Convex lens-induced confinement for imaging single molecules.
Convex lens-induced confinement for imaging single molecules.
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DOI：
10.1021/ac101041s
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发表时间：
2010-07-15
影响因子：
7.4
通讯作者：
Cohen, Adam E.
中科院分区：
文献类型：
--
作者：
Leslie, Sabrina R.;Fields, Alexander P.;Cohen, Adam E.
荧光成像用于研究溶液中或附着在表面上的各种单分子的动力学。这一追求的两个关键挑战是（1）在高水平荧光背景下对固定的单分子进行成像，以及（2）对自由扩散的单分子进行长时间成像。在一项指标上表现良好的策略，在另一项指标上往往表现不佳。在这里，我们对宽视场荧光显微镜进行了简单的修改，可以解决这两个挑战并显着改善单分子成像。凸透镜诱导限制 (CLIC) 技术将分子限制在平凸透镜和平面盖玻片之间形成的纳米级深度的楔形间隙内。成像体积的浅深度导致对背景荧光的抑制比全内反射荧光 (TIRF) 成像高 20 倍。消除面外扩散导致每个分子的扩散限制观察时间比共焦荧光相关光谱长约 10 000 倍。 CLIC 系统还提供了一种确定分子大小的新方法。 CLIC 系统不需要任何纳米加工，也不需要任何定制光学、电子或计算机控制。
Fluorescence imaging is used to study the dynamics of a wide variety of single molecules in solution or attached to a surface. Two key challenges in this pursuit are (1) to image immobilized single molecules in the presence of a high level of fluorescent background and (2) to image freely diffusing single molecules for long times. Strategies that perform well by one measure often perform poorly by the other. Here, we present a simple modification to a wide-field fluorescence microscope that addresses both challenges and dramatically improves single-molecule imaging. The technique of convex lens-induced confinement (CLIC) restricts molecules to a wedge-shaped gap of nanoscale depth, formed between a plano-convex lens and a planar coverslip. The shallow depth of the imaging volume leads to 20-fold greater rejection of background fluorescence than is achieved with total internal reflection fluorescence (TIRF) imaging. Elimination of out-of-plane diffusion leads to an approximately 10 000-fold longer diffusion-limited observation time per molecule than is achieved with confocal fluorescence correlation spectroscopy. The CLIC system also provides a new means to determine molecular size. The CLIC system does not require any nanofabrication, nor any custom optics, electronics, or computer control.
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影响因子：
7.4
作者：
Chiu DT;Lorenz RM;Jeffries GD
通讯作者：
Jeffries GD
影响因子：
64.8
作者：
Bustamante, C;Bryant, Z;Smith, SB
通讯作者：
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DOI：
10.1073/pnas.0507728102
发表时间：
2005-10-25
影响因子：
11.1
作者：
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通讯作者：
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影响因子：
3.5
作者：
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通讯作者：
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影响因子：
5.5
作者：
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通讯作者：
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