High Accuracy 3D Quantum Dot Tracking with Multifocal Plane Microscopy for the Study of Fast Intracellular Dynamics in Live Cells
High Accuracy 3D Quantum Dot Tracking with Multifocal Plane Microscopy for the Study of Fast Intracellular Dynamics in Live Cells
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DOI：
10.1529/biophysj.108.140392
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发表时间：
2008-12-15
影响因子：
3.4
通讯作者：
Ober, Raimund J.
中科院分区：
文献类型：
--
作者：
Ram, Sripad;Prabhat, Prashant;Ober, Raimund J.
活细胞环境中的三维单粒子追踪有望揭示重要的新生物学见解。然而，传统的基于显微镜的成像技术不太适合细胞中单个颗粒的快速三维 (3D) 跟踪。此前，我们开发了一种成像模式多焦点平面显微镜（MUM），用于在活细胞的三个维度上对细胞内动态进行快速成像。在这里，我们介绍一种算法，即 MUM 定位算法 (MUMLA)，用于确定使用 MUM 成像的点源的 3D 位置。我们通过模拟和实验数据验证了 MUMLA，并表明可以在广泛的空间范围内确定量子点的 3D 位置。我们证明 MUMLA 确实提供了确定 3D 位置的最佳精度。我们的分析表明，MUM 克服了传统显微镜深度辨别能力差的问题，从而为活细胞环境中纳米粒子的高精度跟踪铺平了道路。在这里，我们使用 MUM 和 MUMLA 首次报告了 QD 标记抗体分子在活细胞中从质膜到细胞深处分选内体的内吞作用的完整 3D 轨迹。
Single particle tracking in three dimensions in a live cell environment holds the promise of revealing important new biological insights. However, conventional microscopy-based imaging techniques are not well suited for fast three-dimensional (3D) tracking of single particles in cells. Previously we developed an imaging modality multifocal plane microscopy ( MUM) to image fast intracellular dynamics in three dimensions in live cells. Here, we introduce an algorithm, the MUM localization algorithm (MUMLA), to determine the 3D position of a point source that is imaged using MUM. We validate MUMLA through simulated and experimental data and show that the 3D position of quantum dots can be determined over a wide spatial range. We demonstrate that MUMLA indeed provides the best possible accuracy with which the 3D position can be determined. Our analysis shows that MUM overcomes the poor depth discrimination of the conventional microscope, and thereby paves the way for high accuracy tracking of nanoparticles in a live cell environment. Here, using MUM and MUMLA we report for the first time the full 3D trajectories of QD-labeled antibody molecules undergoing endocytosis in live cells from the plasma membrane to the sorting endosome deep inside the cell.