我们感谢迈克尔 Rosbash 在这个方法发展的最初阶段的指导和支持。这项工作由 JST 方案、瑞士国家科学基金会 (31003A_149893 和 31003A_169548)、欧洲研究理事会 (ERC-StG-311194)、诺华医学生物医学研究基金会 (13A39) 和日内瓦大学资助。.

1. 在文化罩下准备库存解决方案
<ol><li>准备400毫升的1x 施耐德活性培养基 (SAM) 优化的ex 体内培养幼体的大脑 (修改从参考24,25) (表 1)。分到5毫升, 在液氮中闪冻 (在2), 存储在-80 ° c。</li>
	<li>通过稀释10x 库存溶液 (从参考26中修改) (表 2), 准备1x 解剖盐水溶液 (DSS)。</li>
	<li>分纤维蛋白原为10毫克, 储存在4° c 为单一用途。 注意: 在层流下称纤维蛋白原, 戴上手套, 因为它是有害的。</li>
	<li>在 SAM (1500 NIH 单位/mg) 和分10µL 中准备凝血酶溶液, 在2中进行闪存冻结, 并保持在-80 ° c。 注意: 在处理凝血酶时戴上手套是有害的。</li>
	<li>分100x 青霉素-链霉素的库存解决方案。保持在-20 ° c。</li>
	<li>将聚四氟乙烯 (PTFE) 膜 (见材料表) 切割成符合玻璃底盘内侧的尺寸。冲洗70% 乙醇, 然后在蒸压 dH2o 在层流下用 UV 消毒和干燥。保持不育的培养皿。一次性使用。 注: PTFE 是一种多孔的柔性膜, 允许气体交换, 防止介质在长期的记录中蒸发。</li>
</ol>2. 延时显微镜设置
注: 以下设置为串联扫描仪倒置共焦显微镜 (见材料表) 配备了共振扫描仪 (8000 Hz)。该系统包括一个高度敏感的光倍增器探测器, 与共振扫描仪一起防止光和漂白。温度和湿度控制在显微镜环境室内 (参见材料表), 它涵盖了大部分的显微镜。
<ol><li>放置一个舞台顶部的湿度室。</li>
	<li>打开温度和湿度控制器, 平衡显微镜室到25° c 和80% 湿度。</li>
	<li>在恒定的黑暗 (DD) 中进行实验, 用不透明的布覆盖显微镜环境室 (除非显微镜室是由不透明的材料制成)。</li>
	<li>如果使用水浸泡目标, 在目标的顶部放置一个水浸微饮水器, 并与 Autoimmersion 目标控制器软件一起编程, 以恒定速率在时间推移成像时分配水。 注: 水浸泡目标连同饮水机或干物镜推荐用于长期实时成像, 因为其他类型的浸泡介质会干燥。</li>
	<li>在舞台上放置一个培养皿支架, 里面的湿度室。</li>
	<li>启动显微镜软件, 从第一对话框中选择共振扫描仪模式。选择 "确定" 以在出现提示时初始化阶段。转到 "配置" 选项卡和 "硬件配置" 以切换相关的激光线路。</li>
	<li>转到 "获取" 选项卡和 "XY" 面板以选择双向模式并设置图像获取参数。 注意: 本文所描述的成像设置 (图 2、图 3和电影 1) 被优化以观察特定荧光记者的昼夜节律表达。以下参数选择最适合我们的规格: 40X 水浸泡目标, 8X 线平均 512 x 512 图像分辨率。对于多色成像, 当一个荧光的发射波长和另一个重叠的激发波长 (这里是黄色荧光蛋白 (YFP) 的波长和番茄激发重叠) 时, 需要序列图像采集。选择 "在栈之间" 的顺序模式, 因为它是最快的, 在 Z 栈的获取过程中, 时钟神经元的运动是微不足道的。显微镜的设置应适合每一个实验的目的。</li>
</ol>3. 幼虫脑外植体培养的建立
注意: 从解剖到植入脑外植体的整个过程需要30分钟。
<ol><li>
在培养罩下制备试剂。
	<ol>
<li>解冻凝血酶的分并保持室温直到嵌入步骤 (步骤 3.3)。一次性使用。</li>
		<li>在37° c 的情况下迅速解冻分, 并在冰上保持一个单一用途。</li>
