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>KH2PO4</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 &#34;suitable for insect cell culture&#34; whenever available</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>C7902</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4.7H2O</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>NaHCO3</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-(&#8722;)-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>&#945;-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&#38;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>NaH2PO4</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, NH2-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 &#956;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&#160;&#181;m, PVDF, 33&#160;mm, gamma sterilized</td>
			<td>Millipore</td>
			<td>SLHV033RS</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex-GV Syringe Filter Unit, 0.22&#160;&#181;m, PVDF, 33&#160;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 &#38; 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. ...

single-cell-resolution-fluorescence-live-imaging-drosophila-circadian

Research

JoVE Journal

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

Magnetic Resonance Spectroscopy of live Drosophila melanogaster using Magic Angle Spinning

High-Resolution Magic Angle Spinning (HRMAS) proton magnetic resonance spectroscopy (1H-MRS) is a novel non-destructive technique that improves spectral line-widths and allows high-resolution spectra to be obtained from extracts, intact cells, cell cultures, and more importantly intact tissue to investigate relationships between metabolites and cellular processes. In vivo HRMAS 1H-MRS studies have yet to be reported in the live fruit fly Drosophila melanogaster. Drosophila, as a simpler genetic organism, allows the multiple biological functions and various evolutionarily conserved signaling pathways to be examined at the whole organism level and it is a useful model for investigating genetics and physiology. To this end, we developed and implemented an in vivo HRMAS 1H-MRS method to investigate live Drosophila at 14.1 T. Here, we outline an HRMAS 1H-MRS protocol for the molecular characterization of Drosophila with a conventional MR spectrometer equipped with an HRMAS probe. This technique is a novel, in vivo, non-destructive Drosophila metabolite measurement approach, which enables the identification of disease biomarkers and thus may contribute to novel therapeutic development.

Magnetic Resonance Spectroscopy of live Drosophila melanogaster using Magic Angle Spinning

This technique enables the use of high-resolution magic angle spinning proton MR spectroscopy (HRMAS 1H-MRS) for molecular characterization of live Drosophila melanogaster with a conventional 14.1 tesla spectrometer equipped with an HRMAS probe.

生活磁共振波谱果蝇</em用魔角旋转

High-Resolution Magic Angle Spinning (HRMAS) proton magnetic resonance spectroscopy (1H-MRS) is a novel non-destructive technique that improves spectral ...

科研

神经科学

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of its convenient genetics and fully sequenced genome. Additionally, the larval nervous system is an ideal model system to study mechanisms of axonal transport as the larval segmental nerves contain bundles of axons with their cell bodies located within the brain and their nerve terminals ending along the length of the body. Here we describe the procedure for visualization of synaptic vesicle proteins within larval segmental nerves. If done correctly, all components of the nervous system, along with associated tissues such as muscles and NMJs, remain intact, undamaged, and ready to be visualized. 3rd instar larvae carrying various mutations are dissected, fixed, incubated with synaptic vesicle antibodies, visualized and compared to wild type larvae. This procedure can be adapted for several different synaptic or neuronal antibodies and changes in the distribution of a variety of proteins can be easily observed within larval segmental nerves.

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster larvae provide an ideal model system to investigate the mechanisms of axonal transport within larval segmental nerves. Using this procedure, 3rd instar larvae carrying various mutations can be compared to wild type larvae.

