Single-molecule high-resolution imaging with photobleaching
Single-molecule high-resolution imaging with photobleaching
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DOI：
10.1073/pnas.0401638101
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发表时间：
2004-04-27
影响因子：
11.1
通讯作者：
Selvin, PR
中科院分区：
文献类型：
--
作者：
Gordon, MP;Ha, T;Selvin, PR
传统的光学显微镜在其分辨能力方面受到瑞利极限的限制，其长度尺度为200 nm。另一方面，诸如荧光共振能量转移的光谱技术不能用于测量>10 nm的距离，从而在光学技术测量10至100 nm尺度的距离的能力方面留下了“空白”。我们以前已经证明了本地化的能力，单个染料分子的精度为1.5 nm，亚秒级的时间分辨率。在这里，我们找到了两个染料的位置，并确定他们的分离与5纳米的精度，使用单荧光染料分子的量子光漂白行为。通过拟合一种染料光漂白前后的图像，我们可以同时定位两种染料并计算它们的分离。因此，我们绕过了瑞利极限，实现了纳米级的分辨率。具体来说，我们通过测量通过连接到固定在表面上的双链DNA分子的末端分离10-20 nm的单个荧光团之间的距离来演示该技术。除了弥补光学分辨率的差距之外，该技术还可用于生物物理学或基因组学应用，包括产生单核苷酸多态性的超高密度图。
Conventional light microscopy is limited in its resolving power by the Rayleigh limit to length scales on the order of 200 nm. On the other hand, spectroscopic techniques such as fluorescence resonance energy transfer cannot be used to measure distances >10 nm, leaving a "gap" in the ability of optical techniques to measure distances on the 10- to 100-nm scale. We have previously demonstrated the ability to localize single dye molecules to a precision of 1.5 nm with subsecond time resolution. Here we locate the position of two dyes and determine their separation with 5-nm precision, using the quantal photobleaching behavior of single fluorescent dye molecules. By fitting images both before and after photobleaching of one of the dyes, we may localize both dyes simultaneously and compute their separation. Hence, we have circumvented the Rayleigh limit and achieved nanometer-scale resolution. Specifically, we demonstrate the technique by measuring the distance between single fluorophores separated by 10-20 nm via attachment to the ends of double-stranded DNA molecules immobilized on a surface. in addition to bridging the gap in optical resolution, this technique may be useful for biophysical or genomic applications, including the generation of super-high-density maps of single-nucleotide polymorphisms.