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首页> 美国卫生研究院文献>Scientific Reports >3D tracking of single nanoparticles and quantum dots in living cells by out-of-focus imaging with diffraction pattern recognition
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3D tracking of single nanoparticles and quantum dots in living cells by out-of-focus imaging with diffraction pattern recognition

机译：通过具有衍射图案识别的离焦成像对活细胞中的单个纳米颗粒和量子点进行3D跟踪

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Live cells are three-dimensional environments where biological molecules move to find their targets and accomplish their functions. However, up to now, most single molecule investigations have been limited to bi-dimensional studies owing to the complexity of 3d-tracking techniques. Here, we present a novel method for three-dimensional localization of single nano-emitters based on automatic recognition of out-of-focus diffraction patterns. Our technique can be applied to track the movements of single molecules in living cells using a conventional epifluorescence microscope. We first demonstrate three-dimensional localization of fluorescent nanobeads over 4 microns depth with accuracy below 2 nm in vitro. Remarkably, we also establish three-dimensional tracking of Quantum Dots, overcoming their anisotropic emission, by adopting a ligation strategy that allows rotational freedom of the emitter combined with proper pattern recognition. We localize commercially available Quantum Dots in living cells with accuracy better than 7 nm over 2 microns depth. We validate our technique by tracking the three-dimensional movements of single protein-conjugated Quantum Dots in living cell. Moreover, we find that important localization errors can occur in off-focus imaging when improperly calibrated and we give indications to avoid them. Finally, we share a Matlab script that allows readily application of our technique by other laboratories.
机译：活细胞是三维环境，生物分子在其中移动以找到其靶标并完成其功能。但是，由于3d跟踪技术的复杂性，到目前为止，大多数单分子研究仅限于二维研究。在这里，我们提出了一种基于自动识别离焦衍射图案的单纳米发射器三维定位的新方法。使用常规的落射荧光显微镜，我们的技术可以应用于追踪活细胞中单分子的运动。我们首先证明了荧光纳米珠的三维定位在4微米深处的体外精度低于2μnm。值得注意的是，我们还通过采用结扎策略建立了量子点的三维跟踪，以克服它们的各向异性发射问题，该策略允许发射器自由旋转并结合适当的模式识别。我们将可商购获得的量子点定位在活细胞中，在2微米深度上的精度优于7nm。我们通过跟踪单个蛋白缀合的量子点在活细胞中的三维运动来验证我们的技术。此外，我们发现，如果校准不当，在离焦成像中可能会发生重要的定位误差，并给出了避免它们的指示。最后，我们共享一个Matlab脚本，该脚本可让其他实验室轻松应用我们的技术。

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