Gardini Lucia, Capitanio Marco, Pavone Francesco S
LENS - European Laboratory for Non-linear Spectroscopy, Via Nello Carrara 1, 50019 Sesto Fiorentino, Italy.
Department of Physics and Astronomy, University of Florence, Via Sansone 1, 50019 Sesto Fiorentino, Italy.
Sci Rep. 2015 Nov 3;5:16088. doi: 10.1038/srep16088.
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.
活细胞是三维环境，生物分子在其中移动以寻找其靶标并完成其功能。然而，到目前为止，由于三维跟踪技术的复杂性，大多数单分子研究仅限于二维研究。在此，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新方法。我们的技术可应用于使用传统落射荧光显微镜跟踪活细胞中单个分子的运动。我们首先在体外证明了荧光纳米珠在超过4微米深度的三维定位，精度低于2纳米。值得注意的是，我们还通过采用一种连接策略建立了量子点的三维跟踪，克服了它们的各向异性发射，该策略允许发射器具有旋转自由度并结合适当的图案识别。我们在活细胞中定位市售量子点，在超过2微米深度时精度优于7纳米。我们通过跟踪活细胞中单个蛋白质偶联量子点的三维运动来验证我们的技术。此外，我们发现当校准不当时，离焦成像中会出现重要的定位误差，并给出了避免这些误差的提示。最后，我们分享了一个Matlab脚本，便于其他实验室应用我们的技术。
