Name: tirfm and ph-sensitive gfp-probes to evaluate neurotransmitter vesicle dynamics in sh-sy5y neuroblastoma cells: cell imaging and data analysis
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Description: Watch this Scientific Journal Video about TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis at JoVE.com

笔者想承认UNIVERSITA德利阿布鲁Studi住宅米兰向埃利安娜迪卡伊拉诺（博士后奖学金）和斯特凡莫雷蒂（博士奖学金）的支持。这项工作是由大学研究计划PUR到CP支持我们要感谢教授杰里米·M.亨利，生物化学学院，布里斯托，英国的大学，为pHluorin建设和Dotti弗朗西斯博士在数据分析的援助，和Silvia Marsicano技术援助。

1.细胞培养和转染<ol><li> SH-SY5Y细胞培养 注意：使用人神经母细胞瘤SH-SY5Y（ATCC＃CRL-2266）15实验已经完成。 SH-SY5Y细胞成长为浮动集群和贴壁细胞的混合物。按照报告的协议（细胞密度，分光比等 ），以有生长牢固地附着在玻璃盖，其用于TIRFM关键细胞说明书。 <ol><li>在开始之前，层流生物安全柜下，使无菌磷酸盐缓冲生理盐水（PBS）和培养基的时机音量。 <ol><li>...

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本文提出了一种协议，以图像和分析囊泡动力学分泌细胞，采用荧光基因编码向量和TIRFM。成功的成像通过TIRFM关键要素是细胞模型和细胞转染与囊泡释放和回收的基因编码的光学指标的选择。 TIRFM非常适合于细胞生长附着于玻璃盖和足够平坦以使膜融合事件的稳定可视化。囊泡最好应分散在细胞使它们的贩卖，融合和胞吞作用可以被成像和量化在单囊泡水平。不幸的是，神?...

神经元之间的化学突触传递是通信中的神经系统的主要机制。它依赖于神经递质通过囊泡融合和检索的动态循环的释放在突触前部位。许多参与囊泡动力学的蛋白质已被鉴定;然而，它们对这一现象的具体贡献仍有待阐明1。 我们的理解是由以下事实最广泛使用的测定法外/胞吞作用并不总是最合适的部分的限制。相关囊泡融合一些研究和动态依靠电生理技术。这种技术提供了一个最佳的时间分辨率和优良调查囊泡至质膜的初始熔化，但无法检测到许多支持突触功能的基本分子事件。电子显微镜，在另一侧，提供了最好的morphologica每个奇异步骤，但是事件的动态方面的升说明不能被捕获，因为样品必须固定以便进行分析。 的新的光记录技术2,3，结合在荧光分子探针发展4-6进步的出现，使得在活细胞中胞吐过程的可视化，从而提供约突触的结构和功能的信息的新的水平。 最初的研究开发活动相关的苯乙烯染料（FM1 - 43和相关有机染料）7,8。国家的最先进的成像技术采用了绿色荧光蛋白（GFP）（pHluorin）拴管腔小泡蛋白9的pH敏感的变体。这些探针通常被关闭，因为低pH值管腔的囊泡当存在时。融合与质膜后，囊泡内部暴露于中性细胞外空间中，pH<html>突然增加，缓解pHluorin的质子依赖淬火和荧光信号迅速出现。如pHluorin的变化的速度比的融合事件，通过监测荧光的增加，囊泡融合与膜可以测量和分析。因为表面pHluorin标签的分子被内吞，所述荧光信号随后返回到基础水平，因此也可以使用相同的构建体并监控囊泡再循环9。 而囊泡标记的pH传感器可以确保只有那些囊泡真正融合与质膜的可视化，成像在高空间和时间分辨率是必需的，以详细描述了所涉及的外/内吞过程的步骤。的光学技术，它提供了必要的时空分辨率为全内反射荧光显微镜（TIRFM），荧光显微镜10的应用程序。 “&gt;全内反射发生在玻璃盖玻片和样品。当光路到达玻璃盖玻片用的入射角比临界角大的界面，激发光不被传递到样品，但完全反射回去。在这些条件下，一个瞬逝光波的形式在界面处，并传播与较少的光密度（样品）的平台。由于渐逝场的强度呈指数衰减的距离从接口（具有穿透深度约100nm）只在最接近于盖滑移的荧光团可被激发而更远离边界都没有。在转染的细胞用GFP-构建体，该深度对应于表示在质膜上的蛋白质或在囊泡结构接近它。如在细胞内部的荧光团可以不被激励时，所述背景荧光最小化，并具有非常高的信号/背景的大鼠的图像IO形成11。 有几个特点使选择的TIRFM技术监测囊泡动态。完美的对比度和高的信号 - 噪声比允许非常低的信号从单个囊泡导出的检测。在各帧的基于芯片的图像采集提供必要检测高动态过程的时间分辨率。最后，细胞的最小曝光的样品中任何其他飞机大大地减少光毒性，并能持久延时录制12。 数据分析仍然这种技术的最具挑战性的和关键的方面。监视囊泡融合的最简单的方法是测量报告荧光蛋白在细胞表面的积累，随着时间的推移13。作为融合的增加，净荧光信号也增加。然而，这种方法可能会低估的过程中，特别是在大的细胞和在静止的条件下，因为内吞作用和漂白过程偏移由于囊泡的胞吐作用的增加的荧光强度。另一种方法是按照每个单个融合事件14。这后一种方法是很敏感的，并且可以揭示关于融合的机制的重要细节。然而，它要求手动选择单一活动，因为完全自动化程序遵循囊泡和登记他们的荧光信号的波动并不总是可用。囊泡动力学的观察要求采样单元在高频率。这会产生大量的数据，可以几乎不进行手动分析。 本文的建议是优化TIRFM成像技术用于监测基底和刺激的神经递质释放，在SH-5YSY神经母细胞瘤细胞系，并描述，一步一步，在实验室中开发的程序来分析数据，既在全细胞和单囊泡水平。