		<li>添加 SAM 到10毫克分的纤维蛋白原到10毫克/毫升, 轻轻地轻拍和孵化在37° c 至少30分钟, 并在嵌入步骤 (步骤 3.3)。一次性使用。 注意: 当纤维蛋白原溶液开始聚合时, 不要孵育超过2小时。</li>
		<li>解冻分100x 股青霉素-链霉素。准备2.5 毫升的 sam 含有1x 青霉素-链霉素 (sam/抗生素)。保持室温。 注: 将剩余的100x 库存青霉素-链霉素储存在4° c 至1月。</li>
		<li>准备一分500µL 从剩下的 SAM。继续冰</li>
		<li>对于长期成像, 将蒸压真空润滑脂应用到玻璃底盘底部的塑料部分 (图 1A), 并在层流下用 UV 消毒。</li>
	</ol>
</li>
	<li>
解剖中枢神经系统和腹神经线。
	<ol>
<li>清洗镊子和 2 x 三井玻璃解剖菜与70% 乙醇。将400µL 的 DSS 注入到4口井和400µL 的 SAM 中。继续冰</li>
		<li>舀出含有幼虫的苍蝇培养基, 用镊子摘取非游荡的第三龄幼虫 (L3)。在 2 DSS 填充井中依次冲洗。把它们放在第三的 DSS 中--在冰上 anesthetizes 幼虫。 注: L3 持续约2天, 在25° c。观察大脑在生理相关的时间范围内, 我们选择使用非游荡 L3 幼虫。其他的时钟神经元子群, 如 l-LNvs 和 DN3s, 开始在游荡 L3 阶段形成, 但它们不一定是循环的 (数据没有显示)。因此, 虽然这是可能的图像周期时钟神经元在这一阶段 (数据没有显示), 重要的是要仔细区分已经功能性的幼虫时钟神经元从开发的数据分析。非游荡的 L3 出现大约4到4.5 天在蛋以后放置在25° c。要使用本协议来研究昼夜节律, 在收集 L3 幼虫之前, 在25° c 的 12 h/12 中, 同步 (entrain) 发育中的幼虫在3天内循环。</li>
		<li>使用内而外的方法27, 在第四 DSS 填充井中用细钳解剖幼体的脑部, 这是没有被用来冲洗幼虫的。<ol>
<li>简言之, 将幼虫切成两半, 用镊子轻轻捏住嘴钩, 然后用另一副钳子将身体内壁卷起, 从而露出大脑。通过切断所有附着在身体其他部位的气管和神经来释放大脑。</li>
			<li>在大约3µL 的 dss 中, 用20µL 吸管 (预冲洗与 dss), 并在 SAM 填充的井中抽吸大脑。重复7大脑的程序。 注意: 应在寒冷的表面进行解剖, 以保持幼虫麻醉和大脑冷冻。例如, 将解剖盘放在连接到水泵的冷却块上, 以循环冰水。解剖程序每只大脑需要三十年代。重要的是要保持眼睛/触角形象光盘连接到大脑和腹神经脊髓完整。撕掉这些部分会损害大脑, 降低文化质量。同时, 不要让大脑暴露在空气中。在吸取第一个大脑之前, 吸管向上和向下的 DSS 包含部分的解剖幼虫, 因为它防止大脑粘在顶端的墙壁。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
植入3D 矩阵和安装 (约20分钟) 的脑外植体。
	<ol>
<li>将一个解剖的大脑浸入纤维蛋白原工作溶液 (纤维蛋白原最终浓度〜3.3 毫克/毫升, 每脑10.5 µL) 如下:</li>
		<li>吸出3.5 µL 的 SAM/抗生素 (在3.2.3 节使用相同的提示)。从解剖井抽出一个解剖的大脑到相同的吸管尖端, 而不配发的 SAM/抗生素介质的尖端。</li>
		<li>将大脑和培养基放在无菌培养皿的盖子上。添加另外3.5 µL 的 SAM/抗生素和3.5 µL 的暖纤维蛋白原/sam 10 毫克/毫升的解决方案的下降, 包含大脑。通过移20µL 吸管尖端, 轻轻地将溶液混合在大脑周围。 注: 在这一步使用吸管而不是镊子, 避免了大脑的压力。</li>
