我们感谢苔丝·拉迈耶女士、露丝·弗兰克女士和迈克尔·穆尼亚克博士的编辑和评论,以及佛罗里达州马克斯·普朗克的Ryohei Yasuda博士对2pFLIM采集软件的介绍。这项工作得到了两个BRAIN倡议奖U01NS094247(H.Z.和T.M.)和R01NS104944(H.Z.和T.M.)的支持,一个R01授予R01NS081071(T.M.),以及R21授予R21NS097856(H.Z.)。所有奖项均来自美国国家神经疾病和中风研究所。

这里描述的所有方法都已获得俄勒冈健康与科学大学的机构动物护理和使用委员会(IACUC)的批准。
1. 2pFLIM 显微镜设置
<ol><li>安装光子定时计数模块(PTCM,材料表),并根据制造商手册连接到计算机(图1)。 注:PTCM 通常是接收激光脉冲计时的"同步"输入和来自光电倍增管 (PMT) 的光子输入的计算机板。它还从双光子成像控制软件接收像素、线条和帧的时钟计时。PTCM 使用时钟信号将单个光子分离到不同的像素和帧中。</li>
	<li>添加具有 >200 MHz 带宽的光电二极管,以测量激光定时。在光道中放置一个标准玻璃盖玻片,将激光的一小部分反射到垂直于光路的光电二极管中(图1)。将光电二极管输出连接到 PTCM 的"同步"输入。 注:许多现代激光器也输出激光正时。对于这些激光器,光电二极管是不必要的,可以直接将激光正时输出连接到PTCM的同步输入。</li>
	<li>
在 tAKAR® 的情况下,使用低噪声、快速 GaAsP PMT 交换 PMT(图 1)。将 PMT 输出连接到 PTCM 的信号输入。
	<ol>
<li>如果需要通过传统的双光子成像通道同时采集强度,则添加可选的信号分路器(图1)。将 PMT 输出连接到信号分路器,并将分路器输出连接到 PTCM 和传统的双光子成像模块。 注:GaAsP PMT 提供比传统双碱 PMT 更快的单光子信号,并允许更精确地确定光子定时。某些型号的 GaAsP PMT 可冷却到环境温度以下的 10~35 °C,从而将暗数抑制到每秒几百以下(通常为 ±200 计数/秒)。这种低噪声水平对于精确测量荧光寿命非常重要,因为噪声光子计数无法从荧光寿命曲线中轻松区分或减去。</li>
	</ol>
</li>
	<li>添加带通荧光发射过滤器,将接受荧光剂的光谱污染(如果有)降至最低。例如,对于 tAKAR®,用于绿色通道的 500 nm × 20 nm 阻隔过滤器用于减少接受器 sREACh 的污染,这是一种深色 (QY =0.07) 黄色荧光蛋白 (YFP)29、30。与控制软件(PTCM 用户手册中所述)连接计时信号,如单个图像像素、线和帧的时钟。安装适当的数据控制和采集软件。 注:一些PTCM制造商 (材料表) 提供其 2pFLIM 成像软件.在这里,使用称为FLIMimage的定制软件,这是由Yasuda实验室(马克斯普朗克佛罗里达州,通过个人沟通)开发的。该软件作为某些双光子采集软件(材料表)的附加用户功能。它在双光子成像过程中以适当的时间控制和与PTCM通信,以获取2pFLIM图像。</li>
</ol>2. 建造电动跑步机
注:定制电动跑步机的设计如图2所示。
<ol><li>使用精细的锯子将泡沫辊 (Φ = 200 mm) 切割至 150 mm 的长度。或者,将泡沫球的两半粘在一起,将胶带放在接缝上。可选择在滚轮上粘附轮廓的橡胶垫,以增加滚子的牵引力。</li>
	<li>在滚子平侧的滚子中心钻一个直径为 1/4 英寸的孔,或者在使用泡沫球时,在球的中间钻一个直径为 1/4 英寸的孔。</li>
	<li>
通过孔安装直径为 1/4 英寸的钢轴。使用与泡沫兼容的胶水将泡沫滚轮/球粘附到轴上。或者,修改两个柔性轴联轴联轴(1/4 英寸内径),以加强轴与泡沫辊/球的耦合。 注:请注意,许多常见的胶水可能会溶解泡沫。<ol>
<li>对于每个轴联轴器,将轴联轴放在其平面和矩形金属板的中心(0.7 mm x 15 mm x 76 mm)。将板焊接到轴联轴器上。在板的中心钻一个 1/4 英寸的孔,以便将修改后的轴联轴安装到轴上,并将两个螺钉孔从中心侧向安装到金属板上。</li>
		<li>将轴联轴接头安装在轴上,与泡沫滚轮/球进行连接。如果使用后者,则稍微弯曲板以适合球的曲率。将螺钉放入侧孔以固定轴上的滚子/球。</li>
	</ol>
</li>
	<li>钻取和敲击笼板中心的 3/8-32 螺纹,然后安装旋转编码器。将螺钉孔钻入直角电机支架的底座,以便将电机连接到柱架上。使用灵活的轴联轴将旋转编码器和电机连接到轴末端。</li>
	<li>使用电机和旋转编码器的柱子将组装的跑步机安装在铝面包板上(图2)。将电机输入连接到速度控制器,并将旋转编码器输出连接到计算机数据采集 (DAQ) 板的模拟输入。 注:旋转角度速度由旋转编码器编码为电压,并使用名为"动物跟踪器"的自定义软件进行数字化,该软件名为"动物跟踪器",用 MATLAB 编写。</li>
