这项工作得到了美国国立卫生研究院拨款R01-HD093827和R03-HD090304（AJT）的支持。

所有动物程序均根据指南进行，并得到康奈尔大学机构动物护理和使用委员会 （#2002-0095） 的批准。本研究使用 8-10 周龄的 AcroSensE 小鼠2 。有关AcroSensE鼠标可用性的信息请求可以提交给通讯作者。
1.精子收集和清洗
<ol><li>从AcroSensE小鼠中收集附睾尾精子（有关此程序的更多详细信息在11中提供）。在37°C下含有0.5mL改良Whitten培养基（MW）的3.5cm组织培养皿中允许15分钟的游出程序。 培养皿应被覆盖以减少MW的蒸发，并且应倾斜，使培养基有足够的深度来覆盖附睾尾部。 注意：改良的Whitten培养基（分子量）包括22mM HEPES，1.2mM MgCl 2,100mM NaCl，4.7mM KCl，1mM丙酮酸，4.8mM乳酸hemi-Ca2+盐，pH 7.35。葡萄糖（5.5mM），NaHCO3（10mM）和2-羟丙基环糊精（CD;3mM）根据需要补充（见材料表）以诱导获能，并且可以针对特定实验修改浓度。此外，可以使用替代甾醇受体代替克罗恩病（例如，牛血清白蛋白或高密度脂蛋白）。</li>
	<li>使用细镊子，从游泳收集皿中取出任何附睾组织。将含有精子的培养基转移到15mL锥形管中，并在室温下将悬浮液在摆动桶转子中以100× g 离心1分钟。使用塑料移液管，从在此步骤中沉淀的任何大组织碎片中取出含有精子的MW上清液。</li>
	<li>通过在37°C下加入MW，使含有精子的MW上清液的总体积达到3mL。 在圆底管中以400× g 离心8分钟（室温下）。从松散颗粒的精子中慢慢除去MW的上清液。</li>
	<li>如果可行，精子应该看起来像一个“蓬松”的颗粒。 通过 用大口径移液管研磨或 轻 轻手动敲击管子（“手指轻弹”）轻轻重悬精子。重新悬浮均匀后，将它们转移到新的圆底管中。使用 10-20 μL 精子悬浮液来计数和评估活力。在MW中稀释精子以供使用。 注意：对于大多数涉及小鼠精子获能的实验，使用5-1000万精子/ mL MW的最终浓度。这里描述的成像方案不需要精子的获能，尽管精子必须获得经历生理相关的顶体胞吐作用的能力。人们可以研究病理性胞吐作用或通过钙离子载体等试剂诱导的胞吐作用，而不会使精子具有能力。此外，人们可以探索非容量细胞中顶体钙响应各种刺激的潜在变化。</li>
	<li>在收集后3-5小时内使用精子，使时间尽可能短，并在实验试验中保持一致。</li>
	<li>使用MW培养基在37°C下执行收集和洗涤的所有步骤，采用尽量减少膜损伤的方法。这包括使用大口径塑料移液器或大孔径移液器吸头，建议在手术的所有阶段使用。</li>
</ol>2. 用于成像的盖玻片培养皿的聚-D-赖氨酸涂层
注意：聚-D-赖氨酸 （PDL） 培养皿应在每次实验前新鲜制备。
<ol><li>在盖玻片培养皿的中心分配0.5μL PDL液滴（0.5mg / mL，参见 材料表）。</li>
	<li>使用10μL刻度吸头，将PDL液滴涂抹在盖玻片上（纵横交错的图案，如 图1B所示）。</li>
	<li>在37°C下干燥10分钟。将涂有PDL的培养皿盖上盖子并保持在37°C直至使用。</li>
</ol>3.毛细管拉动，用于膨化
<ol><li>使用如下设置拉动硼硅酸盐玻璃毛细管（外径，2 mm，内径，1.56 mm，参见 材料表）- 热量：330，拉力：250，速度：250和时间：70。此步骤应针对使用的特定拉拔器进行优化。</li>
	<li>在每次实验之前，用含有3-5倍兴奋剂浓度的刺激溶液加载单个毛细管（以解释溶液在膨化时的快速扩散）。填充前，用细镊子在距末端约 1-2 毫米处掰开毛细管尖端。这应该产生 ∼5 μm 的开口直径。不需要热抛光。</li>
	<li>使用薄塑料移液管，将刺激溶液缓慢注入毛细管中，直到其长度的四分之三充满。</li>
	<li>通过轻弹动毛细管去除填充过程中形成的任何气泡。</li>
	<li>将填充的毛细管安装在显微操纵器上（参见 材料表）。</li>
</ol>4.准备为膨化提供刺激
注意： 为了最大限度地降低用户受伤的风险，在膨化程序操作过程中应佩戴护目镜。
<ol><li>设置单电池输送系统设置，以在 5 psi 下提供 10 秒脉冲。</li>
	<li>将硼硅酸盐毛细管连接到单细胞输送系统移液器支架。确保密封严密。</li>
	<li>在观察毛细管尖端时，在 20 psi 下短吸 2 秒，以确保溶液确实通过尖端末端分配（尖端末端应明显有一小滴）。</li>
</ol>5. 显微镜和图像采集
注意：有多种品牌的显微镜可供选择，可以使用;但是，最低帧速率为 3 帧/秒是可取的。关于温度和环境控制，请注意，应确认培养皿中介质的实际温度，例如使用非接触式红外测温仪。
<ol><li>将80μL稀释的精子加入聚D-赖氨酸包被的35mm盖玻片培养皿的中心。对于大多数涉及小鼠精子获能的实验，使用5-1000万精子/mL的最终浓度。</li>
