Imaging exocytosis in retinal bipolar cells with TIRF microscopy.
Imaging exocytosis in retinal bipolar cells with TIRF microscopy.
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DOI：
10.3791/1305
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发表时间：
2009-06-09
期刊：
影响因子：
--
通讯作者：
Zenisek, David
中科院分区：
文献类型：
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作者：
Joselevitch, Christina;Zenisek, David
全内反射荧光（TIRF）显微镜是一种技术，允许通过选择性成像最接近高折射率物质（如玻璃）的荧光分子来研究细胞膜上发生的事件。在这篇文章中，我们应用这种技术的图像从金鱼视网膜分离的视网膜双极细胞的突触囊泡的胞吐。这些神经元由于其大的轴突终末而非常适合这种研究。通过同时膜片钳双极细胞，可以研究突触前电压和突触释放之间的关系。通过将荧光染料（FM 1-43）和含有高K（+）浓度的林格溶液共同喷到突触末端上，使双极细胞末端内的突触囊泡负载荧光染料。这使细胞去极化并刺激内吞作用和随后的染料摄取进入胞浆能囊泡。在将过量染料洗掉约30分钟后，细胞准备好进行膜片夹持并同时用488 nm激光成像。贴片移液管溶液含有一种基于罗丹明的肽，该肽选择性地与突触带蛋白RIBEYE结合，从而在用561 nm激光对末端成像时特异性地标记带。这允许活动区的精确定位以及突触与突触外事件的分离。
Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective imaging of fluorescent molecules that are closest to a high refractive index substance such as glass. In this article, we apply this technique to image exocytosis of synaptic vesicles in retinal bipolar cells isolated from the goldfish retina. These neurons are very suitable for this kind of study due to their large axon terminals. By simultaneously patch clamping the bipolar cells, it is possible to investigate the relationship between pre-synaptic voltage and synaptic release. Synaptic vesicles inside the bipolar cell terminals are loaded with a fluorescent dye (FM 1-43) by co-puffing the dye and a ringer solution containing a high K(+) concentration onto the synaptic terminals. This depolarizes the cells and stimulates endocytosis and consequent dye uptake into the glutamatergic vesicles. After washing the excess dye away for around 30 minutes, cells are ready for being patch clamped and imaged simultaneously with a 488 nm laser. The patch pipette solution contains a rhodamine-based peptide that binds selectively to the synaptic ribbon protein RIBEYE, thereby labeling ribbons specifically when terminals are imaged with a 561 nm laser. This allows the precise localization of active zones and the separation of synaptic from extra-synaptic events.