	<li>将头板兼容支架安装到右角支架上。在跑步机前面的面包板上安装一个实心支架,并将装配的头板支架与右角支架安装在支架上(图2)。确保头板支架杆与车轴对齐,以便鼠标可以在跑步机上采用适当且舒适的行走位置(图 2C)。</li>
</ol>3. 在小鼠皮质中表达 tAKAR® 传感器
<ol><li>
在子宫电穿孔
	<ol>
<li>在质粒DNA(3⁄4 μg/μL)中加入0.2%的快速绿色染料的最终浓度(用于注射期间的可视化),为IUE制备DNA溶液;传感器结构包含CAG前动、传感器序列和木丘克肝炎病毒转录后反应元件[WPRE]转化增强剂)溶于/稀释在水中或Tris-EDTA中。</li>
		<li>在 E1624为 IUE 准备一只有时位怀孕的雌性小鼠(例如 C57BL/6)。用异构体麻醉小鼠(4%用于诱导,1.5%用于维护,在95%O2与5%CO 2)上,并应用皮下注射,包括5毫克/千克Meloxicam和4mg/kgBupivacaine。用手术刀和剪刀切开腹腔,小心地露出子宫角。</li>
		<li>如前所述,在一个半球的侧心室中注射每个胚胎的1μLDNA溶液。</li>
		<li>将正电极端脚置于皮层,并在 1 Hz 处使用 5 个 100 ms 方脉冲 (38 V),使用电波器,对皮质神经元执行常规 IUE24。 注: 通过改变电极端脚相对于侧心室的位置,可以针对不同的皮质区域进行电穿孔。</li>
	</ol>
</li>
	<li>
立体注射
	<ol>
<li>准备在产后第30天进行老鼠立体手术26。如步骤3.1.2所述,麻醉小鼠,并应用皮下注射含有5毫克/千克卡洛芬的术前镇痛药。</li>
		<li>稀释 AAV 血清型 2/1 (AAV2/1) 将 hSyn-tAKAR®-WPRE 表达到经验测定的定位子 (+1 x 109+1 x10 10 基因组/μL) 注射器过滤 (0.2 μm 纤维素醋酸酯膜) 磷酸盐缓冲盐水。</li>
		<li>在运动皮层的以下坐标下,使用手持钻头在立体显微镜下钻一个直径为 ±500 μm 的孔:0.5 mm 前向布雷格马,1.2 mm 横向到中线。</li>
		<li>将喷油器(例如,带定制柱塞/玻璃移液器支架的油液压操纵器)安装到电动操纵器上。将注射针置于相对于布雷格玛-兰布达平面的 15° 角处。分别沿前后轴和背向轴对角线移动,等效于 700 μm 和 200 μm 的步数。 注:为避免对正上方目标成像场上方的组织造成损伤,AAV 粒子以相对于布雷格玛-兰布达平面的角度注入。</li>
		<li>将注射针的尖端置于钻孔中心的 pia 处,然后缓慢执行上述对角线运动(±25 μm/s)。此过程将注射中心定位在 1.2 mm 前向布雷格马,1.2 mm 横向到中线,0.2 mm 低于 pia。</li>
		<li>注射20 nL稀释的病毒颗粒(±10 nL/min)。等待至少 10 分钟,然后慢慢缩回注射针(±12.5 μm/s)。</li>
		<li>完成立体注射程序,并粘合/缝合皮肤26。</li>
	</ol>
</li>
</ol>4. 颅窗的安装
<ol><li>在产后30至60天,通过IUE(第3.1节)或病毒颗粒的立体注射(第3.2节)对表达tAKAR®的小鼠进行颅状窗口的放置。对于感染病毒颗粒的小鼠,在病毒注射后至少两周实施颅窗口。安装颅窗,如前所述27,28,与以下细节。如步骤3.1.2所述,麻醉小鼠,并在4mg/kg下注射术前镇痛药0.075mg/kgBuprenex和抗炎剂Dexamethasone。</li>
	<li>取出穿孔并缩回颈部肌肉。用组织粘合剂将皮肤边缘粘附到头骨上,以避免手术后颈部肌肉暴露。</li>
	<li>使用手术刀轻轻刮擦,干燥并去除颅骨上的任何骨膜。放置成像头板(8 mm 内径)以环绕预定的成像场。使用氰丙烯酸酯胶水将头板粘到头骨上,然后是牙科丙烯酸水泥。为获得最佳附着力,请确保头板位于裸露和干燥的头骨上。胶水加速器可用于加速硬化。</li>
	<li>使用牙科钻头在预定成像场上方绘制一个直径为 5 mm 的圆(步骤 3.2.3 中指定的坐标),并露出 Dura 母体。</li>
	<li>在杜拉表面涂上一层薄薄的透明聚合物(也称为人造杜拉)以覆盖整个颅面。聚合物将保护和稳定杜拉母体。在杜拉垫上放置无菌圆形盖玻片(直径 5 mm)。用氰丙烯酸胶固定在窗边上,然后涂上牙科丙烯酸水泥。</li>
</ol>5. 在 Vivo 双光子荧光寿命成像显微镜
<ol><li>在颅窗安装后2周或2周以上开始2pFLIM成像(第4节)。在开始成像研究之前,通过频繁处理和擦伤小鼠来适应小鼠,以尽量减少由于压力引起的实验干扰。</li>