	<li>向精子中缓慢加入补充有10mM CaCl2的3mL温（37°C）MW基础培养基。 注意：人们可以在室温下进行实验以减缓细胞过程，但重要的是要注意，由于获能是由脂质交换以及微观和宏观结构域的流动性介导的，因此必须记住，生理相关的获能和顶体胞吐作用是温度依赖性过程。</li>
	<li>将精子与精子一起安装在显微镜上（预热至37°C）。</li>
	<li>使用 488 nm 激光线激发 GCaMP3，并使用 505-550 nm 带通 （BP） 滤光片查看。使用 555 nm 激光线激发 mCherry，并使用 575-615 nm BP 滤光片查看。</li>
	<li>使用显微操纵器（建议将机械手连接到设置显微镜的气台上），将毛细管的尖端放置在感兴趣的细胞平面上方约100μm和5-10μm。 注意：刺激溶液以正常浓度的 3-5 倍制备，以补偿毛细管和细胞之间体积中发生的快速扩散。</li>
	<li>在显微镜上开始成像序列。</li>
	<li>以高帧速率（优选 >10 帧/秒）对精子细胞进行成像。在这里，使用了共振扫描仪，能够以 33 毫秒/帧的速度成像。</li>
	<li>在显微镜上开始图像采集10秒后，激活单细胞递送系统以启动10秒的兴奋剂。</li>
	<li>成像细胞持续时间为10-15分钟。</li>
	<li>使用单细胞递送系统，根据 图1B中提供的位置对单个培养皿中的多个细胞进行成像。 注意：在显微镜下未经处理/未受刺激的精子应大部分呈红色，绿色信号强度较低。精子头应附着在盖玻片上，活精子可以通过其移动的尾巴来识别。在经历AE的精子中，GCaMP3绿色信号最初会增加，如果AE完成，mCherry红色信号和GCaMP3绿色信号都会消失，如 图2所示。mCherry荧光的减少为AE开始的时间提供了更具体的指标，因为Ca2+ 浓度的变化将不断导致GCaMP3荧光强度的变化。</li>
</ol>6. 图像和数据分析
注意：离线图像分析是使用 ImageJ 进行的（参见 材料表）。此前，在导致 AE 的过程中报道了几个中间步骤，包括顶体钙升高 （ACR） 和全膜融合。后者导致 mCherry 荧光的损失，因此代表完全 AE。在一些细胞中，当使用电化学方法进行胞吐作用研究时，观察到类似于尖峰前足事件 （PSF） 的信号（有关详细信息，请参见2）。有几种可选方法可用于分析 AcroSensE 原始数据，以量化 ACR、AE 及其中间步骤。下面介绍一些基本的分析方法。
<ol><li>根据特定的显微镜系统文件扩展名，使用分割通道工具（Image > Colors > Split Channels）为 GCaMP3 和 mCherry 通道生成单独的窗口。</li>
	<li>使用“同步窗口”工具（分析>工具>同步窗口）同步各个窗口。</li>
	<li>在红色通道中选择精子头部周围的感兴趣区域（ROI），以包括顶体区域（图3A，B）。同步工具允许在绿色通道上自动应用相同的区域选择，其中精子仍然太暗而无法看到。</li>
	<li>选择“Z Profiler”插件（Z Profiler > Stacks > Plugins ）或“Time Series Analyzer”（Plugins > Stacks > Time Series Analyzer），以绘制两个通道中每个通道的荧光强度变化随帧数/时间的变化。</li>
	<li>将数据复制到电子表格软件（例如 Microsoft Excel）以进行绘图和进一步分析。 注意：将数据复制到电子表格后，可以提取多个参数进行分析，包括以下参数，如 图 4 所示。<ol><li>熔孔 （FP） 开始时间：测量从时间点 0 到 GCaMP3 信号开始上升的时间点的时间。该参数表示刺激和精子ACR反应之间的延迟。</li>
		<li>AE 开始时间：测量从时间点 0 到 mCherry 信号开始衰减的时间点的时间。该参数表示刺激和精子AE反应之间的延迟。该参数还可用于计算 FP 形成和 AE 响应之间的延迟。</li>
		<li>峰值FP幅度：测量荧光信号从基底到GCaMP3信号上升的峰值。此参数指示 ACR 响应的总增加量。</li>
		<li>mCherry 的损耗率：通过对 mCherry 信号损耗部分的线性或指数拟合来确定此参数（如 图 4B 中的“斜率”所示）。 注意：或者，通过残差 （R） 信号法确定不同时间点的衰减率（例如，R60：持续时间（以秒为单位）到距 mCherry 信号基线 60% 的残余信号）。该参数可用于量化顶体内容物在 AE 时分散的速率。</li>
		<li>mCherry 丢失的信号降低：确定基线 mCherry 荧光强度与 AE 后信号之间的幅度差异。该参数表示在 AE 上分散的顶体内容物的总量。</li>
		<li>将红色和绿色通道的荧光（F）变化绘制为原始数据，或通过初始荧光（F0）对其进行归一化，并将其表示为ΔF / F0。后者可以更好地可视化单个图上红色和绿色的变化（例如，参见 图3D 和 图3H）。 注意：最后，可以比较不同条件下的不同测量参数，以确定顶体钙浓度变化与AE之间的关系。有关各种实验条件比较的示例，请参阅参考文献2。</li>
	</ol></li>
</ol>

