Using fixed fiduciary markers for stage drift correction.
Using fixed fiduciary markers for stage drift correction.
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DOI：
10.1364/oe.20.012177
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发表时间：
2012-05-21
期刊：
影响因子：
3.8
通讯作者：
Selvin PR
中科院分区：
文献类型：
--
作者：
Lee SH;Baday M;Tjioe M;Simonson PD;Zhang R;Cai E;Selvin PR
为了以超分辨率测量纳米特征，需要保持样品的载物台稳定到纳米精度。在这里，我们介绍了一种新的方法，使用传统的设备，是低成本的，不需要密集的计算。在盖玻片上以规则的间隔放置在x、y和z方向上约为1 µ m x 1 µm x 1 µm的可信标记。这些基准标记物易于放下，相对于盖玻片完全静止，是生物相容的，并且不干扰荧光或强度测量。当盖玻片经历漂移（或被故意移动）时，可以使用高斯拟合容易地将基准标记的x-y中心跟踪到1纳米。通过使光稍微离焦聚焦，通过观察衍射环，对于干样品，z轴也可以跟踪到< 5 nm，对于湿样品，z轴也可以跟踪到<17 nm。跟踪基准标记的过程不会干扰可见荧光，因为使用了红外发光二极管（IR-LED）（690和850 nm），并且使用廉价的相机单独检测IR光。盖片的所产生的运动然后可以在事后或通过使用主动稳定器来校正，以校正运动。我们应用这种方法来观察驱动蛋白的步行与8 nm的步骤。
To measure nanometric features with super-resolution requires that the stage, which holds the sample, be stable to nanometric precision. Herein we introduce a new method that uses conventional equipment, is low cost, and does not require intensive computation. Fiduciary markers of approximately 1 µm x 1 µm x 1 µm in x, y, and z dimensions are placed at regular intervals on the coverslip. These fiduciary markers are easy to put down, are completely stationary with respect to the coverslip, are bio-compatible, and do not interfere with fluorescence or intensity measurements. As the coverslip undergoes drift (or is purposely moved), the x-y center of the fiduciary markers can be readily tracked to 1 nanometer using a Gaussian fit. By focusing the light slightly out-of-focus, the z-axis can also be tracked to < 5 nm for dry samples and <17 nm for wet samples by looking at the diffraction rings. The process of tracking the fiduciary markers does not interfere with visible fluorescence because an infrared light emitting diode (IR-LED) (690 and 850 nm) is used, and the IR-light is separately detected using an inexpensive camera. The resulting motion of the coverslip can then be corrected for, either after-the-fact, or by using active stabilizers, to correct for the motion. We applied this method to watch kinesin walking with ≈8 nm steps.