	<li>使用控制双光子激光的软件将双光子激发激光波长设置为 960 nm。</li>
	<li>使用 4% 的异常胶对鼠标进行麻醉。通过尾部捏合和观察呼吸速率确认适当的麻醉。也就是说,不应对尾部捏合有反应,呼吸速率应降至每秒±1次。为了尽量减少不必要的程序时间,并且麻醉只持续两到三分钟,不使用眼部润滑剂。</li>
	<li>将麻醉鼠标转移到电动跑步机(图2C),并将鼠标的表板安装到跑步机设置的头板支架上(详情见图2)。用 70% 乙醇清洁鼠标上的颅面窗盖玻片表面。</li>
	<li>将装有鼠标的电动跑步机置于 2pFLIM 目标下。在颅窗盖玻片和目标之间涂抹一滴蒸馏水。</li>
	<li>让装载的鼠标从麻醉中醒来,在跑步机和显微镜环境中适应至少10分钟。</li>
	<li>导航到外光照明下的喷射位置。在明景下记录基准特征(即血管),以帮助在后续成像过程中对同一感兴趣区域 (ROI) 进行成像。</li>
	<li>除脑组织发出的光外,消除任何入射光。关闭外光光源并关闭 2pFLIM 钻机的外壳。通过打开硬件命令电压控制来激活 2pFLIM PMT。</li>
	<li>使用 2pFLIM 采集软件 FLIMimage 获取 z-stack 2pFLIM 图像,并采用以下推荐设置,用于在醒着的小鼠中成像 tAKAR® 阳性 somata。将平均帧设置为 3 帧,扫描速度设置为 2 ms/线,图像大小设置为 128 x 128 像素,将视野设置为 90*100 μm。根据准备和硬件配置调整映像设置。</li>
	<li>
在 FLIMview 中检查获取的图像(内部开发的自定义软件;请参阅第 6 节)。在步骤 5.9 之后调整成像设置,以优化光子计数并最大限度地减少光漂白。 注:根据特定刺激产生的信号振幅,在 ROI 中,一个可行的集成光子计数,用于对体内 tAKAR® 阳性体进行终生成像,其值为 ±1,000~10,000 光子。<ol>
<li>必要时,使用视野减少、扫描速度降低、激光功率增加和平均帧数增加,以增加集成光子计数并减少寿命估计误差。同时,请务必使用最小的基本激光功率、帧平均和扫描速度,以尽量减少光漂白。</li>
	</ol>
</li>
	<li>使用步骤 5.10 中确定的设置重复 z 堆栈采集,以常规时间间隔(例如,每 30-60 秒)进行映像。在零跑步机速度下获取基线 2pFLIM 图像至少 15 分钟。</li>
	<li>将跑步机转速设置为 ±15 cm/s 15 分钟,同时获取 2pFLIM 图像。关闭跑步机旋转后,继续成像 20 分钟,以评估强制运动停止后 PKA 活动的持续时间。</li>
</ol>6. 分析 2pFLIM 图像
<ol><li>
在 FLIMview 中打开获取的图像,并在 FLIMview 中设置以下参数。 注:参数详细信息在讨论中描述。<ol>
<li>单击 FLIMview 中的单个光子计数 (SPC) 最小和最大范围字段。输入适当的最小值和最大 SPC 范围值,通常分别介于 1.2⁄2 和 10*12 ns 之间。</li>
		<li>单击 FLIMview 中的 t0值字段并输入 t0值(通常为 ±2 ns)。单击 FLIMview 中的生存期亮度最小阈值值字段,并将所需的阈值输入 5±30 光子。</li>
	</ol>
</li>
	<li>单击新的组按钮 (N) 并指定实验组名称。这将生成一个组合来自每个添加的 FLIM 图像的数据的组。</li>
	<li>单击 FLIMview 的ROI 控制模块中的ROI按钮,并围绕 tAKAR® 阳性索马绘制 ROI。通过在 FLIMview 中的 z 堆栈控制滑块中移动z-stack 控制滑块的下限和上限,减小 z 堆栈范围,以尽量减少来自其他 z 深度背景光子的信号污染。</li>
	<li>单击+按钮将 FLIM 图像添加到组中(步骤 6.2)。单击Calc按钮以计算 ROI 和生存期估计误差 (*) 的平均寿命(LT,也称为平均光子发射时间 [MPET])。</li>
	<li>打开慢性 2pFLIM 成像系列中的下一个文件。重复步骤 6.4。请务必调整 ROI 和 z 堆栈范围的位置,以测量相同的 tAKAR® 阳性索马随时间推移,因为随着时间的推移,组织可能会漂移。</li>
	<li>在组控制模块的下拉菜单中选择增量MPET/MPET0。 单击"基线+"字段并输入索引(例如,对于步骤 6.3 中创建的组中的前五个图像,索引为 1 2 3 4 5)。这将定义用于计算基准生存期 (LT0) 的图像。</li>
	<li>单击绘图以生成包含 tAKAR® 的 FLIM 响应 ([LT/LT0) 的图形。"通过相应的基准生存期 (LT 0) 对单个 ROI 的生存期(+LT) 进行规范化更改,可以比较不同 ROI 在运动期间的 PKA 活动。</li>
</ol>