3幼虫节段性神经的可视化 RD龄果蝇幼虫的筹备工作

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

在完整的感觉器官前体细胞的活细胞成像果蝇蛹

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are characteristic to neurons in both normal and diseased brain. Towards this, a great deal of effort has been placed on building high-resolution neuroanatomical maps1-3. With the expansion of molecular genetics and advances in light microscopy has come the ability to query not only neuronal morphologies, but also the molecular and cellular makeup of individual neurons and their associated networks4. Major advances in the ability to mark and manipulate neurons through transgenic and gene targeting technologies in the rodent now allow investigators to 'program' neuronal subsets at will5-6. Arguably, one of the most influential contributions to contemporary neuroscience has been the discovery and cloning of genes encoding fluorescent proteins (FPs) in marine invertebrates7-8, alongside their subsequent engineering to yield an ever-expanding toolbox of vital reporters9. Exploiting cell type-specific promoter activity to drive targeted FP expression in discrete neuronal populations now affords neuroanatomical investigation with genetic precision.
Engineering FP expression in neurons has vastly improved our understanding of brain structure and function. However, imaging individual neurons and their associated networks in deep brain tissues, or in three dimensions, has remained a challenge. Due to high lipid content, nervous tissue is rather opaque and exhibits auto fluorescence. These inherent biophysical properties make it difficult to visualize and image fluorescently labelled neurons at high resolution using standard epifluorescent or confocal microscopy beyond depths of tens of microns. To circumvent this challenge investigators often employ serial thin-section imaging and reconstruction methods10, or 2-photon laser scanning microscopy11. Current drawbacks to these approaches are the associated labor-intensive tissue preparation, or cost-prohibitive instrumentation respectively.
Here, we present a relatively rapid and simple method to visualize fluorescently labelled cells in fixed semi-thick mouse brain slices by optical clearing and imaging. In the attached protocol we describe the methods of: 1) fixing brain tissue in situ via intracardial perfusion, 2) dissection and removal of whole brain, 3) stationary brain embedding in agarose, 4) precision semi-thick slice preparation using new vibratome instrumentation, 5) clearing brain tissue through a glycerol gradient, and 6) mounting on glass slides for light microscopy and z-stack reconstruction (Figure 1).
For preparing brain slices we implemented a relatively new piece of instrumentation called the 'Compresstome' VF-200 (http://www.precisionary.com/products_vf200.html). This instrument is a semi-automated microtome equipped with a motorized advance and blade vibration system with features similar in function to other vibratomes. Unlike other vibratomes, the tissue to be sliced is mounted in an agarose plug within a stainless steel cylinder. The tissue is extruded at desired thicknesses from the cylinder, and cut by the forward advancing vibrating blade. The agarose plug/cylinder system allows for reproducible tissue mounting, alignment, and precision cutting. In our hands, the 'Compresstome' yields high quality tissue slices for electrophysiology, immunohistochemistry, and direct fixed-tissue mounting and imaging. Combined with optical clearing, here we demonstrate the preparation of semi-thick fixed brain slices for high-resolution fluorescent imaging.

Here we describe a rapid and simple method to image fluorescently labeled cells in semi-thick brain slices. By fixing, slicing, and optically clearing brain tissue we describe how standard epifluorescent or confocal imaging can be used to visualize individual cells and neuronal networks within intact nervous tissue.

一个快速高分辨率的荧光成像的方法在半厚的脑片

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are ...

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic development and axonal wiring. Recently the dendritic arborization (DA) sensory neurons of the Drosophila larval peripheral nervous system (PNS) have become powerful genetic models in which to elucidate both general and class-specific mechanisms of neuron differentiation.
There are four main DA neuron classes (I-IV)1. They are named in order of increasing dendrite arbor complexity, and have class-specific differences in the genetic control of their differentiation2-10. The DA sensory system is a practical model to investigate the molecular mechanisms behind the control of dendritic morphology11-13 because: 1) it can take advantage of the powerful genetic tools available in the fruit fly, 2) the DA neuron dendrite arbor spreads out in only 2 dimensions beneath an optically clear larval cuticle making it easy to visualize with high resolution in vivo, 3) the class-specific diversity in dendritic morphology facilitates a comparative analysis to find key elements controlling the formation of simple vs. highly branched dendritic trees, and 4) dendritic arbor stereotypical shapes of different DA neurons facilitate morphometric statistical analyses.
DA neuron activity modifies the output of a larval locomotion central pattern generator14-16. The different DA neuron classes have distinct sensory modalities, and their activation elicits different behavioral responses14,16-20. Furthermore different classes send axonal projections stereotypically into the Drosophila larval central nervous system in the ventral nerve cord (VNC)21. These projections terminate with topographic representations of both DA neuron sensory modality and the position in the body wall of the dendritic field7,22,23. Hence examination of DA axonal projections can be used to elucidate mechanisms underlying topographic mapping7,22,23, as well as the wiring of a simple circuit modulating larval locomotion14-17.
We present here a practical guide to generate and analyze genetic mosaics24 marking DA neurons via MARCM (Mosaic Analysis with a Repressible Cell Marker)1,10,25 and Flp-out22,26,27 techniques (summarized in Fig. 1).

