <ol><li>点击"开始实验。" 注:如果需要，打开"在线处理选项"选项卡，勾选"联机处理掌"和"飞度Gauss2D"（在实验开始前）的原始数据收集过程中监视重建手掌图像。这将有利于数据的质量和持续的相对耗时的数据收集过程的这样的值的评估。 </li><li>视觉监测光转化。 </li><li>增加了405纳米（photoconverting）激光的强度时Dendra2光转化减退（一般收购〜10,000原始图像之后）。 </li><li>如果光转化增幅继续实验。否则，通过点击"停止"当前步骤按钮停止实验（无数据丢失）。 </li><li>保存图像时，数据收集完成后（后〜15分钟）。 </li></ol></li></ol> 3，图像处理，显示和分析<ol><li>在没有被保存到磁盘的在线处理，处理原始图像。 <ol><li>单击处理选项卡上，打开方式选项卡，然后选择"掌"。 </li><li>从四个子选项再次选择​​"掌"。 </li><li>转到文件菜单，打开并查看感兴趣的文件，并点击"选择"。 </li><li>点击"应用"。 注:这将启动峰值/荧光发现和峰值本地化默认OPTI附件。如果有必要，峰也可以使用"PAL-组"选项卡分组。分组分配中出现的多个连续的帧，以一个单一的分子，如果峰值坐标是足够相似的峰。此外，该数据可以使用该软件的基于模型的漂移对齐选项样品漂移进行修正。 </li></ol></li><li>筛选出的峰值本地化数据不佳本地化和采样不足的成分。 <ol><li>丢弃荧光本地化精度，σ&gt;使用"PAL-过滤器"的工具35纳米。 </li><li>丢弃荧光的地区，山峰，D之间的平均距离，过大解决的直径，D的囊泡<ol><li>提取的平均定位精度，σ，从定位精度直方图。 </li><li>计算在d基于σ最大可接受值，香农-奈奎斯特criteron 19，所体现的分辨率逼近20 R = D =[（2.35σ）2 +（2D）2] 1/2。 </li><li>半径D / 2的圆内，在"删除Palm离群"工具设置的门槛，删除了峰比πD2 /（4D 2）较少的邻居包围。 </li></ol></li><li>处理过的PALM数据转换成使用"PAL-渲染"工具手掌图像;参见图1。 </li><li>通过选择"配置文件"功能，量化的大小和分离。 <ol><li>检查关联的行和表格选项，并画出沿图像中的所希望的方向的线。 </li><li>通过确定全宽半最​​大强度量化直径。 </li><li>通过确定的强度分布的峰 - 峰间距量化分离。 </li></ol></li></ol></li></ol>