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作者没有什么可透露的。

该协议演示了使用 FRET-FLIM 传感器 tAKAR® 来可视化头部固定行为小鼠的神经调节触发的 PKA 活动。此应用基于 TAKAR® 体外和体内的广泛测试和表征,以证明获得的 FLIM 信号与生理神经调节事件17相关。在这里,一个在体内的应用,运动诱导的PKA活动在运动皮层,用于描述程序,提供传感器到大脑,动物手术成像,硬件和软件要求的行为和成像数据采集,用于成像数据分析的软件和算法。
<p ...

神经调节,也称为缓慢突触传输,在不同的行为状态(如压力、觉醒、注意力和运动1、2、3)期间对大脑功能施加强有力的控制。4.尽管其重要性,神经调节事件发生的时间和地点的研究仍处于起步阶段。神经调节剂,包括乙酰胆碱、多巴胺、肾上腺素、血清素和许多神经肽,激活G蛋白耦合受体(GPCRs),进而触发细胞内第二信使通路,时间范围很宽从秒到小时。虽然每个神经调节器触发一组不同的信号事件,cAMP/蛋白质激酶A(PKA)通路是许多神经调节器1,5的常见下游通路。cAMP/PKA通路调节神经元兴奋性、突触性传播和可塑性6,7,8,9,因此,调节神经元网络动力学。由于不同的神经元或神经元类型表达不同类型或水平的神经调节器受体10,同一细胞外神经调节器的细胞内效应可能是异质的,因此,必须研究与细胞分辨率。迄今为止,在行为过程中监测体内单个神经元的神经调节事件仍然具有挑战性。
为了研究神经调节的时空动力学,需要一种合适的记录方式。微透析和快速扫描循环伏特经常用于研究神经调节器的释放,但它们缺乏监测细胞事件11、12的空间分辨率。PKA成像与钙动力学一样,在群体成像13中用作神经元电活动的代理,PKA成像可用于以细胞分辨率读取神经元群中的神经调节事件。本协议描述了使用改进的A-激酶活动报告器(AKAR)来监测动物行为期间体内的PKA活动。此处描述的方法允许以亚细胞分辨率同时成像神经元群,其时间分辨率可跟踪生理神经调节事件。
AKAR由一个供体和接受荧光蛋白组成,由PKA磷酸化基质肽和叉头相关(FHA)域连接,与基质14、15的磷酸化丝氨酸或三甲氨酸结合。PKA通路激活后,AKAR的基质肽被磷酸化。因此,FHA 域与磷酸化基质肽结合,从而使两个荧光苷酸接近,称为 AKAR 的闭合状态。磷酸化AKAR的封闭状态导致供体和受体荧光量之间增加Fürster共振能量转移(FRET)。由于磷酸化ARNA的比例与PKA活性16的水平有关,生物样品中的FRET量可用于量化PKA活性16、17、18的含量。 19,20.
早期版本的AKARs主要设计为双色比例成像14。当成像深入到脑组织,比例法遭受信号失真,由于波长依赖光散射17,18,21。如下所述,荧光寿命成像显微镜(FLIM)消除了这个问题,因为FLIM只测量供体荧光量18、21发出的光子。因此,FRET 的 FLIM 定量不受组织深度17的影响。此外,可以使用接受器荧光的"暗"(即低量子收率[QY])变体。这释放了一个颜色通道,以方便通过同步成像第二个传感器或形态标记17,19,20的正交神经元属性的多路测量。
FLIM成像量化荧光在兴奋状态下花费的时间,即荧光寿命18。荧光回到地面状态,从而结束兴奋状态,通常伴随光子的发射。虽然单个兴奋分子的光子发射是随机的,但在人群中,平均荧光寿命是该特定荧光的特性。当纯荧光群同时激发时,产生的荧光将跟随单个指数衰变。此指数衰减的时间常数对应于荧光蛋白的平均荧光寿命,荧光蛋白通常为 1 到 4 纳秒。FRET 也可以将兴奋的供体荧光剂返回到地面状态。在FRET的存在下,供体荧光的荧光寿命缩短。非磷酸化的AVR表现出相对较长的供体荧光寿命。在PKA的磷酸化后,传感器的寿命较短,因为供体和受体的荧光量彼此靠近,FRET增加。因此,在AKARs的种群中荧光寿命的量化代表PKA活性水平。
早期版本的AKARs尚未成功用于单细胞分辨率的体内成像。这主要是由于AKAR传感器对生理活化信号振幅低。最近,通过系统地比较两光子荧光寿命成像显微镜(2pFLIM)的可用AKAR传感器,发现一种名为FLIM-AKAR的传感器性能优于替代传感器。此外,还开发了一系列称为靶向AKARs(tAKARs)的FLIM-AKAR变体,以可视化特定亚细胞位置的PKA活性:微管(tAKAR®)、细胞醇(tAKAR®)、活性素(tAKAR®)、丝状活性素(tAKAR®)、膜(tAKARé)、膜(tAKARé)、微管(tAKAR®)、细胞醇(tAKAR®)、活性素(tAKAR®)、膜(tAKARé)、膜(tAKARé)、微管(tAKAR®)、细胞源性活性素(tAKAR®)、活性剂(tAKAR®)。(tAKAR®),和鼻后密度(tAKAR*)。在tAKARs中,tAKAR®将去甲肾上腺素引起的信号振幅提高了2.7倍。这与神经元中的大多数PKA固定在静止状态22、23的微管上的知识是一致的。tAKAR® 是现有 2pFLIM 的 AVR 中表现最好的。此外,tAKAR®检测到由多个神经调节剂引起的与生理相关的PKA活性,tAKAR®的表达并没有改变神经元功能17。
最近,tAKAR®成功地用于可视化头固定行为小鼠17中的PKA活动。结果表明,强制运动触发了运动、桶和视觉皮质中表层神经元(第1层至3层,距离pia深度为+300μm)的PA活性。运动触发的PKA活性部分依赖于通过β-肾上腺素受体和D1多巴胺受体的信号,但不受D2多巴胺受体拮抗剂的影响。这项工作说明了tAARs使用2pFLIM跟踪体内神经调节事件的能力。
在当前协议中,在强制运动范式期间,在头部固定的醒小鼠中进行 PKA 活动成像的整个方法用六个步骤来描述。首先,在传统的双光子显微镜中增加2pFLIM功能(图1)。第二,建造电动跑步机(图2)。第三,通过子宫电穿孔(IUE)的DNA质粒,或对腺相关病毒(AAV)的立体注射,在小鼠皮层中表达tAKAR®传感器。IUE24、25和立体注射病毒颗粒26的出色手术方案已经发表。我们使用的关键参数如下所述。第四,安装一个颅窗。优秀的方案已经发表颅窗手术27,28。介绍了从标准协议修改的几个步骤。第五,在体内表演2pFLIM。第六,分析2pFLIM图像(图3和图4)。这种方法应该很容易适用于许多其他头部固定的行为范式和大脑区域。