将成像与兴奋剂递送系统相结合以研究活体小鼠精子

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没有作者有与这项工作相关的利益冲突。

在这里，描述了一种基于显微镜的方法，利用新生成的 AcroSensE 小鼠模型进行实时单细胞监测和分析顶体钙动力学与导致 AE 的中间步骤之间的相互作用。与现成的遗传方法（例如与其他小鼠遗传模型杂交或基因编辑）一起，该模型和方法提供了一个强大的系统来研究参与与获能、AE 和受精相关的精子信号通路的各种成分和途径的作用。
关键步骤 在所有精子处理步骤中，重要的是使用大口径塑料移液器或大孔口移液器吸头，并在37°C下执行收集和洗涤的所有步骤，以尽量减少膜损伤。同样，摆动桶转子优于固定角度转子，以避免由于细胞与微量离心管侧壁相互作用而导致膜损坏。如果 Z 位置漂移校正可用，强烈建议应用 Z 位校正硬件/软件，以避免成像精子在成像间隔期间漂移出焦平面。
修改和故障排除 这里描述的方案针对脂质介导的信号传导2,3的研究进行了优化，可以应用于有能力的精子和非有能力的精子。可以使用不同的精子获能方法;例如，白蛋白或环糊精可以作为甾醇受体。此外，还可以添加或省略各种能量基材或碳酸氢盐。然而，请注意，碳酸氢盐会因暴露在空气中而在水性介质中形成，因此测试真正的“无碳酸氢盐”条件需要在制造/制备/处理介质和进行实验的所有步骤中小心使用氮环境。上述方案包括使用10mM钙添加到实验溶液中;然而，钙离子可以省略、螯合（例如，EGTA）或以较低浓度添加，用于钙敏感的实验应用。请注意，降低或省略钙浓度会降低 AE2 时预期的 GCaMP3 信号。此外，人们可以使用其他更适合其实验设计和目标的协议（而不是此处使用的MW）的介质。提供的方案描述了单细胞成像和兴奋剂单细胞递送的使用;然而，通过大量刺激可以实现简化的变化，例如将刺激试剂添加到整个培养皿中，而不是通过膨化。此外，还可以使用具有荧光功能的荧光计或酶标仪，使用 AcroSensE 模型进行全群体实验。
局限性 此处提供的方法需要 AcroSensE 小鼠菌落的可用性、维护和使用。该方法还取决于先进成像系统的可用性，这成本高昂且需要训练有素的人员。对于某些实验应用，可能的局限性在于无法同时测量 AcroSensE 精子中的胞质钙，因为许多可用的合成钙指示剂的波长与 GCaMP3 相似。尽管超出了该协议的范围，但群体水平实验的潜在局限性可能包括AE后mCherry信号的读取和介质中的扩散或来自死亡/透化细胞的GCaMP3信号。为了优化这些方法，人们可以探索采用z聚焦来最大限度地减少分泌的mCherry的背景噪音，和/或利用假处理的对照来确保从死亡或透化精子中定量基线GCaMP。
该方法相对于现有/替代方法的意义 在过去的二十年中，有报道称有几种方法用于实时研究活精子中的AE动力学，包括顶体靶向GFP转基因小鼠7，加载到顶体中的钙染料（例如，Fluo-412），或使用胞吐特异性指示剂，如合成染料FM-6413。然而，与这些方法相比，AcroSensE模型提供了使用最低限度处理的精子的便利性，这些精子不需要任何染料加载或其他潜在的膜扰动程序。重要的是，拥有表达双重指示剂的精子有很大的优势，可以监测顶体钙动力学、膜融合和孔形成，以及 AE 和随之而来的顶体内容物的释放。
该方法在特定研究领域的重要性和潜在应用 请注意，下面的实验应用示例可以作为单细胞或群体检测进行，后者在需要高通量分析时具有显着优势。可以通过与AcroSensE小鼠模型杂交来研究各种小鼠模型中的AE动力学，包括（1）各种钙通道或钙通道亚基的无效或突变形式（例如，与α1E和CatSper-null模型杂交）;（2）介导膜融合的蛋白质的无效或突变形式（例如SNAREs）。此外，还可以进行研究以研究与不同信号通路相关的各种药理学药物如何调节顶体钙和 AE 动力学。此外，还可以研究环境变化如何影响 AE，包括温度、pH 值以及各种离子或小分子的存在或浓度。

