Name: light spot-based assay for analysis of drosophila larval phototaxis
Uploaded: 2019-09-27T14:45:00
Description: Watch this Scientific Journal Video about Light Spot-Based Assay for Analysis of Drosophila Larval Phototaxis at JoVE.com

这项工作得到了中国自然科学基金（31671074）和浙江省高校基础研究基金（2019XZZX003-12）的支持。

1.果蝇幼虫的制备
<ol><li>准备标准介质，包括煮玉米粉（73克）、琼脂（5.6克）、大豆粉（10克）、酵母（17.3克）、糖浆（76 mL）和水（1000 mL）。</li>
	<li>在12小时/12小时光/暗循环的房间里，在标准介质上以25°C升起所有苍蝇。</li>
</ol>2. 准备琼脂板
<ol><li>准备 1.0% 琼脂溶液。在500 mL的烧杯中称量3克琼脂，并加入300 mL的蒸馏水。在断路器上放置箔纸，以防止水蒸发。在微?...

<ol>
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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+visual+pathways+mediate+Drosophila+larval+light+avoidance+and+circadian+clock+entrainment.">Distinct visual pathways mediate Drosophila larval light avoidance and circadian clock entrainment.</a> Journal of Neuroscience. 31, 6527 (2011).</li><li>Keene, A. C., Sprecher, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeing+the+light:+photobehavior+in+fruit+fly+larvae.">Seeing the light: photobehavior in fruit fly larvae.</a> Trends in Neurosciences. 35, (2012).</li><li>Kane, E. 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该协议提出了光斑测定，以测试果蝇幼虫从光中逸出的能力。这种测定允许跟踪幼虫在进入之前、遇到期间和离开光斑后的行为。可以捕获和分析幼虫运动的细节。光斑测定非常简单，具有很强的实用性。整个设备的成本不高。在实验中，LED灯被用作光源。如果需要，可以替换为不同波长的光源。光照强度也可以由 LED 驱动器调整。现场最低光照强度可达1.80 pW/mm2（冷白光）。即使?...

