Name: imaging exocytosis in retinal bipolar cells with tirf microscopy
Uploaded: 2009-06-09T00:00:00
Description: Watch this Scientific Journal Video about Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy at JoVE.com

这项工作是由美国国立卫生研究院批准安永14990支持。

第1部分：夹层和双极细胞的分离<ol><li>准备在表2中列出的解决方案;铃声“（外部）解决方案的pH值应调整到7.4用NaOH和内部解决方案的pH值应调整至7.2与CsOH。光用铝箔保护内部的解决方案，并保持在4 °，直到使用C; </li><li>暗适应至少30分钟前清扫金鱼; </li><li>而动物黑暗的适应，准备5毫升的透明质酸酶（V型透明质酸酶1100单位/毫升在低Ca 2 +林格;西格玛，圣路易斯，密苏里州）和10毫升的L...

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	<li>Axelrod, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+internal+reflection+fluorescence+microscopy+in+cell+biology.">Total internal reflection fluorescence microscopy in cell biology.</a> Traffic. 2, 764-774 (2001).</li><li>Zenisek, D., Steyer, J. A., Almers, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transport,+capture+and+exocytosis+of+single+synaptic+vesicles+at+active+zones.">Transport, capture and exocytosis of single synaptic vesicles at active zones.</a> Nature. 406, 849-854 (2000).</li><li>Zenisek, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicle+association+and+exocytosis+at+ribbon+and+extraribbon+sites+in+retinal+bipolar+cell+presynaptic+terminals.">Vesicle association and exocytosis at ribbon and extraribbon sites in retinal bipolar cell presynaptic terminals.</a> Proc. Natl. Acad. Sci. U. S. A. 105, 4922-4927 (2008).</li><li>Zenisek, D., Horst, N. K., Merrifield, C., Sterling, P., Matthews, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+synaptic+ribbons+in+the+living+cell.">Visualizing synaptic ribbons in the living cell.</a> J. Neurosci. 24, 9752-9759 (2004).</li><li>Axelrod, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+imaging+of+surface+fluorescence+with+very+high+aperture+microscope+objectives.">Selective imaging of surface fluorescence with very high aperture microscope objectives.</a> J. Biomed. Opt. 6, 6-13 (2001).</li><li>Rouze, N. C., Schwartz, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continous+and+transient+vesicle+cycling+at+a+ribbon+synapse.">Continous and transient vesicle cycling at a ribbon synapse.</a> J. Neurosci. 18, 8614-8624 (1998).</li></ol>

客观型TIRF显微镜的优点是：1）激发光在焦平面的客观限制在一个狭小的区域提供优良的光学切片，从而最大限度地减少了集中光线; 2）由于光线与距离成指数下降荧光强度的变化，可以在垂直方向的运动进行监测; 3）通过高数值孔径的目标1,5的高效率的光收集。 该技术的主要缺点是，它是有限的成像事件发生在100纳米的细胞表面，这是大致相当于一个在电子显微镜超薄切片。因此，坚持以玻璃的?...

imaging exocytosis in retinal bipolar cells with tirf microscopy

共有内部反射荧光（TIRF）显微镜是一种技术，使细胞膜发生的事件的研究是最接近高折射指数物质，如玻璃的荧光分子的选择性成像，<sup&gt; 1</sup&gt;。在这篇文章中，我们应用此技术从金鱼视网膜视网膜双极细胞的突触囊泡的胞吐形象。这些神经元是非常适合这种学习，由于其巨大的轴突终端。同时夹紧双极细胞的修补程序，它是可能的调查之间的突触前电压和突触释放的关系<sup&gt; 2,3</sup&gt;。突触小泡内的双极细胞端子装有一个荧光染料（调频1-43 ®）合作膨化染料和林格液含有高K<sup&gt; +</sup&gt;到突触终端的浓度。这种去极化的细胞，刺激成谷氨酸囊泡内吞作用和随之而来的上染率。洗涤多余的染料，大约30分钟后，细胞被夹住的补丁，并同时与488 nm激光成像准备。该补丁吸管解决方案包含一个基于罗丹明的肽结合选择性突触带状蛋白质肉眼<sup&gt; 4</sup&gt;，从而标签色带特别是当终端与561 nm激光成像。这使得活跃区的精确定位和分离的突触的突触事件。

Watch this Scientific Journal Video about Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy at JoVE.com

Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy

在这个视频中，我们演示了如何标记和可视化单突触囊泡的胞吐贩卖和使用总内部反射荧光（TIRF）显微镜在金鱼视网膜双极细胞。

TIRF显微镜视网膜双极细胞的影像胞外分泌

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective ...

Research

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