精子在称为获能1 的过程中获得受精能力。这个过程的一个终点是精子获得经历AE的能力。超过二十年的数据支持哺乳动物精子中存在复杂的多步骤AE模型（总结于2,3）。然而，研究活精子中的AE具有挑战性，目前可用的以足够分辨率监测这一过程的方法很麻烦，需要多个制备步骤4，仅限于检测AE的最后一步（例如，使用PNA5），仅限于测量胞质钙的变化（与顶体钙动力学相反）， 或仅限于细胞溶质钙动力学或 AE6 的测量。
为了克服生理条件下实时AE研究的一些关键局限性，并能够研究钙动力学和AE之间的相互作用，生成了一个独特的小鼠模型。在该小鼠模型中，由基因编码的 Ca2+ 传感器 （GCaMP3） 和 mCherry 组成的融合蛋白被表达并使用 acrosin 启动子和信号转导肽2 靶向顶体。靶向双GCaMP3-mCherry传感器能够使用显微镜和单细胞兴奋剂递送系统，在生理条件下同时实时测量活精子中的钙浓度和顶体内容物状态（图1）。作为顶体基质的一个组成部分，膜融合和 AE 将导致精子中光稳定和 pH 不敏感的 mCherry 荧光丢失，因为这种蛋白质从顶体囊泡中扩散出来。在这方面，该模型反映AE的时间和发生的能力类似于顶体靶向GFP小鼠系7,8,9的益处。
该转基因小鼠品系中使用的 GCaMP3 变体的 KD 约为 400 μM，Ca2+ 的动态范围为 10-4-10-3 M10，适用于该囊泡。我们发现，GCaMP3 的这种特征组合可以揭示质膜和顶体外膜 （OAM） 之间的融合孔形成2。融合孔检测是由于孔径太小而无法使 AcroSensE 蛋白从顶体中分散出来（通过顶体内容物丢失），同时提供膜“通道”，使 Ca2+ 离子能够流入顶体腔，导致 GCaMP3 的荧光强度增加。
明亮的、单体的、非钙敏感的荧光蛋白mCherry支持顶体的可视化，而GCaMP3信号微弱（例如，在Ca2+ 结合之前， 图2），重要的是，它还允许识别适合成像的顶体完整精子细胞。
以下协议描述了独特的AcroSensE小鼠模型的利用以及实验中使用的显微镜方法，以高空间和时间分辨率研究AE和精子钙动力学。

<table><tbody><tr><td>100x oil objective&amp;nbsp;</td><td>Olympus Japan</td><td>UPlanApo,</td><td></td></tr><tr><td>2-hydroxypropyl-b-cyclodextrin&amp;nbsp;</td><td>Sigma</td><td>C0926</td><td></td></tr><tr><td>35 mm coverslip dish, 1.5 thickness</td><td>MatTek Corp.&amp;nbsp;</td><td>P35G-1.5-20-C&amp;nbsp;</td><td></td></tr><tr><td>5 mL round-bottomed tube</td><td>Falcon</td><td>352054</td><td></td></tr><tr><td>Borosilicate glass capilarries</td><td>Sutter Instrument Co. CA USA</td><td>B200-156-10</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>C4901</td><td></td></tr><tr><td>Confocal microscope</td><td>Olympus Japan</td><td>Olympus FluoView&amp;nbsp;</td><td></td></tr><tr><td>Glucose&amp;nbsp;</td><td>Sigma</td><td>G7528</td><td></td></tr><tr><td>Graduated tip&amp;nbsp;</td><td>TipOne, USA Scientific</td><td></td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H7006</td><td></td></tr><tr><td>ImageJ&amp;nbsp;</td><td>National Institutes of Health</td><td></td><td>https://imagej.nih.gov/ij/plugins/index.html</td></tr><tr><td>KCl</td><td>Sigma</td><td>P9541</td><td></td></tr><tr><td>Lactic acid</td><td>Sigma</td><td>G5889</td><td></td></tr><tr><td>Live-Cell Microscope Incubation Systems&amp;nbsp;</td><td>TOKAI HIT Shizuoka, Japan</td><td>Model STX</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M8266</td><td></td></tr><tr><td>Micropipette Puller&amp;nbsp;</td><td>Sutter Instrument Co. CA USA</td><td>Model P-97</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S3014</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>S6297</td><td></td></tr><tr><td>Plastic transfer pipette&amp;nbsp;</td><td>FisherBrand&amp;nbsp;</td><td>13-711-6M</td><td></td></tr><tr><td>Poly-D-lysine&amp;nbsp;</td><td>Sigma</td><td>P7280</td><td></td></tr><tr><td>Pyruvic acid</td><td>Sigma</td><td>107360</td><td></td></tr><tr><td>Single cell delivery system</td><td>Parker, Hauppauge, NY</td><td>Picospritzer III</td><td></td></tr></tbody></table>

real-time imaging of acrosomal calcium dynamics and exocytosis in live mouse sperm