在觅食阶段，果蝇的幼虫表现出明显的避光行为。果蝇幼虫照片一直在调查，特别是在过去50年1，2，3，4，5，6，7 ，8.近年来，尽管事实上1）许多神经元调解幼虫光避免已被确定4，5，9，10，11，122）在突触的分辨率下，幼虫视觉系统的完整连接体已经建立13个，幼虫光突厥背后的神经机制基本仍不清楚。
在研究幼虫光轴时，已经使用了一些行为测定。它们大致可以分为两类：一类涉及空间光梯度，另一类涉及时光梯度。对于空间光梯度测定，竞技场在光明和黑暗中被分成相等数量的截面。竞技场可以分为光明和黑暗两部分2，4或光明和暗象限14，15，甚至可以分为交替的光明和黑暗方块，如棋盘7。通常，琼脂板用于空间光梯度测定，但被分为交替光和暗部分的管子也可以使用10，14。
在旧版本的测定中，光照通常源自幼虫的下方。然而，较新版本的照明主要来自上述，因为幼虫眼（例如，对低或中光强度16敏感的Bolwig器官）包含在不透明的头足骨骨架中，其开口朝向上前。这使得幼虫对来自上前方向的光比从方向7后面的下方对光更敏感。对于时光梯度测定，竞技场中的光强度在空间上是均匀的，但强度会随时间而变化。除了时态方波光（即闪烁开/关或强/弱光3，7），与线性强度斜带一致的时态变化光也使用8测量幼虫对时间变化的光刺激。
第三种类型的光射器测定是定向光景导航，它涉及从上面以45°7的角度进行照明。在Kane等人7号工作之前，在幼虫光刻测定中只计算了幼虫在光和暗区域的数量、转弯频率和足迹长度等粗糙参数。由于同一组的工作，通过分析幼虫光片的高时态分辨率视频记录，在光刻过程中幼虫运动的详细动态（即幼虫身体不同部位的瞬间速度、方向、转弯角度和相应的角速度）已被分析7。因此，已经能够发现更多的幼虫光轴行为的细节。在这些测定中，幼虫在组内测试，因此不排除组效应。
该协议引入了一种光点测定，用于研究幼虫对个体光刺激的行为反应。主要实验装置由视觉刺激系统和红外光成像系统组成。在视觉刺激系统中，LED光源在琼脂板上产生一个直径为2厘米的圆形光点，对幼虫进行测试。可以使用 LED 驱动器调整光照强度。成像系统包括一个红外摄像机，除了三个为摄像机提供照明的 850 nm 红外 LED 外，还可捕获幼虫的行为。相机镜头由 850 nm 带通滤镜覆盖，以阻止视觉刺激系统的光线进入摄像机，同时允许红外光进入摄像机。因此，视觉刺激对成像的干扰是可以避免的。在本次测定中，记录和分析进入光之前、期间和之后的一段时间内个体幼虫快速反应的行为细节。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>850 nm &#177; 3 nm infrared-light-generating LED</td>
			<td>Thorlabs, USA</td>
			<td>PM100A</td>
			<td>Compatible Sensors: Photodiode and Thermal 
			Optical Power Rangea: 100 pW to 200 W 
			Available Sensor Wavelength Rangea: 185 nm-25 &#956;m Display Refresh Rate: 20 Hz 
			Bandwidtha: DC-100 kHz 
			Photodiode Sensor Rangeb: 50 nA-5 mA 
			Thermopile Sensor Rangeb: 1 mV-1 V</td>
		</tr>
		<tr>
			<td>AC to DC converter</td>
			<td>Thorlabs, USA</td>
			<td>S120VC</td>
			<td>Aperture Size: &#216;9.5 mm 
			Wavelength Range: 200-1100 nm 
			Power Range: 50 nW-50 mW 
			Detector Type: Si Photodiode (UV Extended) 
			Linearity: &#177;0.5% 
			Measurement Uncertaintyc: &#177;3% (440-980 nm), &#177;5% (280-439 nm), &#177;7% (200-279 nm, 981-1100 nm)</td>
		</tr>
		<tr>
			<td>band-pass filter</td>
			<td>Thorlabs, USA</td>
			<td>DC2100</td>
			<td>LED Current Range: 0-2 A 
			LED Current Resolution: 1 mA 
			LED Current Accuracy: &#177;20 mA 
			LED Forward Voltage: 24 V 
			Modulation Frequency Range: 0-100 kHz Sine Wave 
			Modulation: Arbitrary</td>
		</tr>
		<tr>
			<td>Collimated LED blue light&#160;</td>
			<td>ELP, China</td>
			<td>USBFHD01M</td>
			<td>Max. Resolution: 1920X1080 
			F6.0 mm 
			Sensor: 1/2.7&#34; CMOS OV2710</td>
		</tr>
		<tr>
			<td>Compact power meter console&#160;</td>
			<td>Ocean Optics, USA</td>
			<td>USB2000+(RAD)</td>
			<td>Dimensions: 89.1 mm x 63.3 mm x 34.4 mm 
			Weight: 190 g 
			Detector: Sony ILX511B (2048-element linear silicon CCD array) 
			Wavelength range: 200-850 nm 
			Integration time: 1 ms &#8211; 65 seconds (20 seconds typical) 
			Dynamic range: 8.5 x 10^7 (system); 1300:1 for a single acquisition 
			Signal-to-noise ratio: 250:1 (full signal) 
			Dark noise: 50 RMS counts 
			Grating: 2 (250 &#8211; 800 nm) 
			Slit: SLIT-50 
			Detector collection lens: L2 
			Order-sorting: OFLV-200-850 
			Optical resolution: ~2.0 nm FWHM 
			Stray light: &#60;0.05% at 600 nm; &#60;0.10% at 435 nm 
			Fiber optic connector: SMA 905 to 0.22 numerical aperture single-strand fiber</td>
		</tr>
		<tr>
			<td>High-Power LED Driver</td>
			<td>Minhongshi, China</td>
			<td>MHS-48XY</td>
			<td>Working voltage: DC12V 
			Central wavelength: 850nm</td>
		</tr>
		<tr>
			<td>high-resolution web camera</td>
			<td>Thorlabs, USA</td>
			<td>MWWHL4</td>
			<td>Color: Warm White 
			Correlated Color Temperature: 3000 K 
			Test Current for Typical LED Power: 1000 mA 
			Maximum Current (CW): 1000 mA 
			Bandwidth (FWHM): N/A 
			Electrical Power: 3000 mW 
			Viewing Angle (Full Angle): 120&#730; 
			Emitter Size: 1 mm x 1 mm 
			Typical Lifetime: &#62;50 000 h 
			Operating Temperature (Non-Condensing): 0 to 40 &#176;C 
			Storage Temperature: -40 to 70 &#176;C 
			Risk Groupa: RG1 &#8211; Low Risk Group</td>
		</tr>
		<tr>
			<td>LED Warm White</td>
			<td>Mega-9, China</td>
			<td>BP850/22K</td>
			<td>&#216;25.4(+0~-0.1) mm 
			Bandwidth: 22&#177;3nm 
			Peak transmittance:80% 
			Central wavelength: 850nm&#177;3nm&#160;</td>
		</tr>
		<tr>
			<td>Spectrometer&#160;</td>
			<td>Noel Danjou</td>
			<td>Amcap9.22</td>
			<td>AMCap&#160;is a still and video capture application with advanced preview and recording features. It is a Desktop application designed for computers running Windows 7 SP1 or later. Most&#160;Video-for-Windowsand&#160;DirectShow-compatible devices are supported whether they are cheap webcams or advanced video capture cards.</td>
		</tr>
		<tr>
			<td>Standard photodiode power sensor&#160;</td>
			<td>Super Dragon, China</td>
			<td>YGY-122000</td>
			<td>Input: AC 100-240V~50/60Hz 0.8A 
			Output: DC 12V 2A</td>
		</tr>
		<tr>
			<td>Thermal power sensor&#160;</td>
			<td>Thorlabs, USA</td>
			<td>M470L3-C1</td>
			<td>Color: Blue 
			Nominal Wavelengtha: 470 nm 
			Bandwidth (FWHM): 25 nm 
			Maximum Current (CW): 1000 mA 
			Forward Voltage: 3.2 V 
			Electrical Power (Max): 3200 mW 
			Emitter Size: 1 mm x 1 mm 
			Typical Lifetime: 100 000 h 
			Operating Temperature (Non-Condensing): 0 to 40 &#176;C 
			Storage Temperature: -40 to 70 &#176;C 
			Risk Groupb: RG2 &#8211; Moderate Risk Group</td>
		</tr>
		<tr>
			<td>Thermal power sensor&#160;</td>
			<td>Thorlabs, USA</td>
			<td>S401C</td>
			<td>Wavelength range: 190 nm-20 &#956;m 
			Optical power range:10 &#956;W-1 W(3 Wb) 
			Input aperture size: &#216;10 mm 
			Active detector area: 10 mm x 10 mm 
			Max optical power density: 500 W/cm2 (Avg.) 
			Linearity: &#177;0.5%</td>
		</tr>
	</tbody>
</table>