		<li>采取2µL 的纤维蛋白原工作解决方案从下降和应用它的片玻璃底部盘作为一个小圆圈。加入0.8 µL 的凝血酶, 并很快与吸管尖端混合。纤维蛋白原被转化为纤维蛋白, 并开始聚合。</li>
		<li>采取的大脑, 坐在纤维蛋白原工作解决方案 (步骤 3.3.1) 之间的一对镊子和位置上的矩阵一旦一个白色基质纤维蛋白是可见的。使大脑前侧向下 (用于成像时钟神经元)。把它尽可能靠近盖子玻璃。纤维蛋白凝块由纤维蛋白层组成。选择一层, 并把它折叠在大脑的顶部, 小而温和的运动, 而不分离矩阵。<ol>
<li>从血块的不同部位重复2到3次, 以稳定大脑。 注意: 当用镊子处理大脑时, 要小心不要把它弄干, 或者把它强加给身体压力。</li>
		</ol>
</li>
		<li>添加20µL 的 SAM/抗生素在嵌入式大脑的顶部停止凝血酶的作用。</li>
		<li>重复这个程序来装剩下的大脑。在一个玻璃底盘上嵌入多达5到6的大脑是可能的。</li>
		<li>对于长期活成像, 增加600µL 的 SAM/抗生素在内井 (玻璃部分)。将预切的聚四氟乙烯膜放在介质顶部, 并将其粘在底部塑料部分 (3.1.5) (图 1A) 上的真空油脂上。</li>
		<li>对于药理学化验, 用2毫升的 SAM/抗生素 (图 1B) 填充该菜。 注意: 避免介质的蒸发是非常重要的, 因为培养基的渗透对神经元的活力至关重要。因此, 在长期的成像实验中, 有必要用 PTFE 膜密封培养皿。在短期实验中, 不需要密封膜, 只要盘子里装满2毫升的培养基, 并在湿润的室内进行监测。</li>
	</ol>
</li>
</ol>4. 延时图像采集
<ol><li>将玻璃底盘放在舞台顶部湿度室内的培养皿上。</li>
	<li>转到 "获取" 选项卡中的 "z 堆栈" 面板, 并从显微镜软件 (2 µm x ~ 40 中的实验显示在图 2和图 3和电影 1) 中设置 z 节步骤。将 z 堆叠的起始位置设置在离片最远的平面上, 在脑外植体的表面, 靠近片。虽然大脑的运动是微不足道的, 在 z 栈的开始和结束处添加额外的 z 部分。</li>
	<li>转到 "标记 & 查找" 面板并设置多位置图像获取。以40X 的目标, 1 大脑半球可以被捕获的视野内</li>
	<li>转到 "采集模式" 面板并选择 xyzt (z 堆栈时间间隔)。转到 "时间采集" 面板, 设置首选时间间隔和频率参数。 注意: 获取所需频率的延时图像。请注意, 长期活成像 (24-72 小时) 的时间间隔短于2小时往往会导致较少的循环神经元, 可能是因为它降低了神经元的生存能力。随着图像采集率的提高, 数据量也随之增大, 使得数据分析和存储更具挑战性。</li>
</ol>5. 短期活体成像对脑外植体的药理治疗
注意: 以下过程与长期成像的程序基本相同, 但不需要聚四氟乙烯膜。为了避免在将化学物质添加到培养物时使大脑暴露在蓝光中, 应将显微镜放在带有红光的暗室里。
<ol><li>进入时间采集面板, 设置所需的时间间隔和扫描次数, 以获得药物应用前的基线数据。开始扫描。</li>
	<li>基线扫描完成后, 抬起显微镜系统的扫描头。卸下舞台顶部湿度控制器的盖子, 并非常小心地卸下玻璃底盘的盖子而不移动它。</li>
	<li>必要时, 删除适当的培养基数量。在培养基中加入实验溶液, 小心和非常轻柔地混合, 不接触到大脑外植体的不育的巴斯德吸管。 注: 对于沐浴应用 pdf, 使用2µM pdf 股票解决方案 (在10% 亚甲基亚砜)。</li>
	<li>更换玻璃底盘和潮湿腔的盖, 以及扫描头。如果使用, 在显微镜室周围重新定位黑色不透明布。</li>
	<li>检查是否大脑处于原位扫描模式的原始位置。必要时进行调整。</li>
	<li>转到 "时间收集" 面板, 并设置所需的时间间隔和扫描次数。开始扫描。 注: 在这里, 我们测试了每小时获取的时间推移图像高达 10 h。</li>
</ol>

