Quantitative Super-Resolution Microscopy of Nanopipette-Deposited Fluorescent Patterns
Quantitative Super-Resolution Microscopy of Nanopipette-Deposited Fluorescent Patterns
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DOI：
10.1021/acsnano.5b02220
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发表时间：
2015-08-01
期刊：
影响因子：
17.1
通讯作者：
Sauer, Markus
中科院分区：
文献类型：
--
作者：
Hennig, Simon;van de Linde, Sebastian;Sauer, Markus
我们描述了一种在掺杂甘油和半胱胺的聚乙烯醇（PVA）导电透明固体层（PVA-G-C层）中以高局部精度沉积微量荧光团标记寡核苷酸的方法。带负电的荧光分子的沉积是通过基于扫描离子电导显微镜 (SICM) 的装置，使用尖端直径类似于 100 nm 的纳米移液器，通过使用通过纳米移液器在两个电极之间流动的离子通量来完成的。为了研究局部沉积过程的精度，我们通过直接随机光学重建显微镜（dSTORM）进行了原位超分辨率显微镜。利用 (dSTORM) 的单分子灵敏度和可靠性，我们确定了沉积在单个点上的荧光分子的数量。所施加的电荷和沉积分子数量的相关性使得能够通过测量递送过程中的电荷来量化递送的分子。我们演示了 3-168 个荧光分子在单个点上的可重复沉积以及荧光结构的创建。 荧光结构高度稳定，可以重复使用多次。
We describe a method for the deposition of minute amounts of fluorophore-labeled oligonucleotides with high local precision in conductive and transparent solid layers of poly(vinyl alcohol) (PVA) doped with glycerin and cysteamine (PVA-G-C layers). Deposition of negatively charged fluorescent molecules was accomplished with a setup based on a scanning ion conductance microscope (SICM) using nanopipettes with tip diameters of similar to 100 nm by using the ion flux flowing between two electrodes through the nanopipette. To investigate the precision of the local deposition process, we performed in situ super-resolution microscopy by direct stochastic optical reconstruction microscopy (dSTORM). Exploiting the single-molecule sensitivity and reliability of (dSTORM, we determine the number of fluorescent molecules deposited in single spots. The correlation of applied charge and number of deposited molecules enables the quantification of delivered molecules by measuring the charge during the delivery process. We demonstrate the reproducible deposition of 3-168 fluorescent molecules in single spots and the creation of fluorescent structures. The fluorescent structures are highly stable and can be reused several times.