顶体胞吐作用 （AE） 是精子的单个胞吐囊泡与质膜融合，是一个复杂的、依赖钙的过程，对受精至关重要。然而，我们对钙信号转导如何调节 AE 的理解仍然不完整。特别是，顶体内钙动力学与导致 AE 的中间步骤之间的相互作用尚不明确。在这里，我们描述了一种方法，该方法提供了对顶体钙动力学及其与膜融合和随后顶体囊泡胞吐作用的关系的空间和时间见解。该方法利用一种新型转基因小鼠，表达顶体靶向胞吐传感器（AcroSensE）。该传感器结合了与 mCherry 融合的基因编码钙指示剂 （GCaMP）。这种融合蛋白专门设计用于同时观察顶体钙动力学和膜融合事件。实时监测活体 AcroSensE 精子中的顶体钙动力学和 AE 是使用高帧率成像和可靶向单个精子的兴奋剂递送系统实现的。该协议还提供了几个用于量化和分析原始数据的基本方法示例。由于 AcroSensE 模型是基因编码的，因此可以通过使用现成的遗传工具（例如与其他小鼠遗传模型杂交或基于基因编辑 （CRISPR） 的方法）来增强其科学意义。通过这种策略，可以解决其他信号通路在精子获能和受精中的作用。总之，这里描述的方法提供了一种方便有效的工具来研究特定亚细胞区室（精子顶体）中的钙动力学，以及这些动力学如何调节导致膜融合和顶体胞吐作用的中间步骤。

图2 提供了一个简化的图示，显示了成功刺激精子后预期的荧光变化的顺序。 图 2 的上图显示了 GCaMP3 荧光强度的变化，其中信号最初变暗（基线顶体钙浓度低于 GCaMP3 KD），当钙离子 通过 融合孔进入时，荧光亮度增加。最后，在 AE 时，由于扩散和传感器向细胞外空间的损失而丢失信号。 图 2 的底板显示了 mCherry 荧光强度的变化，其中初始信号很亮，只有在 AE 和传感器与顶体基质的其他内容物一起扩散时，红色信号才会变暗。
图 3 提供了在单个视场中捕获的用 125 μM 神经节苷脂 GM1 刺激的两个活精子中上述阶段的实际测量数据（有关 GM1 如何调节精子12 中 AE 的更多详细信息）。图3A，B和图3E，F显示了在时间点0（刺激前）在两个精子中测量的红色和绿色信号，其中最初，mCherry信号是明亮的，而GCaMP3是暗淡的。 图3C，图3G和图3D，图3H提供了整个实验期间的Z Profiler分析（原始数据或F/ F 0，分别）。插入面板提供发生 ACR 和 AE 的时间窗口的放大视图。
由于精子具有高度异质性，并且由于各种处理程序对膜损伤非常敏感，因此图 5 提供了两种类型的阴性结果的示例。图5A，C显示了在一个视野中捕获的几个精子。黄色轮廓突出显示了两个精子细胞，它们在时间点 0（刺激前）表现出高 GCaMP3 信号。在本实验中，避免了这样的细胞，因为它们表明顶体囊泡已经经历了一些膜融合过程，或者它们已经经历了一些允许钙泄漏的膜损伤。图5A中的精子用箭头表示（图5A，C中的圆圈ROI）显示初始mCherry信号，但在红色或绿色通道中没有响应（图5B，D，分别显示ImageJ提供的Z-Profiler分析窗口）在用125μM GM1刺激后。尽管精子亚群（例如本例中提供的精子）对刺激没有反应，但这些精子包含在最终分析中，并在百分比反应数据分析2中计算（图3）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65962/65962fig01.jpg"> 图 1：仪器和实验设置。 （A）将活精子细胞放在PDL包被的盖玻片培养皿上，放置在37°C的培养室中。 硼硅酸盐毛细管被拉动并附着在单细胞递送系统上，位于成像区域中心的精子细胞附近，用于直接递送兴奋剂。在刺激期间，绿色 （GCaMP3） 和红色 （mCherry） 通道都以高帧速率（例如 10 帧/秒）连续监控和记录。（B） 盖玻片皿上PDL沉积模式的示意图。（C） 培养皿上可以使用毛细血管刺激精子的特定区域的示意图，以最大限度地减少同一培养皿中先前抽吸的非特异性刺激。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65962/65962fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65962/65962fig02.jpg"> 图2：荧光信号和强度的变化。 上图：GCaMP3 信号的增加表示钙流入顶体，随后由于 AcroSensE 融合蛋白的扩散而导致信号丢失。下图：mCherry信号在初始膜融合期间保持稳定，随后在AcroSensE扩散和顶体胞吐作用（AE）时变暗。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65962/65962fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65962/65962fig03.jpg"> 图3：代表性数据和荧光迹线。对包含两个精子细胞的场进行成像。使用 ImageJ 离线定量绿色/红色通道荧光并在 Excel 中绘制（如步骤 6 中所述）。（A-D）定量细胞 #1 的红色 （A） 和绿色 （B） 通道荧光强度随时间变化，计算选定的 ROI，然后呈现为原始数据 （C） 或归一化为初始信号 （D， F/F0）。（D）中的插入提供了25-125秒时间范围的放大视图。 （E-H）定量细胞#2的红色（E）和绿色（F）通道荧光强度随时间变化，计算选定的ROI，然后呈现为原始数据（G）或归一化为初始信号（H，F/F0）。（H） 中的插入提供了 25-125 秒时间范围的放大视图。比例尺 = 5 μm。所有 x 轴都表示以秒为单位的时间。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65962/65962fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65962/65962fig04.jpg"> 图4：数据分析和定量参数。 （A） 典型的 GCaMP3 荧光强度轨迹图示，显示可量化的参数，包括开始时间（秒）、振幅（荧光强度）和持续时间（秒）。（B） 典型的 mCherry 荧光强度轨迹图示，显示可量化的参数，包括开始时间（秒）、持续时间（秒）、斜率 （F/s）、R60（荧光强度）和振幅（荧光强度）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65962/65962fig04large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65962/65962fig05.jpg"> 图 5：阴性结果。 （A，C） mCherry（红色）和 GCaMP3（绿色）通道显示在单个视野中捕获的多个精子细胞。（A）中的箭头表示精子细胞最初表现出mCherry荧光，但随后没有响应于125μM GM1刺激的荧光强度变化，如Z-Profiler生成的痕迹中观察到的（B）（mCherry）和（D）（GCaMP3）。在刺激之前，黄色矩形内的细胞在时间点 0 表现出升高的 GCaMP3 荧光强度。比例尺 = 5 μm。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65962/65962fig05large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>