<table><tbody><tr><td>0.2 &amp;mu;m cellulose acetate syringe filter</td><td>Nalgene</td><td>190-2520</td><td>Step 3.2.2.</td></tr><tr><td>16x 0.8 NA water-immersion objective</td><td>Nikon</td><td>MRP07220</td><td>Step 5.5.</td></tr><tr><td>3-pin cable</td><td>US digital</td><td>CA-MIC3-SH-NC</td><td>Step 2.5. To connect rotation sensor to the DAQ input of the microscope</td></tr><tr><td>Aluminum bread board</td><td>Thorlabs</td><td>MB1012</td><td>Step 2.5.</td></tr><tr><td>AnimalTracker MATLAB software</td><td>N/A</td><td>N/A</td><td>Step 2.5 and sections 5 - 6. Will be provided upon request to the lead author</td></tr><tr><td>Band-pass barrier filter</td><td>Chroma</td><td>ET500-40m</td><td>Step 1.4.</td></tr><tr><td>Cage plate</td><td>Thorlabs</td><td>CP01</td><td>Step 2.4. Used as mount for rotation sensor</td></tr><tr><td>Carbon steel burrs for micro drill, 0.5 mm tip diameter</td><td>FST</td><td>19007-05</td><td>Steps 3.2.3. and 4.4.</td></tr><tr><td>Circular coverslip (5 mm diameter)</td><td>VWR</td><td>101413-528</td><td>Step 4.5.</td></tr><tr><td>Custom-made injection needle holder</td><td>N/A</td><td>N/A</td><td>Step 3.2.4. Technical details provided upon request to the lead author</td></tr><tr><td>Dental acrylic</td><td>Yates Motloid</td><td>44114</td><td>Steps 4.3. and 4.5.</td></tr><tr><td>Dental drill; Microtorque ii</td><td>Ram products</td><td>66699</td><td>Steps 3.2.3. and 4.4.</td></tr><tr><td>Dowsil transparent polymer</td><td>The Dow Chemical Company</td><td>3-4680</td><td>Step 4.5. Artificial dura</td></tr><tr><td>Electroporation electrode</td><td>Bex</td><td>LF650P5</td><td>Step 3.1.4.</td></tr><tr><td>Electroporator</td><td>Bex</td><td>CUY21</td><td>Step 3.1.4.</td></tr><tr><td>Fast green FCF</td><td>Sigma-aldrich</td><td>F7258-25G</td><td>Step 3.1.1.</td></tr><tr><td>FLIMimage MATLAB software</td><td>N/A</td><td>N/A</td><td>Section 5. Kindly provided by Dr. Ryohei Yasuda, Max Planck Florida</td></tr><tr><td>FLIMview MATLAB software</td><td>N/A</td><td>N/A</td><td>Sections 5. and 6. Will be provided upon request to the lead author</td></tr><tr><td>Foam-compatible glue (Gorilla White Glue)</td><td>Gorilla</td><td>5201204</td><td>Step 2.3.</td></tr><tr><td>Headplate</td><td>N/A</td><td>N/A</td><td>Step 4.3. Technical details provided upon request to the lead author</td></tr><tr><td>Headplate holder</td><td>N/A</td><td>N/A</td><td>Step 2.6. Technical details provided upon request lead author, used in combination with mounting post bracket and right-angled bracket</td></tr><tr><td>Hydraulic micromanipulator</td><td>Narishige</td><td>MO-10</td><td>Step 3.2.4.</td></tr><tr><td>Krazy glue</td><td>Krazy glue</td><td>KG82648R</td><td>Step 4.3. Cyanoacrylate-based glue</td></tr><tr><td>Low-noise fast photomultiplier tube</td><td>Hamamatsu</td><td>H7422PA-40 or H10769PA-40</td><td>Step 1.3.</td></tr><tr><td>MATLAB 2012b</td><td>Mathworks</td><td>N/A</td><td>Steps 2.6, and sections 5, and 6. Used to run microscope acquisition and data analysis software</td></tr><tr><td>Motor</td><td>Zhengke</td><td>ZGA37RG</td><td>Step 2.4.</td></tr><tr><td>Motor speed controller</td><td>Elenker</td><td>EK-G00015A1-1</td><td>Step 2.5.</td></tr><tr><td>Motorized micromanipulator</td><td>Sutter</td><td>MP-285</td><td>Step 3.2.4.</td></tr><tr><td>Mounting base</td><td>Thorlabs</td><td>BA1S</td><td>Step 2.5. Used for posts for motor and sensor in combination with PH4 and TR2</td></tr><tr><td>Mounting post</td><td>Thorlabs</td><td>P14</td><td>Step 2.6. Used for headplate holder post in combination with PB2</td></tr><tr><td>Mounting post base</td><td>Thorlabs</td><td>PB2</td><td>Step 2.6. Used for headplate holder post in combination with P14</td></tr><tr><td>Mounting post bracket</td><td>Thorlabs</td><td>C1515</td><td>Step 2.6. Used in combination with right-angle bracket and headplate holder</td></tr><tr><td>Optical post</td><td>Thorlabs</td><td>TR2</td><td>Step 2.5. Used for posts for motor and sensor in combination with BA1S and PH4</td></tr><tr><td>Phosphate-buffered saline</td><td>&amp;Nu;/&amp;Alpha;</td><td>&amp;Nu;/&amp;Alpha;</td><td>Step 3.2.2. Protocol: Cold Spring Harbor Protocols 2006, doi: 10.1101/pbd.rec8247</td></tr><tr><td>Photodiode</td><td>Thorlabs</td><td>FDS010</td><td>Step 1.2.</td></tr><tr><td>Photon timing counting module</td><td>Becker and Hickl</td><td>SPC-150</td><td>Step 1.1.</td></tr><tr><td>Plasmid: tAKAR&amp;alpha; (CAG-tAKAR&amp;alpha;-WPRE)</td><td>Addgene</td><td>119913</td><td>Step 3.1.3.</td></tr><tr><td>Post holder</td><td>Thorlabs</td><td>PH4</td><td>Step 2.5. Used for posts for motor and sensor in combination with BA1S and TR2</td></tr><tr><td>Right-angle bracket</td><td>Thorlabs</td><td>AB90</td><td>Step 2.6 Used in combination with mounting post bracket and headplate holder</td></tr><tr><td>Rotation encoder</td><td>US digital</td><td>MA3-A10-250-N</td><td>Step 2.4.</td></tr><tr><td>Rubber mat</td><td>Rubber-Cal</td><td>B01DCR5LUG</td><td>Step 2.1.</td></tr><tr><td>Shaft coupling (1/4 inch x 1/4 inch)</td><td>McMaster</td><td>6208K433</td><td>Steps 2.3. and 2.4.</td></tr><tr><td>ScanImage 3.6</td><td>Svoboda Lab/Vidrio Technology</td><td>N/A</td><td>Steps 5.9. and 6.1.</td></tr><tr><td>Signal splitter</td><td>Becker and Hickl</td><td>HPM-CON-02</td><td>Step 1.3.1.</td></tr><tr><td>Stainless steel axle (diameter 1/4 inch, L = 12 inch)</td><td>McMaster</td><td>1327K66</td><td>Step 2.3.</td></tr><tr><td>Stereotaxic alignment systsem</td><td>David kopf</td><td>1900</td><td>Steps 3.2. and 4.1. modified; Sutter micromanipulator, custom-made injection needle holder, hydraulic micromanipulator</td></tr><tr><td>Two-photon microscope</td><td>N/A</td><td>N/A</td><td>Section 5. Built based on Modular in vivo multiphoton microscopy system (MIMMS) from HHMI Janelia Research Campus (https://www.janelia.org/open-science/mimms)</td></tr><tr><td>Vetbond tissue adhesive</td><td>3M</td><td>14006</td><td>Step 3.2.6.</td></tr><tr><td>Virus: tAKAR&amp;alpha; (AAV2/1 hSyn-tAKAR&amp;alpha;-WPRE)</td><td>Addgene</td><td>119921</td><td>Step 3.2.2.</td></tr><tr><td>White PE foam roller (8 inch x 12 inch)</td><td>Fabrication enterprises INC.</td><td>30-2261</td><td>Step 2.1.1.</td></tr><tr><td>White polystyrene fom ball halves</td><td>GrahamSweet</td><td>200mm diameter 2 hollow halves</td><td>Step 2.1.1.</td></tr><tr><td>Zipkicker</td><td>PACER</td><td>PT29</td><td>Step 4.3. Hardening accelerator</td></tr></tbody></table>

