立体暗场干涉测量：一种用于单分子亚纳米力谱和单细胞三维跟踪的通用定位方法
Stereo Darkfield Interferometry : a versatile localization method for subnanometer force spectroscopy of single molecules and 3D-tracking of single cells
摘要：
超分辨率3D跟踪，例如PSF工程或倏逝场成像，长期以来一直用于跟踪微粒并提高单分子力光谱测量的通量。然而，目前的方法存在两个缺点。首先，与光学镊子或AFM相比，它们缺乏精度。其次，它们的信号对位置的依赖性是复杂的，从而需要耗时的校准步骤。在这里，我们介绍了一种新的光学技术，该技术可以解决这两个问题，并允许对稀释颗粒进行简单，通用和高效的3D跟踪，同时在所有三个空间方向上提供亚纳米级的帧到帧精度。其原理是将立体视觉和干涉测量相结合，使得通过两个干涉条纹图案之间的距离来测量z（轴向）位置。该立体技术的线性度使得对查找表的需要变得简单，而结构化干涉图案提高了精度。另一方面，该PSF的扩展空间足迹最大化了每帧检测到的光子数量，而不需要花哨的相机，也不需要复杂的硬件。因此，由于其简单性和多功能性，我们相信SDI（立体暗场干涉）技术有潜力显着提高3D跟踪的传播。我们证明了这种技术的效率在各种单分子测量感谢磁镊子。特别是，我们证明了精确的量化的两态动力学涉及轴向步骤短至1纳米。然后，我们表明SDI可以直接嵌入到商业目标中，提供了一种在3D中跟踪多个单细胞的方法。
英文摘要：
Super-resolutive 3D tracking, such as PSF engineering or evanescent field imaging has long been used to track microparticles and to enhance the throughput of single molecules force spectroscopic measurements. However, current methods present two drawbacks. First, they lack precision compared with optical tweezers or AFM. Second, the dependence of their signal upon the position is complex creating the need for a time-consuming calibration step. Here, we introduce a new optical technique that circumvents both issues and allows for a simple, versatile and efficient 3D tracking of diluted particles while offering a sub-nanometer frame-to-frame precision in all three spatial directions. The principle is to combine stereoscopy and interferometry, such that the z (axial) position is measured through the distance between two interferometric fringe patterns. The linearity of this stereoscopy technique alleviates the need for lookup tables while the structured interferometric pattern enhances precision. On the other hand, the extended spatial footprint of this PSF maximizes the number of photons detected per frame without the need of fancy cameras, nor the need for complex hardware. Hence, thanks to its simplicity and versatility, we believe that SDI (Stereo Darkfield Interferometry) technology has the potential to significantly enhance the spreading of 3D tracking. We demonstrate the efficiency of this technique on various single-molecule measurements thanks to magnetic tweezers. In particular we demonstrate the precise quantification of two-state dynamics involving axial steps as short as 1 nm. We then show that SDI can be directly embedded in a commercial objective providing a means to track multiple single cells in 3D .
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