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作者没有什么可透露的。

在这里, 我们描述了长期荧光时间推移显微镜的培养幼体大脑的方法。这类实验的成功与否取决于多种因素, 如培养的健康、对大脑外植体的固定化方法、荧光强度和报告者的信噪比、时间和空间分辨率, 以及外植体的可及性。这些因素可以相互排斥。例如, 增加的时间推移频率影响到文化的健康, 和固定的大脑外植体可能阻碍气体交换。因此, 找到一个快乐的媒体 (没有双关语) 的现象正在研究中是...

生物钟帮助生物体适应地球 24 h 自转产生的周期性环境变化。互锁转录-平移反馈回路通常基础上的分子机械的昼夜生物钟的各种物种1。由时钟包含的神经元组成的昼夜节律起搏器电路集成了由环境提示 (如光/暗 (LD) 和温度循环) 所传达的时间信息, 以协调每天过多的生理和行为进程2,3。分子节律与神经元输入和输出的协调对于昼夜节律回路的运行至关重要, 但仍只部分了解。
在果蝇中, 在分子时钟的核心, 时钟/周期 (赤 CYC) heterodimer 激活句点(每) 和永恒(tim) 的转录。每和 TIM 形成一个复杂的, 并进入细胞核, 在那里他们抑制转录活动的时钟/CYC, 因此他们自己的转录。转录后和后转化的规则导致时钟/CYC 介导的转录和压制之间的延迟/TIM, 确保产生约 24 h 分子振荡1,3,4.大约150神经元含有这些分子时钟形成一个电路来控制成人苍蝇的昼夜行为5。一个更简单而功能完整的昼夜节律电路, 由3组时钟神经元组成, 5 腹侧神经元 (LNvs; 4 个 pdf 阳性 LNvs 和一个 pdf 阴性 LNv, 见下), 2 背神经 1s (DN1s) 和2背神经元 2s (DN2s)-存在于幼虫大脑6,7。
简单的幼虫昼夜节律电路提供了一个很好的模型来研究神经元间的交流和分子发条之间的相互作用。利用我们新开发的每 TDT 的荧光报告器, 它模拟每个蛋白质的水平和它的亚细胞位置, 我们试图在幼虫昼夜节律回路中表征不同时钟神经元子群的分子发条的动力学特性8. 此外, 知道神经肽颜料分散因子 (PDF) 的关键作用, 4 LNvs 在调节神经元水平的昼夜节律9,10,11, 我们想检查直接PDF 对分子时钟的影响。为此, 我们开发了一种方法, 监测昼夜节律基因表达节律的幼虫脑外植体多天的时间推移共聚焦显微镜。该协议也适用于药理测试, 以检验 PDF 或其他化合物对每 TDT 的水平的影响。因此, 这种适应包括使脑外植体培养可用于药物应用, 增加时间分辨率和成像时间更短。
不同发育阶段的果蝇大脑的前体培养已经建立12,13,14,15,16,17 ,18。虽然这些协议已被用于成像各种生物现象, 其中一些是不兼容的成像在单细胞分辨率或不支持的文化超过几个小时。在果蝇中对昼夜节律神经元进行长期实时成像的替代方法包括分子节律的生物荧光成像19、20、21和荧光成像钙指示物与轻的板料显微学22,23。虽然生物荧光成像可以实现更高的时间分辨率和光片显微镜可以适应在体内成像, 他们是有限的空间分辨率和要求专门的显微镜系统。
本文所描述的方法是在多天的单细胞分辨率下, 针对全脑培养中的荧光信号进行可视化。这种轻便和多才多艺的方法可以适应于图像培养成人苍蝇脑和药理实验研究许多不同的问题, 在果蝇神经生物学。