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作者什么都没有透露。

Palm和相关超分辨率荧光显微技术，最近成为一个有价值的补充，以更好地建立表格光学显微镜和电子显微镜（EM）22-24。 PALM的积极属性包括相对简单的样品制备和最小的样品扰动。原则上，PALM也可以用于研究活细胞。大概PALM的主要负属性是费时的数据采集，从而阻碍了显著更快的动力学过程研究在活细胞中。此外，Palm是相对较新，因此适当的，稳健的荧光基团和样品制备和数据采集和分析的协议没有得到充分开发和/或记录3。 在这里，我们描述了适合于单色，基于Palm的固定，培养细胞囊泡研究的协议。该协议也是一个很好的起点，同样的发展定向，双色PALM研究。本协议的效力是由我们的PALM数据的两个关键属性的支持。首先，泪点在我们的重建Palm和互补的广角，图像中的分布非常相似。在分配一个显著的区别 - 偶尔的分辨率衍射极限的泪点进入泪点多由PALM - PALM反映的显着增强分辨率，并进一步强调了技术的有效性和实用性。 其次，我们的PALM结果有较好的一致性，和/或一致的，与其他相关的结果。例如，用EM获得的海马神经元的图象偶然揭示切片通过DCV值，并且直流电压的直径从切片推断是〜70-100毫微米11。我们PALM的数据和EM数据之间的显着协议是令人欣慰的两种方法。 PALM仍正在测试，和EM一般须了解必要的苛刻样品制备的关注。同样，活海马神经元同时表达有针对性地DCV值绿色和红色的嵌合体衍射极限的电影显示，重叠的绿色和红色的泪点进行持续的联动25。造成这一结果的最直接的解释 - 红色和绿色信号从同一DCV出现 - 是与Palm，这表明衍射极限的泪点绝大多数代表个人DCV值一致。联动的那么简单解释 - 红色和绿色信号从集群DCV值出现 - 在很大程度上是无效的棕榈。 这里所描述的协议有其他积极的属性。特别地，样品制备是相对简单和相当类似的适合于常规荧光显微术，有两个明显的例外:（1）囊泡必须贴有photoconvertible或光活化的荧光团，以及（2）固定的细胞最好安装在水性介质中。这种相对simpliciTY反映了一个事实，即实验很短，因而样品不需要进行标记与基准珠（以方便漂移修正），它可以是棘手26。另外，水疱通常的标签有许多荧光;因此，实现充分的荧光标记的密度非常简单。与此相反，对于其他结构，这可能是非常困难的3。图像采集也相对比较简单。然而，PALM确实需要电子和光学组件是昂贵的，尖端的，因为PALM信号本质上是非常弱的。 PALM中心的周围的处理，分析和PALM图像的显示可能是最复杂的方面。如这里强调，实施PALM这些方面需要几个微妙的问题，包括分辨率和定位精度之间采样和分辨率之间的关系，并区分的理解。因此，总体而言，PALM studies不约而同地都相当复杂，但这是由分辨率增强，很可能是显著平衡。

许多细胞过程，依赖于生物分子的准确，高效的囊泡介导拐卖到特定的亚细胞的目的地。一个突出的例子是突触组装，这是之前长程，囊泡介导突触传递的成分从生物合成中的神经元胞体部位潜在的远端前和突触后位点1。 荧光显微镜是一个强大的和流行的研究囊泡运输的方法。该技术的优势包括其敏感性，特异性，并与实时成像2的兼容性。不幸的是，直到最近，这项技术已经遭受一个主要弱点，衍射极限分辨率2，妨碍结构，尺寸比〜250纳米更小的研究。近日，在荧光显微镜的横向分辨率超过了衍射障碍，推出的超分辨率荧光显微显微术技术，诸如PALM 3。 PALM的横向分辨率，几十纳米，非常适合小泡，其中有尺寸，通常介于50〜250 nm的4的研究。因此，这是目前可以使用的荧光，以其无数的优势，阐明囊泡以前无法进入的属性，包括其贩运到特定的亚细胞位点的某些方面的频谱。 PALM是不容易实现，并成功的战略往往必须针对系统所研究的类型。在这里，我们描述了如何实现囊状结构的PALM研究，我们证明我们的DCV值的海马神经元的情况下方法的有效性。特别是，我们使用Palm解决假说DCV值的贩运到海马神经元突触是由DCV集群5-8介导的。 囊泡簇介​​导的贩运在发展Ñ突触eurons是一个有趣的可能性，因为它可以促进突触快速稳定和装配9,10。 DCV值的聚集贩卖的支持者举出突触外荧光泪点外源性窝藏的DCV货物作为证据支持集群6大明显的大小。然而，这些泪点出现在使用衍射限制的荧光显微镜技术，它不适合于从由聚类所产生的因衍射鉴别晶粒尺寸效应所产生的图像。 要解决此问题，我们收集了常规的宽视场荧光和海马神经元表达有针对性地DCV值嵌合体PALM图像。这些图像的分析表明，推定的突触外DCV簇在常规图像中&gt; 92％的被解析为80纳米（个人DCV尺寸）11泪点在PALM图像。因此，这些数据的聚类假设基本上无效应用于DCV值在显影hippocaMPAL神经元。