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

The dendritic arborization sensory neurons of the Drosophila larval peripheral nervous system are useful models to elucidate both general and neuron class-specific mechanisms of neuron differentiation. We present a practical guide to generate and analyze dendritic arborization neuron genetic mosaics.

形态分析果蝇幼虫外周感觉神经元树突和轴突使用遗传马赛克

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

高分辨率实时成像发展中的神经上皮细胞的行为

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of existing mouse lines for genetic cell lineage tracing: a tamoxifen-inducible Cre line and a Cre reporter line expressing dsRed upon Cre-mediated recombination. By using a relatively low level of tamoxifen, we were able to induce recombination in a small number of cells, permitting us to follow individual cell divisions. Additionally, we observed the transcriptional response to Sonic Hedgehog (Shh) signaling using an Olig2-eGFP transgenic line 1-3 and we monitored formation of cilia by infecting the cultured slice with virus expressing the cilia marker, Sstr3-GFP 4. In order to image the neuroepithelium, we harvested embryos at E8.5, isolated the neural tube, mounted the neural slice in proper culturing conditions into the imaging chamber and performed time-lapse confocal imaging. Our ex vivo live imaging method enables us to trace single cell divisions to assess the relative timing of primary cilia formation and Shh response in a physiologically relevant manner. This method can be easily adapted using distinct fluorescent markers and provides the field the tools with which to monitor cell behavior in situ and in real time.

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Here we develop the tools necessary for ex vivo live imaging to trace single cell divisions in the mouse E8.5 neuroepithelium

体外实时成像在小鼠神经上皮单细胞分裂

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of ...

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 ...

Super-resolution Imaging of Neuronal Dense-core Vesicles

Detection of fluorescence provides the foundation for many widely utilized and rapidly advancing microscopy techniques employed in modern biological and medical applications. Strengths of fluorescence include its sensitivity, specificity, and compatibility with live imaging. Unfortunately, conventional forms of fluorescence microscopy suffer from one major weakness, diffraction-limited resolution in the imaging plane, which hampers studies of structures with dimensions smaller than ~250 nm. Recently, this limitation has been overcome with the introduction of super-resolution fluorescence microscopy techniques, such as photoactivated localization microscopy (PALM). Unlike its conventional counterparts, PALM can produce images with a lateral resolution of tens of nanometers. It is thus now possible to use fluorescence, with its myriad strengths, to elucidate a spectrum of previously inaccessible attributes of cellular structure and organization.
Unfortunately, PALM is not trivial to implement, and successful strategies often must be tailored to the type of system under study. In this article, we show how to implement single-color PALM studies of vesicular structures in fixed, cultured neurons. PALM is ideally suited to the study of vesicles, which have dimensions that typically range from ~50-250 nm. Key steps in our approach include labeling neurons with photoconvertible (green to red) chimeras of vesicle cargo, collecting sparsely sampled raw images with a super-resolution microscopy system, and processing the raw images to produce a high-resolution PALM image. We also demonstrate the efficacy of our approach by presenting exceptionally well-resolved images of dense-core vesicles (DCVs) in cultured hippocampal neurons, which refute the hypothesis that extrasynaptic trafficking of DCVs is mediated largely by DCV clusters.

We describe how to implement photoactivated localization microscopy (PALM)-based studies of vesicles in fixed, cultured neurons. Key components of our protocol include labeling vesicles with photoconvertible chimeras, collecting sparsely sampled raw images with a super-resolution microscopy system, and processing the raw images to produce a super-resolution image.

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 ...

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 ...