<table><tbody><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>P5655</td><td>I am not sure they are exactly the same ones we have in the lab. I chose &quot;suitable for insect cell culture&quot; whenever available</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>C7902</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>230391</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S5886</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S5761</td><td></td></tr><tr><td>D-(+) Glucose</td><td>Sigma-Aldrich</td><td>G7021</td><td></td></tr><tr><td>Yeast extract</td><td>Sigma-Aldrich</td><td>Y1000</td><td></td></tr><tr><td>Insulin</td><td>Sigma-Aldrich</td><td>I0516-5ML</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma-Aldrich</td><td>P4333</td><td></td></tr><tr><td>BIS-TRIS</td><td>Sigma-Aldrich</td><td>B4429</td><td></td></tr><tr><td>L-(&amp;minus;)-Malic acid</td><td>Sigma-Aldrich</td><td>M7397</td><td></td></tr><tr><td>D-(+)-Trehalose dihydrate</td><td>Sigma-Aldrich</td><td>T0167</td><td></td></tr><tr><td>Succinic acid</td><td>Sigma-Aldrich</td><td>S9512</td><td></td></tr><tr><td>Fumaric acid</td><td>Sigma-Aldrich</td><td>F8509</td><td></td></tr><tr><td>&amp;alpha;-Ketoglutaric acid</td><td>Sigma-Aldrich</td><td>K1128</td><td></td></tr><tr><td>Non-heat-inactivated, Foetal Calf Serum (FCS) Mycoplasma and Virus screened</td><td>BioConcept Ltd. Amimed</td><td>2-01F30-I</td><td></td></tr><tr><td>HEPES-KOH, pH 7.4</td><td>E&amp;amp;K Scientific Products</td><td>EK-654011</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P5405</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S5011</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S7903</td><td></td></tr><tr><td>Fibrinogen from bovine plasma</td><td>Calbiochem (Merck)</td><td>341573-1GM</td><td>CAUTION: Harmful by inhalation, in contact with skin and if swallowed. Manipulate under laminar flow</td></tr><tr><td>Thrombin from bovine plasma</td><td>Sigma-Aldrich</td><td>T9549</td><td>CAUTION: Health Hazard, use gloves</td></tr><tr><td>PDF, NH&lt;sub&gt;2&lt;/sub&gt;-NSELINSLLSLPKNMNDA-OH</td><td>Chi Scientific</td><td>custom made</td><td></td></tr><tr><td>Vaccum grease</td><td>Sigma-Aldrich</td><td>18405</td><td></td></tr><tr><td>35 mm Dish, No. 1.5 Coverslip, 20 mm Glass Diameter, Uncoated</td><td>MatTek</td><td>P35G-1.5-20-C</td><td></td></tr><tr><td>Corning Falcon Easy-Grip Tissue Culture Dishes</td><td>fisherscientific</td><td>08-772A</td><td></td></tr><tr><td>Sterile 500 mL Steritop-GP 33 mm threaded bottle top filter, 0.22 &amp;mu;m</td><td>Millipore</td><td>SCGPS05RE</td><td></td></tr><tr><td>Polytetrafluoroethylene (PTFE) film</td><td>Dupont</td><td>200A Teflon FEP Film</td><td></td></tr><tr><td>Millex-HV Syringe Filter Unit, 0.45&amp;nbsp;&amp;micro;m, PVDF, 33&amp;nbsp;mm, gamma sterilized</td><td>Millipore</td><td>SLHV033RS</td><td></td></tr><tr><td>Millex-GV Syringe Filter Unit, 0.22&amp;nbsp;&amp;micro;m, PVDF, 33&amp;nbsp;mm, gamma sterilized</td><td>Millipore</td><td>SLGV033RS</td><td></td></tr><tr><td>Three-well glass dissection dish</td><td>Any company</td><td></td><td></td></tr><tr><td>Fine forceps, size 5, Dumont</td><td>Fine Science Tools</td><td>11254-20</td><td></td></tr><tr><td>Tandem scanner inverted TCS SP5 confocal microscope, with resonant scanner and HyD photo-multiplier detectors</td><td>Leica microsystem CMS GmbH</td><td></td><td></td></tr><tr><td>Temperature control chamber</td><td>Life Imaging Services</td><td>The CUBE &amp;amp; BOX temperature control system, custom designed</td><td></td></tr><tr><td>Stage-top humidity controller</td><td>Life Imaging Services</td><td>custom made</td><td></td></tr><tr><td>Water Immersion Micro Dispenser: dispenser, extended micro-pump MP6 series and Autoimmersion Objective Controller software</td><td>Leica microsystem CMS GmbH</td><td></td><td></td></tr><tr><td>SUM-stack creation and 3D correction drift plugin</td><td>ImageJ software</td><td></td><td></td></tr><tr><td>10x iterative deconvolution</td><td>AutoQuant and Imaris software</td><td></td><td></td></tr></tbody></table>

