Real-time multi-parameter spectroscopy and localization in three-dimensional single-particle tracking
Real-time multi-parameter spectroscopy and localization in three-dimensional single-particle tracking
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DOI：
10.1098/rsif.2008.0313.focus
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发表时间：
2009-02-06
影响因子：
3.9
通讯作者：
Gratton, Enrico
中科院分区：
文献类型：
--
作者：
Hellriegel, Christian;Gratton, Enrico
在光学显微镜中跟踪单个粒子已经被应用于从材料科学到生物物理学再到单分子水平的研究中。该技术本质上绕过了系综平均，因此可以直接揭示所涉及的动态过程的机械细节。这些过程的范围从平移和旋转运动到光谱动力学。我们区分了传统的对象后验跟踪（例如从图像序列）和实验中更精细的“动态”跟踪技术。在这种技术中，通过反馈算法，显微镜的观察体积相对于运动物体保持中心。与图像跟踪相比，该方法具有一系列优势，从优越的时空分辨率（2-50 nm和1-32 ms）到推断物体在三维空间中移动几微米时的附加数据（例如荧光寿命、发射光谱、极化、强度动力学）的能力。在这篇文章中，我们描述了在双光子激光扫描显微镜上实现的跟踪技术的原理，并用实验数据说明了它的能力，从用不同染料标记的颗粒在液体中移动到活细胞中荧光标记的小蛋白质组装的表征。
Tracking of single particles in optical microscopy has been employed in studies ranging from material sciences to biophysics down to the level of single molecules. The technique intrinsically circumvents ensemble averaging and may therefore reveal directly mechanistic details of the involved dynamic processes. Such processes range from translational and rotational motion to spectral dynamics. We distinguish between conventional a posteriori tracking of objects (e.g. from the sequences of images) and the experimentally more refined 'on- the-fly' tracking technique. In this technique, the observation volume of the microscope is kept centred with respect to the moving object via a feedback algorithm. This approach brings a series of advantages in comparison with the tracking from images, ranging from a superior spatio-temporal resolution (2-50 nm and 1-32 ms) to the capability of inferring additional data (e.g. fluorescence lifetime, emission spectrum, polarization, intensity dynamics) from an object as it moves over several microns in three dimensions. In this contribution, we describe the principle of the tracking technique as implemented on a two-photon laser scanning microscope and illustrate its capabilities with experimental data, from particles labelled with different dyes moving in a liquid to the characterization of small fluorescently labelled protein assemblies in living cells.