light spot-based assay for analysis of drosophila larval phototaxis

在觅食阶段，果蝇的幼虫表现出明显的避光行为。果蝇幼虫光毒可作为研究动物回避行为的模型。该协议引入了一种光点测定，以研究幼虫光法行为。实验装置包括两个主要部分：产生光斑的视觉刺激系统，以及记录幼虫避光过程的红外光成像系统。这种测定允许跟踪幼虫在进入之前、遇到期间和离开光斑后的行为。可以使用此方法捕获和分析幼虫运动的细节，包括减速、暂停、头部铸造和车削。

根据该协议，光斑测定用于研究在12小时/12小时光/暗循环室中标准介质上以25°C升起的第三星幼虫的光避光行为。在25.5°C下使用光斑测定对单个w1118幼虫进行了测试。460 nm LED 产生的光斑的平均光照强度为 0.59 μW/cm2。使用SOS软件和自定义编写的脚本12、17记录和分析幼虫进入和离开光点的整个过程。图2和<stron...

Watch this Scientific Journal Video about Light Spot-Based Assay for Analysis of Drosophila Larval Phototaxis at JoVE.com

Light Spot-Based Assay for Analysis of Drosophila Larval Phototaxis

该协议引入了一种光点测定，以调查果蝇幼虫光法行为。在此测定中，光点产生为光刺激，而幼虫避光过程由红外光成像系统记录。

光斑分析分析果蝇拉瓦尔光轴

The larvae of Drosophila melanogaster show obvious light-avoiding behavior during the foraging stage. Drosophila larval phototaxis can be used as a model ...