single-cell resolution fluorescence live imaging of drosophila circadian clocks in larval brain culture

昼夜节律起搏器电路协调节律的行为和生理输出与环境的提示, 如昼夜循环。在每个起搏器神经元的分子时钟产生的基因表达的昼夜节律, 这基础的节律性神经元功能的运作的电路。不同类型的起搏器神经元的单个分子振荡器的性质及其与神经元信号相互作用的研究能更好地了解昼夜节律起搏器电路。在这里, 我们提出了一个时间推移荧光显微镜的方法来监测时钟神经元的分子发条的培养果蝇幼虫脑。这种方法允许多天记录的节奏, 基因编码荧光昼夜记者在单细胞的决议。这种设置可以结合药理操作, 密切分析分子时钟对各种化合物的实时响应。除了昼夜节律之外, 这种多用途方法与强大的果蝇基因技术结合, 为研究活体脑组织中的不同神经元或分子过程提供了可能性。

在这里, 我们展示了长期记录的代表性的结果, 一个昼夜荧光记者在前体内幼虫脑培养, 和实时成像结果的 PDF 沐浴应用的记者表达。
非游荡 L3 幼虫表达分子时钟记者每 TDT 和 UAS-mCD8::YFP 驱动的时钟神经元驱动程序时钟(856)-gal4 (图 1C) 在 LD 中夹带. 大脑被解剖, 并建立了长期的文化在最后LD 周?...

Watch this Scientific Journal Video about Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture at JoVE.com

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

该协议的目的是建立活体果蝇幼虫脑培养优化监测昼夜节律分子节律与长期荧光时间推移成像。并对该方法在药理实验中的应用进行了探讨。

单细胞分辨荧光活成像在幼虫脑培养中的果蝇昼夜节律时钟

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. ...

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Neuroscience

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

7.5K 次观看.  University of Geneva. 该协议的目的是建立活体果蝇幼虫脑培养优化监测昼夜节律分子节律与长期荧光时间推移成像。并对该方法在药理实验中的应用进行了探讨。

视频: Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

Live-imaging of the Drosophila Pupal Eye

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track during live cell imaging. These processes include: retinal rotation, cell growth and organismal movement. Additionally, irregularities in the topology of the epithelium, including subtle bumps and folds often introduced as the pupa is prepared for imaging, make it challenging to acquire in-focus images of more than a few ommatidia in a single focal plane. The workflow outlined here remedies these issues, allowing easy analysis of cellular processes during Drosophila pupal eye development. Appropriately-staged pupae are arranged in an imaging rig that can be easily assembled in most laboratories. Ubiquitin-DE-Cadherin:GFP and GMR-GAL4&#8211;driven UAS-&#945;-catenin:GFP are used to visualize cell boundaries in the eye epithelium 1-3. After deconvolution is applied to fluorescent images captured at multiple focal planes, maximum projection images are generated for each time point and enhanced using image editing software. Alignment algorithms are used to quickly stabilize superfluous motion, making individual cell behavior easier to track.