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		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Zeiss PALM</td>
			<td style="width:169px;">Carl Zeiss, Inc.</td>
			<td style="width:119px;">Elyra PS.1</td>
			<td style="width:572px;">With Zeiss Efficient Navigation (ZEN) software and fluorescence filter Set 77 HE GFP/mRFP/Alexa633&#160;</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:245px;">Lipofectamine 2000 Transfection Reagent</td>
			<td style="width:169px;">Life Technoligies</td>
			<td style="width:119px;">11668-019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Minimum Essential Medium</td>
			<td style="width:169px;">Life Technologies</td>
			<td style="width:119px;">11095-080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Phosphate-Buffered Saline</td>
			<td style="width:169px;">Life Technologies</td>
			<td style="width:119px;">10010049</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:245px;">Paraformaldehyde</td>
			<td style="width:169px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">19208</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Sucrose</td>
			<td style="width:169px;">Sigma-Aldrich</td>
			<td style="width:119px;">S-8501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">growth glass coverslips 18mm 1.5D</td>
			<td style="width:169px;">Fisher Scientific</td>
			<td style="width:119px;">NC0059095</td>
			<td></td>
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	</tbody>
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super-resolution imaging of neuronal dense-core vesicles

<html>荧光的检测提供了在现代生物学和医学应用中采用的许多广泛应用和迅速发展的显微镜技术的基础。荧光的优势包括其敏感性，特异性，并与实时成像的兼容性。不幸的是，荧光显微镜常规形式遭受一个主要弱点，衍射极限分辨率的成像平面，妨碍结构，尺寸比〜250纳米更小的研究。近日，这一限制已被克服，推出超分辨率荧光显微镜技术，如光敏定位显微镜（PALM）。不同于其传统的对应，PALM可以用几十纳米的横向分辨率的影像。因此，这是目前可以使用的荧光，以其无数的优势，阐明细胞结构和组织以前无法进入属性的频谱。 _content"&gt;不幸的是，PALM是不平凡的实施和成功的战略往往必须针对系统所研究的类型。在这篇文章中，我们将展示如何实现固定，培养的神经元囊泡结构的单色PALM研究。 PALM是非常适合的囊泡，它的外形尺寸使通常为50〜250纳米。在我们的方法的关键步骤包括对研究神经元标记与photoconvertible（绿色到红色）的囊泡货物嵌合体，采集稀疏采样的原始图像与超高分辨率显微镜系统，以及处理原始图像以产生一个高分辨率的手掌图像。我们还证明我们的方法的功效通过在培养的海马神经元呈现致密核心囊泡的格外好分辨图像（DCV值），该反驳这一假设的DCV值的突触外贩卖很大程度上是由DCV簇介导的。