使用光斑测定，可以在进入光区域之前、期间和之后跟踪幼虫的行为，从而分析幼虫运动的细节。这种测定简单易做。光响应只测试一次，以便排除光自适应的任何可能影响。要准备加糖板，缓慢而均匀地覆盖一个15厘米的培养盘的底部，用大约4毫米厚的热1%agar层。让板在室温下冷却，直到加糖凝固。要设置视觉刺激系统，请将准备好的 LED 光源夹在铁架上，注意光线朝桌面向下投射。稍微倾斜气缸，直到圆柱体平面和垂直平面之间的角度约为 10 度。将 470 纳米蓝色 LED 灯连接到大功率 LED 驱动器的 LED 插头，然后打开驱动器。转动驾驶员右上角的旋钮以选择通道 470 纳米并单击 LED。当屏幕显示检查时，桌面上将显示一个蓝色光斑。单击"确定"并转动旋钮以调整灯光的强度。上下移动光源的位置，将光源的直径调整为两厘米。旋转旋钮，根据实验要求选择光强。使用带标准光电二极管功率传感器的紧凑型功率计控制台测量现场的最大光功率和最小光功率。光功率需要在实际实验中在黑暗中测量。然后记录光功率，测量三次，并使用平均值。要设置想象系统，请用比桌面上光点高出约 10 厘米的铁夹住一个高分辨率网络摄像机，然后调整相机镜头朝桌面的方向。通过 USB 接口将相机连接到运行 Windows 7 的计算机，然后将阿加格板放在相机下方的桌面上。打开 AMCap 9.22 软件，光斑将自动显示在 AMCap 窗口中。向左或向左移动相机，以确保光点靠近窗口中心。使用夹子将 850 加或减 3 纳米带通滤波器固定到摄像机正下方的 5 到 7 毫米。将三个红外光生成 LED，其中央波长为 850 纳米，均匀地围绕距板边缘约 5 厘米的 agar 板周围，LED 镜头朝向板的 70 度角。然后通过交流变频器将 LED 连接到电源。在 AMCap 软件的菜单中，选择选项、视频设备和捕获格式，将捕获视频的像素大小设置为 800 x 600，帧速率设置为每秒 60 帧。取下滤镜，在相机下面放置一把标尺。调整摄像机对焦，直到比例线清晰且与视频视场宽度平行，然后单击捕获、设置和视频捕获以选择保存路径。然后单击"开始录制"以记录对应于 600 像素的实际距离，并计算每个像素与实际距离的比率。在将幼虫引入成像设置之前，请获取光位置的简短视频，并命名未过滤的控制背景视频光区域之一。将滤光片放回相机镜头上，以尽可能低的设置打开远离实验装置的灯，同时仍可以清晰观察幼虫。用勺子从培养基中去除一些幼虫，然后轻轻地选择第三个星幼虫。用蒸馏水清洗幼虫，将幼虫放在加糖板的中心。轻轻去除幼虫中多余的水，并关闭房间灯。在打开红外 LED 灯之前，让幼虫在黑暗环境中适应两分钟。需要在黑暗中进行以下避光行为实验。为了方便演示，我们在明亮的灯光下拍摄。轻轻地将幼虫刷到盘子的中心。当幼虫开始直爬行时，旋转板，使幼虫朝光斑方向移动。单击"捕获"、设置和视频捕获以选择保存路径，然后单击"开始录制"录制。允许幼虫爬向、进入和离开光斑，直到它几乎离开视野，然后单击停止录制。将滤光片移离摄像机，获取光斑位置的简短视频。然后命名后检测未过滤的视频光区域二，并将此视频与光区域一个视频进行比较，以确保光斑位置不变。在这里，可以观察到有代表性的幼虫的尾部速度、身体弯曲角度和身体弯曲的角度速度。与父母控制相比，与父母控制相比，在具有八胺神经反应抑制的八巴胺神经元的第三星幼虫中，幼虫头铸件的大小，因此，最大身体弯曲角度显著减小，表明 Tdc2-Gal4 神经元是正常幼虫光反应所必需的。要成功处理图像，请确保相机不会阻挡光线，并且光斑是圆形的，靠近窗口的中心。按照这个程序，可以进行光遗传学来观察幼虫在激活特定神经元时的反应。利用这种测定，可以分析幼虫运动的细节，从而可以研究环境和内部因素对幼虫避光行为的远大影响。

Research

JoVE Journal

Biology

Light Spot-Based Assay for Analysis of Drosophila Larval Phototaxis

Dissection of Larval CNS in Drosophila Melanogaster

在果蝇幼虫中枢神经系统的剖析

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

科研

生物学

Drosophila Larval NMJ Dissection

果蝇幼虫NMJ夹层

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

Drosophila Larval NMJ Immunohistochemistry

Drosophila Larval NMJ Immunohistochemistry

果蝇幼虫NMJ免疫

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

在幼虫的监测心脏功能果蝇生理研究

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained  and  ...

Preparation of Drosophila S2 cells for Light Microscopy

Preparation of Drosophila S2 cells for Light Microscopy

制备果蝇 S2细胞，光学显微镜

The ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature ...

Dissection and Staining of Drosophila Larval Ovaries

Dissection and Staining of Drosophila Larval Ovaries

夹层和染色果蝇幼虫卵巢

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations

从解离分析准备胚胎和幼体斑马鱼骨肌纤维

The zebrafish has  proven to be a valuable model system for exploring skeletal muscle function and  for studying human muscle diseases. Despite  the many ...

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

考试<em&gt;果蝇</em幼虫气管终端细胞光镜

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific ...