Method for high frequency tracking and sub-nm sample stabilization in single molecule fluorescence microscopy.

While fluorescence microscopes and atomic force microscopes are widely used to visualize, track, and manipulate single biomolecules, the resolution of these methods is limited by sample drift. To minimize drift, active feedback methods have recently been used to stabilize single molecule microscopes on the sub-nanometer scale. However, these methods require high intensity lasers which limits their application in single molecule fluorescence measurements. Furthermore, these feedback methods do not track user-defined regions of the sample, but rather monitor the relative displacement of an unknown point on a fiducial marker, which limits their use in biological force measurements. To overcome these limitations, we have developed a novel method to image, track and stabilize a sample using low laser intensities. We demonstrate the capabilities of our approach by tracking a user-chosen point on a fiducial marker at 8.6 kHz and stabilizing it with sub-nanometer resolution. We further showcase the application of our method in single molecule fluorescence microscopy by imaging and stabilizing individual fluorescently-tagged streptavidin proteins under biologically relevant conditions. We anticipate that our method can be easily used to improve the resolution of a wide range of single molecule fluorescence microscopy and integrated force-fluorescence applications.

虽然荧光显微镜和原子力显微镜被广泛用于对单个生物分子进行可视化、追踪和操作，但这些方法的分辨率受到样品漂移的限制。为了使漂移最小化，最近已使用主动反馈方法在亚纳米尺度上稳定单分子显微镜。然而，这些方法需要高强度激光，这限制了它们在单分子荧光测量中的应用。此外，这些反馈方法不是追踪样品中用户定义的区域，而是监测基准标记上一个未知点的相对位移，这限制了它们在生物力测量中的使用。为了克服这些限制，我们开发了一种使用低激光强度对样品进行成像、追踪和稳定的新方法。我们通过以8.6千赫兹的频率追踪基准标记上一个用户选定的点并以亚纳米分辨率稳定它，展示了我们方法的能力。我们还通过在生物相关条件下对单个荧光标记的链霉亲和素蛋白进行成像和稳定，展示了我们的方法在单分子荧光显微镜中的应用。我们预计我们的方法可以很容易地用于提高多种单分子荧光显微镜以及集成力 - 荧光应用的分辨率。