Dedicated Setup for the Photoconversion of Fluorescent Dyes for Functional Electron Microscopy
Dedicated Setup for the Photoconversion of Fluorescent Dyes for Functional Electron Microscopy
复制标题
用于功能电子显微镜荧光染料光转换的专用装置
DOI：
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发表时间：
2019
期刊：
影响因子：
--
通讯作者：
Vincenzo Marra
中科院分区：
文献类型：
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作者：
K. Dobson;Carmel L. Howe;Yuri Nishimura;Vincenzo Marra
在这里，我们描述了一个具有成本效益的设置为有针对性的光转换成电子密度的荧光信号。这种方法为精细神经元结构的形态和功能提供了宝贵的见解。该技术依赖于由激发的荧光团介导的二氨基联苯胺（DAB）的局部氧化。本文详细描述了如何构建一个简单的光电转换设置，可以提高这种成熟技术的可靠性和吞吐量。这里描述的系统特别适合于厚的神经元组织，其中光穿透和氧扩散可能限制DAB氧化。为了证明该系统，我们使用相关光和电子显微镜（CLEM）可视化功能标记的单个突触囊泡释放到一个确定的层5神经元在急性皮层切片。该设置大大简化了光转换工作流程，增加了光照明的深度，改善了感兴趣区域的目标定位，并减少了处理每个样品所需的时间。我们已经广泛地测试了FM 1- 43 FX和荧光黄的光转换的设置，两者都在473 nm激发。原则上，该系统可以适用于任何染料或纳米颗粒能够氧化DAB时，由特定的光波长激发。
Here, we describe a cost-effective setup for targeted photoconversion of fluorescent signals into electron dense ones. This approach has offered invaluable insights in the morphology and function of fine neuronal structures. The technique relies on the localized oxidation of diaminobenzidine (DAB) mediated by excited fluorophores. This paper includes a detailed description of how to build a simple photoconversion setup that can increase reliability and throughput of this well-established technique. The system described here, is particularly well-suited for thick neuronal tissue, where light penetration and oxygen diffusion may be limiting DAB oxidation. To demonstrate the system, we use Correlative Light and Electron Microscopy (CLEM) to visualize functionally-labelled individual synaptic vesicles released onto an identified layer 5 neuron in an acute cortical slice. The setup significantly simplifies the photoconversion workflow, increasing the depth of photoillumination, improving the targeting of the region of interest and reducing the time required to process each individual samples. We have tested this setup extensively for the photoconversion of FM 1-43FX and Lucifer Yellow both excited at 473 nm. In principle, the system can be adapted to any dye or nanoparticle able to oxidize DAB when excited by a specific light wavelength.
FM1-43 果蝇神经肌肉接头处的光转换和电子显微镜分析。
影响因子：
0.8
作者：
Sabeva,NadezhdaS;Bykhovskaia,Maria
通讯作者：
Bykhovskaia,Maria
成熟海马神经元的突触外囊泡回收。
影响因子：
16.6
作者：
Ratnayaka A
通讯作者：
Ratnayaka A