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3D single molecule tracking of quantum-dot labeled antibody molecules using multifocal plane microscopy

机译：使用多焦点平面显微镜对量子点标记的抗体分子进行3D单分子跟踪

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摘要

Single molecule tracking in three dimensions (3D) in a live cell environment promises to reveal important new insights into cell biological mechanisms. However, classical microscopy techniques suffer from poor depth discrimination which severely limits single molecule tracking in 3D with high temporal and spatial resolution. We introduced a novel imaging modality, multifocal plane microscopy (MUM) for the study of subcellular dynamics in 3D. We have shown that MUM provides a powerful approach with which single molecules can be tracked in 3D in live cells. MUM allows for the simultaneous imaging at different focal planes, thereby ensuring that trajectories can be imaged continuously at high temporal resolution. A critical requirement for 3D single molecule tracking as well as localization based 3D super-resolution imaging is high 3D localization accuracy. MUM overcomes the depth discrimination problem of classical microscopy based approaches and supports high accuracy 3D localization of singe molecule/particles. In this way, MUM opens the way for high precision 3D single molecule tracking and 3D super-resolution imaging within a live cell environment. We have used MUM to reveal complex intracel-lular pathways that could not be imaged with classical approaches. In particular we have tracked quantum dot labeled antibody molecules in the exo/endocytic pathway from the cell interior to the plasma membrane at the single molecule level. Here, we present a brief review of these results.
机译：在活细胞环境中对三维（3D）进行单分子跟踪有望揭示出对细胞生物学机制的重要新见解。但是，经典的显微镜技术受深度分辨能力差的困扰，这严重限制了3D具有高时空分辨率的单分子跟踪。我们介绍了一种新颖的成像方式，多焦点平面显微镜（MUM），用于研究3D中的亚细胞动力学。我们已经证明MUM提供了一种强大的方法，利用该方法可以在活细胞中以3D方式跟踪单个分子。 MUM允许在不同的焦平面上同时成像，从而确保可以在高时间分辨率下连续成像轨迹。 3D单分子跟踪以及基于定位的3D超分辨率成像的关键要求是高3D定位精度。 MUM克服了传统的基于显微镜的方法的深度判别问题，并支持单分子/粒子的高精度3D定位。这样，MUM为活细胞环境中的高精度3D单分子跟踪和3D超分辨率成像开辟了道路。我们已经使用MUM揭示了经典方法无法成像的复杂细胞内通路。特别是，我们已经跟踪了从单分子水平到细胞内部到质膜的胞外/内吞途径中量子点标记的抗体分子。在这里，我们对这些结果进行简要回顾。

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