High-resolution colocalization of single dye molecules by fluorescence lifetime imaging microscopy
High-resolution colocalization of single dye molecules by fluorescence lifetime imaging microscopy
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DOI：
10.1021/ac025576g
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发表时间：
2002-07-15
影响因子：
7.4
通讯作者：
Sauer, M
中科院分区：
文献类型：
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作者：
Heilemann, M;Herten, DP;Sauer, M
传统的荧光显微镜可以用来确定物体在空间中的位置，当这些物体之间的距离大于几百纳米时，受到光的衍射极限的限制。基于荧光共振能量转移技术的荧光显微镜/光谱学可用于测量低于10 nm的分离距离。为了填补这些基本限制之间的差距，我们已经开发了一种替代技术的高分辨率共定位的荧光染料。该技术是基于荧光寿命成像。在有利的条件下，该方法可用于区分，并测量之间的距离，两个染料分子，是小于30 nm的距离。为了证明该方法，Cy 5和JF 9（罗丹明衍生物）分子统计吸附在玻璃表面上的混合物的寿命图像被采集和分析。由于这两种分子种类的荧光寿命不同（对于Cy 5，tau（f）= 2.0 μ s，对于JF 9，tau（f）= 4.0 ns），因此可以使用模式识别技术将两种染料类型的荧光的贡献分配给每个图像像素。由于两种染料类型可以使用相同的激光波长激发，因此测量没有色差。所呈现的结果展示了仅基于荧光寿命的单个常规荧光染料之间的第一高精度距离测量。
Conventional fluorescence microscopy can be used to determine the positions of objects in space when those objects are separated by distances greater than several hundred nanometers, as restricted by the diffraction limit of light. Fluorescence microscopy/spectroscopy based on fluorescence resonance energy-transfer techniques can be used to measure separation distances below similar to10 nm. To fill the gap between these fundamental limits, we have developed an alternative technique for high-resolution colocalization of fluorescent dyes. The technique is based on fluorescence lifetime imaging. Under favorable conditions, the method can be used to distinguish, and to measure the distance between, two dye molecules that are less than 30 nm apart. To demonstrate the method, lifetime images of a mixture of Cy5 and JF9 (rhodamine derivative) molecules statistically adsorbed on a glass surface were acquired and analyzed. Since these two molecular species differ in fluorescence lifetime (for Cy5, tau(f) = 2.0 us, and for JF9, tau(f) = 4.0 ns), it is possible to assign the contribution of fluorescence of the two dye types to each image pixel using a pattern recognition technique. Since both dye types can be excited using the same laser wavelength, the measurement is free of chromatic aberrations. The results presented demonstrate the first high-precision distance measurements between single conventional fluorescent dyes based solely on fluorescence lifetime.