Watch this Scientific Journal Video about A Live Cell Imaging Technique to Study Calcium Signaling and Acrosome Exocytosis in Mouse Sperm at JoVE.com

作者聚焦：一种研究小鼠精子中钙信号传导和顶体胞吐作用的活细胞成像技术 

这里描述的AcroSensE小鼠模型和活细胞成像方法为研究精子顶体亚细胞区室中的钙动力学以及它们如何调节导致膜融合和顶体胞吐作用的中间步骤提供了一种新方法。

活小鼠精子顶体钙动力学和胞吐作用的实时成像

Acrosome exocytosis (AE), in which the sperm&#39;s single exocytotic vesicle fuses with the plasma membrane, is a complex, calcium-dependent process ...

我们的研究目标是更好地了解调节精子功能的分子机制，包括获能和受精等过程。具体来说，我们正在探索膜脂质如何激活钙信号传导和顶体胞吐作用。该协议解决了我们对获能，精子激活，钙信号传导和顶体胞吐作用之间相互作用的理解差距。该协议的优点包括在生理条件下对精子进行成像，以及它能够同时监测钙信号传导和顶体胞吐作用，而无需复杂的预加载或站立程序。

real-time-imaging-acrosomal-calcium-dynamics-exocytosis-live-mouse

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

Real-Time Imaging of Acrosomal Calcium Dynamics and Exocytosis in Live Mouse Sperm

926 次观看.  Cornell University. 我们的研究目标是更好地了解调节精子功能的分子机制，包括获能和受精等过程。具体来说，我们正在探索膜脂质如何激活钙信号传导和顶体胞吐作用。该协议解决了我们对获能，精子激活，钙信号传导和顶体胞吐作用之间相互作用的理解差距。该协议的优点包括在生理条件下对精子进行成像，以及它能够同时监测钙信号传导和顶体胞吐作用，而无需复杂的预加载或站?...

视频: 作者聚焦：一种研究小鼠精子中钙信号传导和顶体胞吐作用的活细胞成像技术 

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

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

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

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

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

科研

神经科学

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

技术成像钙 2 +信号在人类精子

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

生物学

Live Imaging of Early Cardiac Progenitors in the Mouse Embryo

The first steps of heart development imply drastic changes in cell behavior and differentiation. While analysis of fixed embryos allows studying in detail specific developmental stages in a still snapshot, live imaging captures dynamic morphogenetic events, such as cell migration, shape changes, and differentiation, by imaging the embryo as it develops. This complements fixed analysis and expands the understanding of how organs develop during embryogenesis. Despite its advantages, live imaging is rarely used in mouse models because of its technical challenges. Early mouse embryos are sensitive when cultured ex vivo and require efficient handling. To facilitate a broader use of live imaging in mouse developmental research, this paper presents a detailed protocol for two-photon live microscopy that allows long-term acquisition in mouse embryos. In addition to the protocol, tips are provided on embryo handling and culture optimization. This will help understand key events in early mouse organogenesis, enhancing the understanding of cardiovascular progenitor biology.

