Hoopmann Peer, Rizzoli Silvio O, Betz William J
Cold Spring Harb Protoc. 2012 Jan 1;2012(1):84-6. doi: 10.1101/pdb.prot067611.
The synaptic vesicle is the essential organelle of the synapse. Many approaches for studying synaptic vesicle recycling have been devised, one of which, the styryl (FM) dye, is well suited for this purpose. The FM dyes have a unique set of properties that allows them to selectively label recycling vesicles: They reversibly stain, but do not permeate, membranes; hence they can specifically label membrane-bound organelles. Their quantum yield is drastically higher when bound to membranes than when in aqueous solution. FM dyes can also be used as endocytic markers in electron microscopy (EM) through a procedure termed photoconversion (or photooxidation), as described here. Fluorescent dye molecules generate free radicals (reactive oxygen species) when subjected to strong illumination. These short-lived radicals readily oxidize any molecules found in the immediate vicinity of the fluorophore. When photoconversion of FM dyes is performed while the preparation is bathing in diaminobenzidine (DAB), a dark brown precipitate forms after the DAB is oxidized. Thus, illumination turns FM-labeled organelles into dark electron-dense ones. The technique results in a substantial increase in the resolution of FM dye labeling studies (with the obvious caveat that it is restricted to fixed preparations).
突触小泡是突触的基本细胞器。人们设计了许多研究突触小泡循环的方法，其中之一，即苯乙烯基（FM）染料，非常适合此目的。FM染料具有一系列独特的特性，使其能够选择性地标记循环小泡：它们可逆地染色，但不渗透膜；因此它们可以特异性地标记膜结合细胞器。当与膜结合时，它们的量子产率比在水溶液中时大幅提高。如本文所述，FM染料还可通过一种称为光转化（或光氧化）的程序用作电子显微镜（EM）中的内吞标记物。荧光染料分子在受到强光照射时会产生自由基（活性氧）。这些短寿命的自由基很容易氧化荧光团附近的任何分子。当在二氨基联苯胺（DAB）中浸泡标本时进行FM染料的光转化，DAB被氧化后会形成深棕色沉淀。因此，光照会将FM标记的细胞器变成暗电子致密的细胞器。该技术显著提高了FM染料标记研究的分辨率（明显的限制是它仅限于固定标本）。