visualizing protein kinase a activity in head-fixed behaving mice using in vivo two-photon fluorescence lifetime imaging microscopy

神经调节对大脑功能有强大的控制作用。神经调节系统功能障碍导致神经和精神疾病。尽管技术非常重要,但用细胞分辨率跟踪神经调节事件的技术才刚刚开始出现。神经调节剂,如多巴胺、去甲肾上腺素、乙酰胆碱和血清素,通过各自的G蛋白耦合受体触发细胞内信号事件,以调节神经元兴奋性、突触通信和其他神经元功能,从而调节神经元网络中的信息处理。上述神经调节器收敛到cAMP/蛋白激酶A(PKA)通路。因此,在体内,具有单细胞分辨率的PKA成像被开发为神经调节事件的读出,其方式类似于神经元电活动的钙成像。在此,提出一种方法,在头部固定行为小鼠皮层的各个神经元水平上可视化PKA活动。为此,使用了一个改进的A-激酶活动报告器(AKAR),称为tAKAR®,它基于Fürster共振能量转移(FRET)。这种基因编码的PKA传感器通过DNA质粒的子宫电穿孔(IUE)或腺相关病毒(AAV)的立体注射引入运动皮层。FRET 变化使用双光子荧光寿命成像显微镜 (2pFLIM) 进行成像,该显微镜比比例 FRET 测量具有优势,用于量化光散射脑组织中的 FRET 信号。为了研究强迫运动期间的PKA活动,tAKAR®通过清醒、头部固定的小鼠皮层上方的慢性颅窗成像,这些小鼠在速度控制的电动跑步机上运行或休息。这种成像方法将适用于许多其他大脑区域,以研究相应的行为诱导的PKA活动和其他基于FLIM的传感器进行体内成像。