We present a detailed protocol for mouse embryo culture and imaging that enables 3D + time imaging of cardiac progenitor cells. This video-toolkit addresses the key skills required for successful live imaging otherwise hard to acquire from text-only publications.

小鼠胚胎中早期心脏祖细胞的实时成像

The first steps of heart development imply drastic changes in cell behavior and differentiation. While analysis of fixed embryos allows studying in detail ...

发育生物学

The Mouse Cremaster Muscle Preparation for Intravital Imaging of the Microcirculation

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic by-products. This balance is achieved by the movement of blood through the microcirculation, which encompasses the smallest branches of the vascular supply throughout all tissues and organs. Arterioles branch from arteries to form networks that control the distribution and magnitude of oxygenated blood flowing into the multitude of capillaries intimately associated with parenchymal cells. Capillaries provide a large surface area for diffusional exchange between tissue cells and the blood supply. Venules collect capillary effluent and converge as they return deoxygenated blood towards the heart. To observe these processes in real time requires an experimental approach for visualizing and manipulating the living microcirculation.
The cremaster muscle of rats was first used as a model for studying inflammation using histology and electron microscopy post mortem1,2. 
The first in vivo report of the exposed intact rat cremaster muscle investigated microvascular responses to vasoactive drugs using reflected light3. However 
curvature of the muscle and lack of focused illumination limited the usefulness of this preparation. The major breakthrough entailed opening the muscle, 
detaching it from the testicle and spreading it radially as a flat sheet for transillumination under a compound microscope4. While shown to be a valuable 
preparation to study the physiology of the microcirculation in rats5 and hamsters6, the cremaster muscle in mice7 has proven particularly useful in dissecting 
cellular pathways involved in regulating microvascular function8-11 and real-time imaging of intercellular signaling12. 
The cremaster muscle is derived from the internal oblique and transverse abdominus muscles as the testes descend through the inguinal canal13. 
It serves to support (Greek: cremaster = suspender) and maintain temperature of the testes. As described here, the cremaster muscle is prepared as a thin flat sheet 
for outstanding optical resolution. With the mouse maintained at a stable body temperature and plane of anesthesia, surgical preparation involves freeing the muscle 
from surrounding tissue and the testes, spreading it onto transparent pedestal of silastic rubber and securing the edges with insect pins while irrigating it 
continuously with physiological salt solution. The present protocol utilizes transgenic mice expressing GCaMP2 in arteriolar endothelial cells. GCaMP2 is a 
genetically encoded fluorescent calcium indicator molecule12. Widefield imaging and an intensified charge-coupled device camera enable in vivo study of 
calcium signaling in the arteriolar endothelium.

A tissue preparation is described for visualization and experimental manipulation of the living microcirculation. In anesthetized male mice, the thin, highly vascularized cremaster muscle is prepared for intravital microscopy to study microvascular networks including arterioles, capillaries and venules. This preparation is readily adapted for rats and hamsters.

鼠标提睾肌肌肉制备的微循环活体成像

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic ...

Tracking Calcium in the Early Developmental Phases of C. elegans

In Vivo Visualization of Calcium Transients during Fertilization and Early Development in C. elegans

Calcium is an important signaling molecule during the oocyte-to-embryo transition (OET) and early embryogenesis. The hermaphroditic nematode Caenorhabditis elegans provides several unique advantages for the study of the OET as it is transparent and has an ordered gonad that produces one mature oocyte every ~23 min at 20 &#176;C. We have modified the genetically encoded calcium indicator jGCaMP7s to fluorescently indicate the moment of fertilization within a living organism. We have termed this reporter &#34;CaFE&#34; for Calcium during Fertilization in C. elegans. The CaFE reporter was&#160;engineered into a safe harbor locus in single copy, has no significant impact on physiology or fecundity, and produces a robust signal upon fertilization. Here, a series of protocols is presented for utilizing the CaFE reporter as an in vivo tool for dissecting the OET and embryogenesis. We include methods to synchronize worms, examine the effects of RNAi knockdown, mount worms for imaging, and to visualize calcium in oocytes and embryos. Additionally, we present the generation of additional worm strains to aid in this type of analysis. Demonstrating the utility of the CaFE reporter to visualize the timing of fertilization, we report that double ovulation occurs when ipp-5 is targeted by RNAi and that only the first oocyte undergoes immediate fertilization. Furthermore, the discovery of single-cell calcium transients during early embryogenesis is reported here, demonstrating that the CaFE reporter persists into early development. Importantly, the CaFE reporter in worms is simple enough to use for incorporation into course-based undergraduate research (CURE) laboratory classes. The CaFE reporter, coupled with the ordered gonad and ease of RNAi in worms, facilitates inquiry into the cell-cell dynamics required to regulate internal fertilization and early embryogenesis.

