Opazo Felipe, Rizzoli Silvio O
STED Microscopy of Synaptic Function, European Neuroscience Institute Göttingen.
J Vis Exp. 2010 Feb 15(36):1790. doi: 10.3791/1790.
The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The vesicles are then retrieved from the plasma membrane (endocytosis) and grouped together with the general pool of vesicles within the nerve terminal, until they undergo a new exo- and endocytosis cycle (vesicle recycling). These processes have been studied using a variety of techniques such as electron microscopy, electrophysiology recordings, amperometry and capacitance measurements. Importantly, during the last two decades a number of fluorescently labeled markers emerged, allowing optical techniques to track vesicles in their recycling dynamics. One of the most commonly used markers is the styryl or FM dye; structurally, all FM dyes contain a hydrophilic head and a lipophilic tail connected through an aromatic ring and one or more double bonds (Fig. 1B). A classical FM dye experiment to label a pool of vesicles consists in bathing the preparation (Fig. 1Ai) with the dye during the stimulation of the nerve (electrically or with high K(+)). This induces vesicle recycling and the subsequent loading of the dye into recently endocytosed vesicles (Fig. 1A(i-iii;)). After loading the vesicles with dye, a second round of stimulation in a dye-free bath would trigger the FM release through exocytosis (Fig. 1A(iv-v;)), process that can be followed by monitoring the fluorescence intensity decrease (destaining). Although FM dyes have contributed greatly to the field of vesicle recycling, it is not possible to determine the exact localization or morphology of individual vesicles by using conventional fluorescence microscopy. For that reason, we explain here how FM dyes can also be used as endocytic markers using electron microscopy, through photoconversion. The photoconversion technique exploits the property of fluorescent dyes to generate reactive oxygen species under intense illumination. Fluorescently labeled preparations are submerged in a solution containing diaminobenzidine (DAB) and illuminated. Reactive species generated by the dye molecules oxidize the DAB, which forms a stable, insoluble precipitate that has a dark appearance and can be easily distinguished in electron microscopy. As DAB is only oxidized in the immediate vicinity of fluorescent molecules (as the reactive oxygen species are short-lived), the technique ensures that only fluorescently labeled structures are going to contain the electron-dense precipitate. The technique thus allows the study of the exact location and morphology of actively recycling organelles.
突触小泡与质膜的融合（胞吐作用）是神经递质释放和神经元通讯过程中必不可少的一步。然后，小泡从质膜回收（胞吞作用），并与神经末梢内的小泡总库聚集在一起，直到它们经历新的胞吐和胞吞循环（小泡循环利用）。人们使用了多种技术来研究这些过程，如电子显微镜、电生理记录、安培测量和电容测量。重要的是，在过去二十年中出现了许多荧光标记物，使得光学技术能够追踪小泡的循环利用动态。最常用的标记物之一是苯乙烯基或FM染料；从结构上看，所有FM染料都含有一个亲水头部和一个通过芳环及一个或多个双键相连的亲脂尾部（图1B）。一个经典的用FM染料标记小泡库的实验是在神经受到刺激（电刺激或用高钾溶液刺激）时，用染料浸泡标本（图1Ai）。这会诱导小泡循环利用，并使染料随后加载到最近内吞的小泡中（图1A(i-iii)）。在用染料加载小泡后，在无染料的浴液中进行第二轮刺激会触发通过胞吐作用释放FM（图1A(iv-v)），这个过程可以通过监测荧光强度降低（褪色）来跟踪。尽管FM染料对小泡循环利用领域做出了巨大贡献，但使用传统荧光显微镜无法确定单个小泡的确切定位或形态。因此，我们在此解释如何通过光转化利用电子显微镜将FM染料用作胞吞标记物。光转化技术利用了荧光染料在强光照射下产生活性氧的特性。将荧光标记的标本浸入含有二氨基联苯胺（DAB）的溶液中并进行照射。染料分子产生的活性物质会氧化DAB，形成一种稳定的、不溶性沉淀，其外观呈深色，在电子显微镜下很容易区分。由于DAB仅在荧光分子的紧邻区域被氧化（因为活性氧寿命很短），该技术确保只有荧光标记的结构会含有电子致密沉淀。因此，该技术可以研究活跃循环利用的细胞器的确切位置和形态。