FRET-FLIM 传感器允许可视化许多不同的信号通路,包括神经调节中涉及的 cAMP/PKA 通路。当前协议利用最近开发的 takaR® 传感器与 2pFLIM 相结合,在头部固定行为小鼠中可视化 PKA 活动。大多数现有的双光子显微镜可以通过添加三到四个组件来升级,如图 1所示(另请参阅第 1 节)。为了在2pFLIM采集的图像中可视化FRET,对每像素收集的光子计时的直方图图进行了?...

Watch this Scientific Journal Video about Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy at JoVE.com

Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy

提出了一个程序,以可视化蛋白激酶A活动在头部固定,行为小鼠。改进的A-激酶活动报告器tAKAR®在皮质神经元中表达,并通过颅窗口进行成像。双光子荧光寿命成像显微镜用于在强制运动期间可视化体内的PKA活动。

可视化蛋白质激酶 在头部固定行为小鼠中使用 Vivo 双光子荧光寿命成像显微镜的活性

Neuromodulation exerts powerful control over brain function. Dysfunction of neuromodulatory systems results in neurological and psychiatric disorders. ...

我们的方法利用了一种新的蛋白激酶一种称为目标A激酶活动报告器阿尔法或塔卡的传感器

visualizing-protein-kinase-activity-head-fixed-behaving-mice-using

Research

JoVE Journal

Neuroscience

8.4K 次观看.  Oregon Health & Science University. 我们的方法利用了一种新的蛋白激酶一种称为目标A激酶活动报告器阿尔法或塔卡的传感器

视频: Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy

Two-photon Calcium Imaging in Mice Navigating a Virtual Reality Environment

In recent years, two-photon imaging has become an invaluable tool in neuroscience, as it allows for chronic measurement of the activity of genetically identified cells during behavior1-6. Here we describe methods to perform two-photon imaging in mouse cortex while the animal navigates a virtual reality environment. We focus on the aspects of the experimental procedures that are key to imaging in a behaving animal in a brightly lit virtual environment. The key problems that arise in this experimental setup that we here address are: minimizing brain motion related artifacts, minimizing light leak from the virtual reality projection system, and minimizing laser induced tissue damage. We also provide sample software to control the virtual reality environment and to do pupil tracking. With these procedures and resources it should be possible to convert a conventional two-photon microscope for use in behaving mice.

Here we describe the experimental procedures involved in two-photon imaging of mouse cortex during behavior in a virtual reality environment.

在小鼠导航虚拟现实环境的双光子钙成像

In recent years, two-photon imaging has become an invaluable tool in neuroscience, as it allows for chronic measurement of the activity of genetically ...

科研

行为学

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution . The method consists in implanting a cranial imaging window, injecting a fluorescent calcium indicator dye that can be taken up by large numbers of neurons and finally recording the activity of neurons with time lapse calcium imaging using an in-vivo two photon microscope. Co-injection of astrocyte-specific dyes allows one to differentiate neurons from astrocytes. The technique can be performed in mice expressing fluorescent molecules in specific subpopulations of neurons to better understand the network interactions of different groups of cells.

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution .

在体内2光子层钙成像对小鼠的2 / 3

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  ...

生物学

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

在体内双光子成像的经验依赖的分子在大脑皮质神经元的变化

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

神经科学

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

同时双光子<em&gt;在体内</em&gt;突触输入的影像和突触后目标中的鼠标Retrosplenial的Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

In Vivo Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located in. Although traditional imaging techniques are useful in identifying protein interactions and morphology in fixed tissue, they are not able to give insight as to how quickly cells are able to bind and uptake proteins, and once activated how their morphology changes in vivo. In the present study, we ask 2 major questions: 1) what is the time-course of fibroblast activation via toll-like receptor-4 (TLR4) and lipopolysaccharide (LPS) interaction and 2) how do these cells behave once activated? Using 2-photon imaging, we have developed a novel technique to assess the ability of LPS-FITC to bind to its cognate receptor, TLR4, expressed on peripheral fibroblasts in the genetic reporter mouse line; FSP1cre; tdTomatolox-stop-lox&#160;in vivo. This unique approach allows researchers to create in-depth, time-lapse videos and/or pictures of proteins interacting with live cells that allows one to have an increased level of granularity in understanding how proteins can alter cellular behavior.

Morphological changes occur in immune responsive fibroblast cells following activation and promote alterations in cellular recruitment. Utilizing 2-photon imaging in conjunction with a genetically engineered Fibroblast-specific protein 1 (FSP1)-cre; tdTomato floxed-stop-floxed (TB/TB) mouse line and green fluorescently tagged lipopolysaccharide-FITC, we can illustrate highly specific uptake of lipopolysaccharide in dermal fibroblasts and morphological changes in vivo.

在 Vivo 双色 2 光子成像中,基因标记报告器细胞在皮肤中

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located ...