Here, we present protocols and tools for visualizing calcium during fertilization and early embryogenesis using a genetically encoded calcium reporter expressed in the germline of the model nematode C. elegans.

Calcium is an important signaling molecule during the oocyte-to-embryo transition (OET) and early embryogenesis. The hermaphroditic nematode ...

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of individual cells. Since an increase of intracellular calcium (Ca2+) in the cytosol often occurs because of the electrical activity, or in response to a myriad of other stimuli, this process can be monitored by the imaging of cells loaded with fluorescent calcium-sensitive dyes.&#160; However, it is difficult to image this response in an individual cell type within whole tissue because these dyes are taken up by all cell types within the tissue. In contrast, genetically encoded calcium indicators (GECIs) can be expressed by an individual cell type and fluoresce in response to an increase of intracellular Ca2+, thus permitting the imaging of Ca2+ signaling in entire populations of individual cell types. Here, we apply the use of the GECIs GCaMP3/6 to the mouse neuromuscular junction, a tripartite synapse between motor neurons, skeletal muscle, and terminal/perisynaptic Schwann cells. We demonstrate the utility of this technique in classic ex vivo tissue preparations. Using an optical splitter, we perform dual-wavelength imaging of dynamic Ca2+ signals and a static label of the neuromuscular junction (NMJ) in an approach that could be easily adapted to monitor two cell-specific GECI or genetically encoded voltage indicators (GEVI) simultaneously. Finally, we discuss the routines used to capture spatial maps of fluorescence intensity. Together, these optical, transgenic, and analytic techniques can be employed to study the biological activity of distinct cell subpopulations at the NMJ in a wide variety of contexts.

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

Here we present a protocol to image calcium signaling in populations of individual cell types at the murine neuromuscular junction.

前体小鼠膈肌三方突触细胞特异性钙信号的影像学研究

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of ...

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Spermatozoa are male reproductive cells especially designed to reach, recognize and fuse with the egg. To perform these tasks, sperm cells must be prepared to face a constantly changing environment and to overcome several physical barriers. Being in essence transcriptionally and translationally silent, these motile cells rely profoundly on diverse signaling mechanisms to orient themselves and swim in a directed fashion, and to contend with challenging environmental conditions during their journey to find the egg. In particular, Ca2+-mediated signaling is pivotal for several sperm functions: activation of motility, capacitation (a complex process that prepares sperm for the acrosome reaction) and the acrosome reaction (an exocytotic event that allows sperm-egg fusion). The use of fluorescent dyes to track intracellular fluctuations of this ion is of remarkable importance due to their ease of application, sensitivity, and versatility of detection. Using one single dye-loading protocol we utilize four different fluorometric techniques to monitor sperm Ca2+ dynamics. Each technique provides distinct information that enables spatial and/or temporal resolution, generating data both at single cell and cell population levels.

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Intracellular Ca2+ dynamics are very important in sperm physiology and Ca2+-sensitive fluorescent dyes constitute a versatile tool to study them. Population experiments (fluorometry and stopped flow fluorometry) and single cell experiments (flow cytometry and single cell imaging) are used to track spatio-temporal [Ca2+] changes in human sperm cells.

测量细胞内Ca<sup&gt; +</sup在人类精子的变化采用4项技术:传统的荧光，停流荧光，流式细胞术及单细胞成像

Spermatozoa  are male reproductive cells especially designed to reach, recognize and fuse  with the egg. To perform these tasks, sperm cells must be ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

活体显微镜成像的亚细胞结构的活体小鼠表达荧光蛋白

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in the brain sense various biological conditions in the periphery and transmit the signals to neurons. Microglia, immune cells in the brain, are involved in synaptic development and plasticity. Therefore, the contribution of microglia to neural circuit construction in response to the internal state of the body should be tested critically by intravital imaging of the relationship between microglial dynamics and neuronal activity.
Here, we describe a technique for the simultaneous imaging of microglial dynamics and neuronal activity in awake mice. Adeno-associated virus encoding R-CaMP, a gene-encoded calcium indicator of red fluorescence protein, was injected into layer 2/3 of the primary visual cortex in CX3CR1-EGFP transgenic mice expressing EGFP in microglia. After viral injection, a cranial window was installed onto the brain surface of the injected region. In vivo two-photon imaging in awake mice 4 weeks after the surgery demonstrated that neural activity and microglial dynamics could be recorded simultaneously at the sub-second temporal resolution. This technique can uncover the coordination between microglial dynamics and neuronal activity, with the former responding to peripheral immunological states and the latter encoding the internal brain states.

Here, we describe a protocol combining adeno-associated virus injection with cranial window implantation for simultaneous imaging of microglial dynamics and neuronal activity in awake mice.

清醒小鼠小胶质细胞动力学和神经元活动的同时成像

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in ...

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 ...

活小鼠精子顶体钙动力学和胞吐作用的实时成像

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