Dual objective fluorescence microscopy for single molecule imaging applications.
Dual objective fluorescence microscopy for single molecule imaging applications.
复制标题
用于单分子成像应用的双物镜荧光显微镜。
DOI：
10.1117/12.808259
复制
发表时间：
2009
期刊：
影响因子：
--
通讯作者：
Ober,RaimundJ
中科院分区：
文献类型：
--
作者：
Ram,Sripad;Prabhat,Prashant;Ward,ESally;Ober,RaimundJ
荧光显微镜是研究细胞生物过程的宝贵工具。在最近的过去，在单分子水平上对细胞过程进行成像已经引起了极大的兴趣。单分子实验消除了系综平均效应，并提供了通常无法通过批量实验获得的信息。单分子成像应用中的主要要求之一是从单分子检测到足够数量的光子。这不仅对单分子的视觉识别很重要，而且在所获得数据的定量分析中也起着至关重要的作用。在这里，我们展示了使用双目标成像配置单分子研究。该配置使用两个相对的物镜，其中一个物镜处于倒置位置，另一个物镜处于直立位置。相对物镜的使用已经在4pi共焦显微镜和I5 M中得到证明，以实现与共焦/宽视场显微镜相比的高轴向分辨率。在这里，我们证明了双物镜成像配置提供了更高的光子收集效率相比，一个普通的显微镜在给定的照明条件。因此，通过相对的物镜成像时，单分子可以比通过常规光学显微镜成像时更准确地定位。引入分析工具来估计单分子的2D位置，并表征它们可以被确定的准确性。
Fluorescence microscopy is an invaluable tool for studying biological processes in cells. In the recent past there has been significant interest in imaging cellular processes at the single molecule level. Single molecule experiments remove ensemble averaging effects and provide information that is typically not accessible through bulk experiments. One of the major requirements in single molecule imaging applications is that a sufficient number of photons be detected from the single molecule. This is not only important for the visual identification of single molecules, but also plays a crucial role in the quantitative analysis of the acquired data. Here, we demonstrate the use of a dual objective imaging configuration for single molecule studies. The configuration uses two opposing objective lenses, where one of the objectives is in an inverted position and the other objective is in an upright position. The use of opposing objective lenses has been previously demonstrated in 4pi confocal microscopy and I5M to achieve high axial resolution when compared to confocal/widefield microscopes. Here we demonstrate that the dual objective imaging configuration provides higher photon collection efficiency when compared to a regular microscope for a given illumination condition. As a result, single molecules can be localized with better accuracy when imaged through opposing objective lenses than when imaged through a regular optical microscope. Analytical tools are introduced to estimate the 2D location of single molecules and to characterize the accuracy with which they can be determined.