图1示出的成像和处理的一个终产物。在此PALM图像，局部荧光团的横向坐标显示使用质心的显示模式，以及相关的直流电压的超分辨率图像是使用高斯显示模式显示。 图2A显示了类似的广角和八天的体外海马神经元表达的tPA-Dendra2的胞体和近端过程PALM图像。图像的重要特征包括（1）广泛的，一对1的对应，和重叠，泪点之间在常规和PALM的图像，（2）显著较小尺寸的PALM泪点，以及一个（3）偶尔分辨率单广角泪成多个PALM泪点。 图2B显示DCV值的手掌图像沿着第二海马神经元表达的tPA-Dendra2的过程的一部分。重要此图像的特征包括（1）小的直径，并均匀的外观，泪点，和（2）不频繁观察密切apposed泪点/推定的直流电压集群。 图3概述了确定两个或更多个DCV值足够接近包括一个簇的一个简单的定量方法;在系统中的聚类是普遍的，更复杂的方法，基于分布函数，可用于定量21。简单的方法是基于生成泪点强度（在图形中示出），它可以被用来量化DCV分离和DCV宽度的线进行访问。如果分离显著超过宽度（如图例中讨论的），本DCV值不"接触"，而如果分离是大致相同的宽度，DCV值可能联系。基于这个标准，在A组的DCV值被列为"不接触"，而那些在B组被列为CON<html>圆通。使用这种方法，我们把一个上界 〜8％，在发展中国家海马神经元突触外DCV值的接触相关的集群。我们的直流电压的大小和簇的数据总结于表1中 。 <img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/51394/51394fig1highres.jpg" src="/files/ftp_upload/51394/51394fig1.jpg" /> 一个直流电压（红色）的高斯呈现PALM形象图1。覆盖和重心显示模式手掌图像显示单个光转换荧光团（白点）中所包含的直流电压坐标。前者模式显示荧光基团与呈现高斯函数通过其定位精度决定的宽度，而后者显示的荧光团/峰为点定位在他们的（X，Y）坐标。酒吧= 0.1微米。T ="_blank"&gt;点击这里查看大图。 <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/51394/51394fig2highres.jpg" src="/files/ftp_upload/51394/51394fig2.jpg" /> 图2:重叠相加广角（绿色）和一个发展中的海马神经元表达的tPA-Dendra2的胞体和近端过程PALM（红色）图像（A）重叠的区域出现黄色/橙色。 DCV值衣料另一小区的表达的tPA-Dendra2的过程的分区域的PALM图像（B）。酒吧= 5和2微米。 <a href="https://www.jove.com/files/ftp_upload/51394/51394fig2highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图3" fo:content-width="5in" fo:src="/files/ftp_upload/51394/51394fig3highres.jpg" src="/files/ftp_upload/51394/51394fig3.jpg" /> 图3。覆盖（<html>总结广角（绿色）和PALM（红色）的泪点中的显影海马神经元表达的tPA-Dendra2图像A和B），在这两种板，宽视场图像显示了一个单一的大泪点，而相关联的手掌图像可解决此单一泪小管成两个更窄泪点。该图显示在面板中的（左图）和B组（右图）获得的泪点类似颜色编码的强度分布。 ;来自手掌图像的面板中的显示，泪点有全宽半最大强度是〜他们的峰 - 峰间距的40％的配置文件因此，这两个泪点不接触，并没有归类为推定的集群。与此相反，在面板B显示了来自于对PALM图像的轮廓，该泪点具有半宽度最大的强度本质上是相同的峰 - 峰间距;因此，这些被归类为一个假定的集群。酒吧= 0.2微米。/ 51394fig3highres.jpg"目标="_blank"&gt;点击这里查看大图。 <table><tbody><tr><td> 参数 </td><td> 结果 </td><td> DCV值分析数 </td></tr><tr><td>上界上聚集DCV值</td><td> 7.9±5.9％ </td><td> 618 </td></tr><tr><td>平均直径DCV </td><td> 77±21 nm的</td><td> 60 </td></tr></tbody></table> 表1。直流电压的大小和簇的数据汇总。

Watch this Scientific Journal Video about Super-resolution Imaging of Neuronal Dense-core Vesicles at JoVE.com

Super-resolution Imaging of Neuronal Dense-core Vesicles

我们描述了如何实现光敏定位显微镜（PALM）为基础的固定，培养的神经元囊泡的研究。我们的协议的关键组件包括标签囊泡photoconvertible嵌合体，采集稀疏采样的原始图像的超分辨率显微镜系统，以及处理原始图像来产生超分辨率图像。

神经元的密集核心囊泡的超分辨率成像

Detection of fluorescence provides the foundation for many widely utilized and rapidly advancing microscopy techniques employed in modern biological and ...

super-resolution-imaging-of-neuronal-dense-core-vesicles

Nanyang Technological University

Research

JoVE Journal

Neuroscience

9.7K 次观看.  Lewis & Clark College. 我们描述了如何实现光敏定位显微镜（PALM）为基础的固定，培养的神经元囊泡的研究。我们的协议的关键组件包括标签囊泡photoconvertible嵌合体，采集稀疏采样的原始图像的超分辨率显微镜系统，以及处理原始图像来产生超分辨率图像。 