<table border="0" cellpadding="0" cellspacing="0" width="1414">
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			<td height="21" style="height:21px;width:259px;">Equipment</td>
			<td style="width:199px;"></td>
			<td style="width:119px;"></td>
			<td style="width:371px;"></td>
			<td style="width:468px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Axio Observer Z1</td>
			<td style="width:199px;">Zeiss</td>
			<td style="width:119px;">491912-9850-000</td>
			<td>inverted microscope 
			<a href="http://www.zeiss.com/microscopy/en_de/products/light-microscopes/axio-observer-for-biology.html#introduction">http://www.zeiss.com/microscopy/en_de/products/light-microscopes/axio-observer-for-biology.html#introduction</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Multiline Argon Laser Lasos 77</td>
			<td style="width:199px;">Lasos</td>
			<td style="width:119px;">00000-1312-752</td>
			<td>multi-line (458/488/514 nm), 100mW argon-ion laser 
			<a href="http://www.lasos.com/products/argon-ion-laser">http://www.lasos.com/products/argon-ion-laser</a></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:259px;">Laser TIRF slider</td>
			<td style="width:199px;">Zeiss</td>
			<td style="width:119px;">423681-9901-000</td>
			<td><a href="http://www.zeiss.com/microscopy/en_de/products/imaging-systems/single-molecular-imaging-laser-tirf-3.html">http://www.zeiss.com/microscopy/en_de/products/imaging-systems/single-molecular-imaging-laser-tirf-3.html</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">100x Objective</td>
			<td style="width:199px;">Zeiss</td>
			<td>421190-9900-000</td>
			<td>Oil, NA 1.45 Alpha-Plan 
			<a href="https://www.micro-shop.zeiss.com/?l=en&amp;p=us&amp;f=o&amp;a=v&amp;m=a&amp;id=421190-9900-000&amp;ss=1">https://www.micro-shop.zeiss.com/?l=en&#38;p=us&#38;f=o&#38;a=v&#38;m=a&#38;id=421 
			190-9900-000&#38;ss=1</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">CCD Camera RetigaSRV Fast 1394&#160;</td>
			<td style="width:199px;">QImaging</td>
			<td style="width:119px;"></td>
			<td><a href="http://www.qimaging.com/products/datasheets/Retiga-SRV.pdf">http://www.qimaging.com/products/datasheets/Retiga-SRV.pdf</a></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:259px;">LAMBDA 10-3 optical filter changer with SmartShutter</td>
			<td style="width:199px;">Sutter Instrument Company</td>
			<td style="width:119px;"></td>
			<td><a href="http://www.sutter.com/IMAGING/lambda103.html">http://www.sutter.com/IMAGING/lambda103.html</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;"></td>
			<td style="width:199px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:259px;">Software</td>
			<td style="width:199px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Image ProPlus 6.3 Software</td>
			<td style="width:199px;">Media Cybernetics</td>
			<td style="width:119px;"></td>
			<td>spot selection, ROI selection, fluorescence intensity determination 
			<a href="http://www.mediacy.com/index.aspx?page=IPP">http://www.mediacy.com/index.aspx?page=IPP</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Excel</td>
			<td style="width:199px;">Microsoft</td>
			<td style="width:119px;"></td>
			<td>photobleaching correction, whole-cell and single-vesicle analyses 
			<a href="http://office.microsoft.com/it-it/excel/">http://office.microsoft.com/it-it/excel/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GraphPad Prism 4.00&#160;</td>
			<td>GraphPad Software, Inc.</td>
			<td style="width:119px;"></td>
			<td>statistical analysis 
			<a href="http://www.graphpad.com/scientific-software/prism/">http://www.graphpad.com/scientific-software/prism/</a></td>
		</tr>
	</tbody>
</table>

tirfm and ph-sensitive gfp-probes to evaluate neurotransmitter vesicle dynamics in sh-sy5y neuroblastoma cells: cell imaging and data analysis