Live-imaging of the Drosophila Pupal Eye

This protocol presents an efficient method for imaging the live Drosophila pupal eye neuroepithelium. This method compensates for tissue movement and uneven topology, enhances visualization of cell boundaries through the use of multiple GFP-tagged junction proteins, and uses an easily-assembled imaging rig.

实时成像<em&gt;果蝇</em&gt;蛹眼

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track ...

科研

发育生物学

Use of Drosophila S2 Cells for Live Imaging of Cell Division

Drosophila S2 cells are an important tool in studying mitosis in tissue culture, providing molecular insights into this fundamental cellular process in a rapid and high-throughput manner. S2 cells have proven amenable to both fixed- and live-cell imaging applications. Notably, live-cell imaging can yield valuable information about how loss or knockdown of a gene can affect the kinetics and dynamics of key events during cell division, including mitotic spindle assembly, chromosome congression, and segregation, as well as overall cell cycle timing. Here we utilize S2 cells stably transfected with fluorescently tagged mCherry:&#945;-tubulin to mark the mitotic spindle and GFP:CENP-A (referred to as &#8216;CID&#8217; gene in Drosophila) to mark the centromere to analyze the effects of key mitotic genes on the timing of cell divisions, from prophase (specifically at Nuclear Envelope Breakdown; NEBD) to the onset of anaphase. This imaging protocol also allows for the visualization of the spindle microtubule and chromosome dynamics throughout mitosis. Herein, we aim to provide a simple yet comprehensive protocol that will allow readers to easily adapt S2 cells for live imaging experiments. Results obtained from such experiments should expand our understanding of genes involved in the cell division by defining their role in several simultaneous and dynamic events. Observations made in this cell culture system can be validated and further investigated in vivo using the impressive toolkit of genetic approaches in flies.

Use of Drosophila S2 Cells for Live Imaging of Cell Division

Cell divisions can be visualized in real time using fluorescently tagged proteins and time-lapse microscopy. Using the protocol presented here, users can analyze cell division timing dynamics, mitotic spindle assembly, and chromosome congression and segregation. Defects in these events following RNA interference (RNAi)-mediated gene knockdown can be assessed and quantified.

使用果蝇S2细胞进行细胞分裂的活成像

Drosophila S2 cells are an important tool in studying mitosis in tissue culture, providing molecular insights into this fundamental cellular process in a ...

遗传学

Live Imaging of Drosophila Larval Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.

Live Imaging of Drosophila Larval Neuroblasts

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

的实时成像<em&gt;果蝇</em&gt;幼虫神经母细胞

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

神经科学

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

发展和成人的制备果蝇大脑和视网膜实时成像

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

生物学

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

在完整的果蝇幼虫体内的亚细胞分辨率成像

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

来自 黑腹果蝇 第三龄幼虫的活脑外植体成像

Live-Cell Imaging of Drosophila melanogaster Third Instar Larval Brains

Drosophila neural stem cells (neuroblasts, NBs hereafter) undergo asymmetric divisions, regenerating the self-renewing neuroblast, while also forming a differentiating ganglion mother cell (GMC), which will undergo one additional division to give rise to two neurons or glia. Studies in NBs have uncovered the molecular mechanisms underlying cell polarity, spindle orientation, neural stem cell self-renewal, and differentiation. These asymmetric cell divisions are readily observable via live-cell imaging, making larval NBs ideally suited for investigating the spatiotemporal dynamics of asymmetric cell division in living tissue. When properly dissected and imaged in nutrient-supplemented medium, NBs in explant brains robustly divide for 12-20 h. Previously described methods are technically difficult and may be challenging to those new to the field. Here, a protocol is described for the preparation, dissection, mounting, and imaging of live third-instar larval brain explants using fat body supplements. Potential problems are also discussed, and examples are provided for how this technique can be used.