生物工程

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.

在体内果蝇前运动神经末端的单分子跟踪

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

小鼠视网膜的经瞳双光子体内成像

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

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 ...

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the other hand, two-photon imaging can resolve the activity of local neural circuits at the single-cell level. It is critical to make a large cranial window to perform multiple-scale analysis using both imaging techniques in the same mouse. To achieve this, one must remove a large section of the skull and cover the exposed cortical surface with transparent materials. Previously, glass skulls and polymer-based cranial windows have been developed for this purpose, but these materials are not easily fabricated. The present protocol describes a simple method for making a large cranial window consisting of commercially available polyvinylidene chloride (PVDC) wrapping film, a transparent silicone plug, and a cover glass. For imaging the dorsal surface of an entire hemisphere, the window size was approximately 6 x 3 mm2. Severe brain vibrations were not observed regardless of such a large window. Importantly, the condition of the brain surface did not deteriorate for more than one month. Wide-field imaging of a mouse expressing a genetically-encoded calcium indicator (GECI), GCaMP6f, specifically in astrocytes, revealed synchronized responses in a few millimeters. Two-photon imaging of the same mouse showed prominent calcium responses in individual astrocytes over several seconds. Furthermore, a thin layer of an adeno-associated virus was applied to the PVDC film and successfully expressed GECI in cortical neurons over the cranial window. This technique is reliable and cost-effective for making a large cranial window and facilitates the investigation of the neural and glial dynamics and their interactions during behavior at the macroscopic and microscopic levels.

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

The present protocol describes making a large (6 x 3 mm2) cranial window using food wrap, transparent silicone, and cover glass. This cranial window allows in vivo wide-field and two-photon calcium imaging experiments in the same mouse.

体内 使用大颅窗的小鼠宽场和双光子钙成像

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the ...

Real-Time Monitoring of Aurora kinase A Activation using Conformational FRET Biosensors in Live Cells

Epithelial cancers are often hallmarked by the overexpression of the Ser/Thr kinase Aurora A/AURKA. AURKA is a multifunctional protein that activates upon its autophosphorylation on Thr288. AURKA abundance peaks in mitosis, where it controls the stability and the fidelity of the mitotic spindle, and the overall efficiency of mitosis. Although well characterized at the structural level, a consistent monitoring of the activation of AURKA throughout the cell cycle is lacking. A possible solution consists in using genetically-encoded F&#246;rster&#8217;s Resonance Energy Transfer (FRET) biosensors to gain insight into the autophosphorylation of AURKA with sufficient spatiotemporal resolution. Here, we describe a protocol to engineer FRET biosensors detecting Thr288 autophosphorylation, and how to follow this modification during mitosis. First, we provide an overview of possible donor/acceptor FRET pairs, and we show possible cloning and insertion methods of AURKA FRET biosensors in mammalian cells. Then, we provide a step-by-step analysis for rapid FRET measurements by fluorescence lifetime imaging microscopy (FLIM) on a custom-built setup. However, this protocol is also applicable to alternative commercial solutions available. We conclude by considering the most appropriate FRET controls for an AURKA-based biosensor, and by highlighting potential future improvements to further increase the sensitivity of this tool.

The activation of the multifunctional Ser/Thr kinase AURKA is hallmarked by its autophosphorylation on Thr288. Fluorescent probes relying on FRET can discriminate between its inactive and activated states. Here, we illustrate some strategies for probe engineering, together with a rapid FRET protocol to follow the kinase activation throughout mitosis.

使用活细胞中的构象FRET生物传感器实时监测极光激酶A活化

Epithelial cancers are often hallmarked by the overexpression of the Ser/Thr kinase Aurora A/AURKA. AURKA is a multifunctional protein that activates upon ...

可视化蛋白质激酶在头部固定行为小鼠中使用 Vivo 双光子荧光寿命成像显微镜的活性

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在小鼠导航虚拟现实环境的双光子钙成像

在体内2光子层钙成像对小鼠的2 / 3

在体内双光子成像的经验依赖的分子在大脑皮质神经元的变化

同时双光子

在 Vivo 双色 2 光子成像中,基因标记报告器细胞在皮肤中

在体内果蝇前运动神经末端的单分子跟踪

小鼠视网膜的经瞳双光子体内成像

清醒小鼠小胶质细胞动力学和神经元活动的同时成像

体内使用大颅窗的小鼠宽场和双光子钙成像

使用活细胞中的构象FRET生物传感器实时监测极光激酶A活化

可视化蛋白质激酶 在头部固定行为小鼠中使用 Vivo 双光子荧光寿命成像显微镜的活性

副本

摘要

探索更多视频

此视频中的章节

在小鼠导航虚拟现实环境的双光子钙成像

在体内2光子层钙成像对小鼠的2 / 3

在体内双光子成像的经验依赖的分子在大脑皮质神经元的变化

同时双光子

在 Vivo 双色 2 光子成像中,基因标记报告器细胞在皮肤中

在体内果蝇前运动神经末端的单分子跟踪

小鼠视网膜的经瞳双光子体内成像

清醒小鼠小胶质细胞动力学和神经元活动的同时成像

体内 使用大颅窗的小鼠宽场和双光子钙成像

使用活细胞中的构象FRET生物传感器实时监测极光激酶A活化

可视化蛋白质激酶在头部固定行为小鼠中使用 Vivo 双光子荧光寿命成像显微镜的活性

体内使用大颅窗的小鼠宽场和双光子钙成像