突触囊泡融合，通过和检索的动态循环释放神经递质的化学突触。 实时监测突触活动和解剖的不同步骤外-内吞作用的单囊泡水平对于理解在健康和疾病突触功能至关重要。 遗传编码的PH敏感的探针直接靶向突触小泡和全内反射荧光显微镜（TIRFM）提供必要遵循囊泡动力学的时空分辨率。通过全内反射所产生的渐逝场只能激发置于一薄层（&lt;150纳米），其上的细胞粘附在玻璃盖之上的荧光团，正是外 - 内吞作用的过程发生。所得高对比度的图像非常适合囊泡跟踪和融合事件的定量分析。 在这个协议中，SH-SY5Y人N-euroblastoma细胞被提出作为一种有价值的模型为研究，因为他们的平坦面神经递质释放在通过TIRFM单囊泡水平，并分散囊泡的存在。被提供的方法用于生长的SH-SY5Y以贴壁细胞和与突触-pHluorin染它们，以及执行TIRFM和成像技术。最后，战略目标选择，计数和分析，在全细胞和单囊泡融合水平的事件呈现。 为了验证成像过程和数据分析方法，pHluorin标记的囊泡的动力学下休息了分析和刺激（去极化钾浓度）的条件。膜去极化增加的融合事件的频率，并导致记录在全细胞的净荧光信号的并行加注。单囊泡分析揭示聚变事件行为（上升峰的高度和宽度）的修饰。这些数据表明，第在钾去极化不仅诱导大量神经递质释放，而且修改囊泡融合和再循环的机制。 用适当的荧光探针，该技术可以在不同的蜂窝系统可以采用解剖构和刺激分泌的机制。

描述的全内反射荧光成像和数据分析程序的目的是研究在蜂窝系统囊泡动态。这种技术可以被用于确定信号分子和药物对聚变事件和神经递质的囊泡动力学17的影响。使用GFP标记质膜蛋白，所述TIRFM分析已被用来表征在胶质GFP标记谷氨酸转运和上皮细胞18,19的构贩卖。 为了验证报告的成像过程和数据分析策略，融合事件被记录在基础和刺激条件（钾引起的去极?...

Watch this Scientific Journal Video about TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis at JoVE.com

TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis

This paper provides a method for investigating neurotransmitter vesicle dynamics in neuroblastoma cells, using a synaptobrevin2-pHluorin construct and Total Internal Reflection Fluorescence Microscopy. The strategy developed for image processing and data analysis is also reported.

TIRFM和pH敏感的GFP-探针来评估神经递质囊泡动力学SH-SY5Y神经母细胞瘤：细胞成像和数据分析

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real ...

Research

JoVE Journal

Neuroscience

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

Live-cell Imaging of Sensory Organ Precursor 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 ...

科研

神经科学

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

嗅鞘细胞移植周围神经损伤后，以评估功能恢复

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory ...

Visualization of Endosome Dynamics in Living Nerve Terminals with Four-dimensional Fluorescence Imaging

内吞体动力学在生活神经末梢与四维荧光成像可视化

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

器官切片文化研究少突胶质细胞动力学和髓鞘

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

神经干细胞和祖细胞来自成年小鼠脑室下区细胞分选和实时成像的细胞周期动力学

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

分析突触调制<em&gt;果蝇</em&gt;曝光时间延长后，光光感受器

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at ...

Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

背根神经节分离与原代培养对神经递质释放的研究

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral ...

In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages

iPSC-巨噬细胞死亡神经母细胞的体外定量成像检测

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. ...

Measuring Membrane Lipid Turnover with the pH-sensitive Fluorescent Lipid Analog ND6

使用 pH 敏感荧光脂质类似物 ND6 测量膜脂质周转率

Exo-/endocytosis is a common process mediating the exchange of biomolecules between cells and their environment and among different cells. Specialized ...

Analyses of Actin Dynamics, Clutch Coupling and Traction Force for Growth Cone Advance

肌动蛋白动力学、离合器耦合和牵引力分析，促进生长锥体前进

To establish functional networks, neurons must migrate to their appropriate destinations and then extend axons toward their target cells. These processes ...