作者聚焦：研究不对称细胞分裂动力学：果蝇幼虫脑外植体实时成像的方案 

Here, we discuss a workflow to prepare, dissect, mount, and image live explant brains from Drosophila melanogaster third instar larvae to observe the cellular and subcellular dynamics under physiological conditions.

黑腹果蝇第三龄幼虫脑的活细胞成像

Drosophila neural stem cells (neuroblasts, NBs hereafter) undergo asymmetric divisions, regenerating the self-renewing neuroblast, while also forming a ...

Real-Time Imaging of Germline Stem Cells in Drosophila Ex Vivo Germaria Culture

Extended Live Imaging of Female Drosophila melanogaster Germline Stem Cell Niches

Live imaging methods allow the analysis of dynamic cellular processes in detail and in real-time. The Drosophila ovary represents an excellent model to explore the dynamics of a myriad of developmental processes, such as cell division, stemness, differentiation, migration, apoptosis, autophagy, cellular adhesion, etc., over time. Recently, we have implemented an extended ex vivo culture and live imaging of the female Drosophila GSC niche. Using a Drosophila line harboring a GFP::Par-1 transgene as an example, this method allows the visualization of the GSCs&#39; asymmetric division within their niche and the description of the changes in the spectrosome morphology along the cell cycle. Here, we present a detailed protocol for the ex vivo culture of Drosophila germaria, enabling prolonged visualization of the female GSC niche. Importantly, this protocol is broadly applicable to live imaging GSCs with multiple fluorescently tagged proteins of interest that are available in stock centers and/or in the Drosophila research community.

Here, we provide a detailed protocol to perform live imaging of the asymmetric division of germline stem cells (GSCs) in the Drosophila ovarian niche. We use a transgenic line that ubiquitously expresses a green fluorescent protein (GFP) fusion of the spectrosome protein Par-1.

Live imaging methods allow the analysis of dynamic cellular processes in detail and in real-time. The Drosophila ovary represents an excellent model to ...

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model animal for endocrine research, Drosophila melanogaster, which has sophisticated genetic tools and genome information, is being increasingly used. This article describes a method for the calcium imaging of Drosophila brain explants. This method enables the detection of the direct signaling of a hormone to the brain. It is well known that many peptide hormones act through G-protein-coupled receptors (GPCRs), whose activation causes an increase in the intracellular Ca2+concentration. Neural activation also elevates intracellular Ca2+ levels, from both Ca2+ influx and the release of Ca2+ stored in the endoplasmic reticulum (ER). A calcium sensor, GCaMP, can monitor these Ca2+ changes. In this method, GCaMP is expressed in the neurons of interest, and the GCaMP-expressing larval brain is dissected and cultured ex vivo. The test peptide is then applied to the brain explant, and the fluorescent changes in GCaMP are detected using a spinning disc confocal microscope equipped with a CCD camera. Using this method, any water-soluble molecule can be tested, and various cellular events associated with neural activation can be imaged using the appropriate fluorescent indicators. Moreover, by modifying the imaging chamber, this method can be used to image other Drosophila organs or the organs of other animals.

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

This paper describes a protocol for ex vivo calcium imaging of the Drosophila brain. In this method, natural or synthetic compounds can be applied to the buffer to test their ability to activate particular neurons in the brain.

前体钙成像用于可视化大脑对果蝇内分泌信号的反应

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model ...

单细胞分辨荧光活成像在幼虫脑培养中的果蝇昼夜节律时钟

摘要

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实时成像

使用果蝇S2细胞进行细胞分裂的活成像

的实时成像

发展和成人的制备果蝇大脑和视网膜实时成像

在完整的果蝇幼虫体内的亚细胞分辨率成像

黑腹果蝇第三龄幼虫脑的活细胞成像

黑腹果蝇第三龄幼虫脑的活细胞成像

黑腹果蝇第三龄幼虫脑的活细胞成像

Extended Live Imaging of Female Drosophila melanogaster Germline Stem Cell Niches

前体钙成像用于可视化大脑对果蝇内分泌信号的反应