视频: Super-resolution Imaging of Neuronal Dense-core Vesicles

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

C.在体内神经元钙成像技术线虫

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

科研

神经科学

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

The mechanisms controlling stress-induced phenotypic plasticity in animals are frequently complex and difficult to study in vivo. A classic example of stress-induced plasticity is the dauer stage of C. elegans. Dauers are an alternative developmental larval stage formed under conditions of low concentrations of bacterial food and high concentrations of a dauer pheromone. Dauers display extensive developmental and behavioral plasticity. For example, a set of four inner-labial quadrant (IL2Q) neurons undergo extensive reversible remodeling during dauer formation. Utilizing the well-known environmental pathways regulating dauer entry, a previously established method for the production of crude dauer pheromone from large-scale liquid nematode cultures is demonstrated. With this method, a concentration of 50,000 - 75,000 nematodes/ml of liquid culture is sufficient to produce a highly potent crude dauer pheromone. The crude pheromone potency is determined by a dose-response bioassay. Finally, the methods used for in vivo time-lapse imaging of the IL2Qs during dauer formation are described.

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

Following exposure to specific environmental stressors, the nematode Caenorhabditis elegans undergoes extensive phenotypic plasticity to enter into a stress-resistant &#8216;dauer&#8217; juvenile stage. We present methods for the controlled induction and imaging of neuroplasticity during dauer.

在永久性幼虫特有的神经重构的C语言活体成像线虫

The mechanisms controlling stress-induced phenotypic plasticity in animals are frequently complex and difficult to study in vivo. A classic example of ...

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the experimental time, and increases the number of animals that are utilized per study. This limitation specifically impacts protocols such as calcium imaging that require prolonged pre-incubation with bath-applied dyes. Exponential bacterial growth within 3 - 4 h after slicing is tightly correlated with a decrease in tissue health. This study describes a method for&#160;limiting the proliferation of bacteria in acute preparations to maintain viable neuronal tissue for prolonged periods of time (&#62;24 h) without the need for antibiotics, sterile procedures, or tissue culture media containing growth factors. By cycling the extracellular fluid through UV irradiation and keeping the tissue in a custom holding chamber at 15 - 16 &#176;C, the tissue shows no difference in electrophysiological properties, or calcium signaling through intracellular calcium dyes at &#62;24 h postdissection. These methods will not only extend experimental time for those using acute neuronal tissue, but will reduce the number of animals required to complete experimental goals, and will set a gold standard for acute neuronal tissue incubation.

Once removed from the body, neuronal tissue is greatly affected by environmental conditions, leading to eventual degradation of the tissue after 6 - 8 h. Using a unique incubation method, which closely monitors and regulates the extracellular environment of the tissue, tissue viability can be significantly extended for &#62;24 h.

对于电生理和钙成像急性神经组织的长时间培养

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a closed-loop event-triggered manner. Although many studies are focusing on the possible benefits and side-effects of TES in healthy and pathologic brains, there are still many fundamental open questions regarding the mechanism of action of the stimulation. Therefore, there is a clear need for a robust and reproducible method to test the acute and the chronic effects of TES in rodents. TES can be combined with regular behavioral, electrophysiological, and imaging techniques to investigate neuronal networks in vivo. The implantation of transcranial stimulation electrodes does not impose extra constraints on the experimental design while it offers a versatile, flexible tool to manipulate brain activity. Here we provide a detailed, step-by-step protocol to fabricate and implant transcranial stimulation electrodes to influence brain activity in a temporally constrained manner for months.

This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving rats.

大鼠慢性经颅电刺激及 Intracortical 记录

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

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+ ...

Quantitative Approaches for Scoring in vivo Neuronal Aggregate and Organelle Extrusion in Large Exopher Vesicles in C. elegans

