Direct Evidence of Lack of Colocalisation of Fluorescently Labelled Gold Labels Used in Correlative Light Electron Microscopy.
Direct Evidence of Lack of Colocalisation of Fluorescently Labelled Gold Labels Used in Correlative Light Electron Microscopy.
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DOI：
10.1038/srep44666
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发表时间：
2017-03-20
影响因子：
4.6
通讯作者：
Gersen H
中科院分区：
文献类型：
--
作者：
Miles BT;Greenwood AB;Benito-Alifonso D;Tanner H;Galan MC;Verkade P;Gersen H
荧光标记的纳米颗粒通常用于相关光电子显微镜(CLEM)，以结合两个独立的显微镜平台的能力：荧光显微镜(LM)和电子显微镜(EM)。固有的假设是，在光学显微镜下观察到的荧光标记与在EM中观察到的电子致密纳米粒子很好地共焦。在这里，我们通过将单分子荧光成像与对单个金纳米颗粒的散射的光学检测相结合，表明对于标记为Alexa-633的10 nm金纳米颗粒的商业生产样品，在Alexa-633的荧光签名和与金纳米颗粒相关的散射之间实际上不存在共定位。这表明，附着的金纳米颗粒对荧光信号的猝灭程度为~95%，或较小的可能性，即络合物已经解离。在任何一种情况下，观察到的荧光信号实际上来自大量未标记的荧光团；使得这些标记可能无效并误导该领域。
Fluorescently labelled nanoparticles are routinely used in Correlative Light Electron Microscopy (CLEM) to combine the capabilities of two separate microscope platforms: fluorescent light microscopy (LM) and electron microscopy (EM). The inherent assumption is that the fluorescent label observed under LM colocalises well with the electron dense nanoparticle observed in EM. Herein we show, by combining single molecule fluorescent imaging with optical detection of the scattering from single gold nanoparticles, that for a commercially produced sample of 10 nm gold nanoparticles tagged to Alexa-633 there is in fact no colocalisation between the fluorescent signatures of Alexa-633 and the scattering associated with the gold nanoparticle. This shows that the attached gold nanoparticle quenches the fluorescent signal by ~95%, or less likely that the complex has dissociated. In either scenario, the observed fluorescent signal in fact arises from a large population of untagged fluorophores; rendering these labels potentially ineffective and misleading to the field.