Imaging intracellular quantum dots: fluorescence microscopy and transmission electron microscopy.
Imaging intracellular quantum dots: fluorescence microscopy and transmission electron microscopy.
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细胞内量子点成像：荧光显微镜和透射电子显微镜。
DOI：
10.1007/978-1-62703-468-5_2
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发表时间：
2013
期刊：
影响因子：
--
通讯作者：
Payne,ChristineK
中科院分区：
文献类型：
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作者：
Szymanski,CraigJ;Yi,Hong;Liu,JoshuaL;Wright,ElizabethR;Payne,ChristineK
对于大多数细胞内应用，量子点(QD)和其他纳米颗粒需要传递和靶向。尽管取得了许多进展，但细胞内的递送和靶向仍然效率低下，许多量子点仍然结合在质膜上，而不是内化到细胞内。这些细胞外量子点产生的荧光导致背景信号与细胞内感兴趣的量子点竞争。我们提出了两种方法来减少和识别由质膜结合量子点产生的信号。第一种方法是光物理方法，使用细胞外猝灭剂来极大地减少细胞外量子点的荧光信号。这种方法与活细胞中快速、宽视场、荧光成像兼容。给出了两种细胞外猝灭剂QSY-21和台盼蓝，与655 nm发射量子点结合使用的结果。胞外猝灭剂的使用可以扩展到各种荧光团。第二种方法使用透射电子显微镜(TEM)对树脂包埋细胞的薄片(60-70 nm)成像。切片细胞和高分辨率电子显微镜的使用使得区分质膜结合和细胞内量子点成为可能。为了克服使用透射电子显微镜成像细胞中单个量子点的困难，我们使用了一种银增强方法，显著提高了透射电子显微镜图像中量子点的对比度。
Quantum dots (QDs) and other nanoparticles require delivery and targeting for most intracellular applications. Despite many advances, intracellular delivery and targeting remains inefficient with many QDs remaining bound to the plasma membrane rather than internalized into the cell. The fluorescence resulting from these extracellular QDs results in a background signal that competes with intracellular QDs of interest. We present two methods for the reduction and discrimination of signal resulting from plasma membrane-bound QDs. The first method, a photophysical approach, uses an extracellular quencher to greatly reduce the fluorescence signal from extracellular QDs. This method is compatible with fast, widefield, fluorescence imaging in live cells. Results are presented for two extracellular quenchers, QSY-21 and trypan blue, used in combination with 655 nm emitting QDs. The use of an extracellular quencher can be extended to a wide variety of fluorophores. The second method uses transmission electron microscopy (TEM) to image thin (60–70 nm) slices of resin-embedded cells. The use of sectioned cells and high-resolution TEM makes it possible to discriminate between plasma membrane-bound and intracellular QDs. To overcome the difficulties associated with using TEM to image individual QDs in cells, we have utilized a silver enhancement method that significantly improves the contrast of QDs in TEM images.