Toxicity of misfolded proteins and mitochondrial dysfunction are pivotal factors that promote age-associated functional neuronal decline and neurodegenerative disease across species. Although these neurotoxic challenges have long been considered to be cell-intrinsic, considerable evidence now supports that misfolded human disease proteins originating in one neuron can appear in neighboring cells, a phenomenon proposed to promote pathology spread in human neurodegenerative disease.
C. elegans adult neurons that express aggregating proteins can extrude large (~4 &#181;m) membrane-surrounded vesicles that can include the aggregated protein, mitochondria, and lysosomes. These large vesicles are called &#8220;exophers&#8221; and are distinct from exosomes (which are about 100x smaller and have different biogenesis). Throwing out cellular debris in exophers may occur by a conserved mechanism that constitutes a fundamental, but formerly unrecognized, branch of neuronal proteostasis and mitochondrial quality control, relevant to processes by which aggregates spread in human neurodegenerative diseases.
While exophers have been mostly studied in animals that express high copy transgenic mCherry within touch neurons, these protocols are equally useful in the study of exophergenesis using fluorescently tagged organelles or other proteins of interest in various classes of neurons.
Described here are the physical features of C. elegans exophers, strategies for their detection, identification criteria, optimal timing for quantitation, and animal growth protocols that control for stresses that can modulate exopher production levels. Together, details of protocols outlined here should serve to establish a standard for quantitative analysis of exophers across laboratories. This document seeks to serve as a resource in the field for laboratories seeking to elaborate molecular mechanisms by which exophers are produced and by which exophers are reacted to by neighboring and distant cells.

Quantitative Approaches for Scoring in vivo Neuronal Aggregate and Organelle Extrusion in Large Exopher Vesicles in C. elegans

This protocol describes approaches for detection and quantitation of large aggregate and/or organelle extrusions (~4 &#181;m) produced by C. elegans cells in the form of membrane-bound exophers. We describe strains, growth conditions, scoring criteria, timing, and microscopy considerations needed to facilitate dissection of this debris expulsion mechanism.

C. elegans 中大型外层囊中体内神经聚合和有机体挤出评分的定量方法

Toxicity of misfolded proteins and mitochondrial dysfunction are pivotal factors that promote age-associated functional neuronal decline and ...

Super-Resolution Imaging to Study Co-Localization of Proteins and Synaptic Markers in Primary Neurons

Synapses are the functional elements of neurons and their defects or losses are at the basis of several neurodegenerative and neurological disorders. Imaging studies are widely used to investigate their function and plasticity in physiological and pathological conditions. Because of their size and structure, localization studies of proteins require high-resolution imaging techniques. In this protocol, we describe a procedure to study in primary neurons the co-localization of target proteins with synaptic markers at a super-resolution level using structured illumination microscopy (SIM). SIM is a patterned-light illumination technique that doubles the spatial resolution of wide-field microscopy, reaching a detail of around 100 nm. The protocol indicates the required controls and settings for robust co-localization studies and an overview of the statistical methods to analyze the imaging data properly.

This protocol shows how to employ super-resolution microscopy to study protein co-localization in primary neuronal cultures.

超分辨率成像研究原发神经元中蛋白质和突触标记的共同定位

Synapses are the functional elements of neurons and their defects or losses are at the basis of several neurodegenerative and neurological disorders. ...

In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively thin to properly visualize and access neurons for patch-clamping or imaging, and in vitro examination of brain circuitry is limited to only what is physically present in the acute slice. To maintain the benefits of in vitro slice experimentation while preserving a larger portion of presynaptic nuclei, we developed a novel slice preparation. This &#8220;wedge slice&#8221; was designed for patch-clamp electrophysiology recordings to characterize the diverse monaural, sound-driven inputs to medial olivocochlear (MOC) neurons in the brainstem. These neurons receive their primary afferent excitatory and inhibitory inputs from neurons activated by stimuli in the contralateral ear and corresponding cochlear nucleus (CN). An asymmetrical brain slice was designed which is thickest in the rostro-caudal domain at the lateral edge of one hemisphere and then thins towards the lateral edge of the opposite hemisphere. This slice contains, on the thick side, the auditory nerve root conveying information about auditory stimuli to the brain, the intrinsic CN circuitry, and both the disynaptic excitatory and trisynaptic inhibitory afferent pathways that converge on contralateral MOC neurons. Recording is performed from MOC neurons on the thin side of the slice, where they are visualized using DIC optics for typical patch-clamp experiments. Direct stimulation of the auditory nerve is performed as it enters the auditory brainstem, allowing for intrinsic CN circuit activity and synaptic plasticity to occur at synapses upstream of MOC neurons. With this technique, one can mimic in vivo circuit activation as closely as possible within the slice. This wedge slice preparation is applicable to other brain circuits where circuit analyses would benefit from preservation of upstream connectivity and long-range inputs, in combination with the technical advantages of in vitro slice physiology.

Integration of diverse synaptic inputs to neurons is best measured in a preparation that preserves all pre-synaptic nuclei for natural timing and circuit plasticity, but brain slices typically sever many connections. We developed a modified brain slice to mimic in vivo circuit activity while maintaining in vitro experimentation capability.

体外楔形切片准备模拟在Vivvo神经元电路连接

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively ...

Preparing Adult Drosophila melanogaster for Whole Brain Imaging during Behavior and Stimuli Responses

We present a method developed specifically to image the whole Drosophila brain during ongoing behavior such as walking. Head fixation and dissection are optimized to minimize their impact on behavior. This is first achieved by using a holder that minimizes movement hindrances. The back of the fly&#8217;s head is glued to this holder at an angle that allows optical access to the whole brain while retaining the fly&#8217;s ability to walk, groom, smell, taste and see. The back of the head is dissected to remove tissues in the optical path and muscles responsible for head movement artefacts. The fly brain can subsequently be imaged to record brain activity, for instance using calcium or voltage indicators, during specific behaviors such as walking or grooming, and in response to different stimuli. Once the challenging dissection, which requires considerable practice, has been mastered, this technique allows to record rich data sets relating whole brain activity to behavior and stimulus responses.

Preparing Adult Drosophila melanogaster for Whole Brain Imaging during Behavior and Stimuli Responses

We present a method specifically tailored to image the whole brain of adult Drosophila during behavior and in response to stimuli. The head is positioned to allow optical access to the whole brain, while the fly can move its legs and the antennae, the tip of the proboscis, and the eyes can receive sensory stimuli.

在行为和刺激反应期间为整个大脑成像准备成人德罗索菲拉褪黑麦片加斯特

We present a method developed specifically to image the whole Drosophila brain during ongoing behavior such as walking. Head fixation and dissection are ...

ROS Live Cell Imaging During Neuronal Development

Reactive oxygen species (ROS) are well-established signaling molecules, which are important in normal development, homeostasis, and physiology. Among the different ROS, hydrogen peroxide (H2O2) is best characterized with respect to roles in cellular signaling. H2O2 has been implicated during the development in several species. For example, a transient increase in H2O2 has been detected in zebrafish embryos during the first days following fertilization. Furthermore, depleting an important cellular H2O2 source, NADPH oxidase (NOX), impairs nervous system development such as the differentiation, axonal growth, and guidance of retinal ganglion cells (RGCs) both in&#160;vivo and in vitro. Here, we describe a method for imaging intracellular H2O2 levels in cultured zebrafish neurons and whole larvae during development using the genetically encoded H2O2-specific biosensor, roGFP2-Orp1. This probe can be transiently or stably expressed in zebrafish larvae. Furthermore, the ratiometric readout diminishes the probability of detecting artifacts due to differential gene expression or volume effects. First, we demonstrate how to isolate and culture RGCs derived from zebrafish embryos that transiently express roGFP2-Orp1. Then, we use whole larvae to monitor H2O2 levels at the tissue level. The sensor has been validated by the addition of H2O2. Additionally, this methodology could be used to measure H2O2 levels in specific cell types and tissues by generating transgenic animals with tissue-specific biosensor expression. As zebrafish facilitate genetic and developmental manipulations, the approach demonstrated here could serve as a pipeline to test the role of H2O2 during neuronal and general embryonic development in vertebrates.

This protocol describes the use of a genetically encoded hydrogen peroxide (H2O2)-biosensor in cultured zebrafish neurons and larvae for assessing the physiological signaling roles of H2O2 during nervous system development. It can be applied to different cell types and modified with experimental treatments to study reactive oxygen species (ROS) in general development.

神经元发育过程中的 ROS 活细胞成像

Reactive oxygen species (ROS) are well-established signaling molecules, which are important in normal development, homeostasis, and physiology. Among the ...