基于后向散射激光形成的散斑图案的样本漂移估计方法。
Sample drift estimation method based on speckle patterns formed by backscattered laser light.
作者信息
Chen Shih-Ya, Heintzmann Rainer, Cremer Christoph
机构信息
Institute of Molecular Biology, Mainz, Germany.
Institute of Physical Chemistry and Abbe Center of Photonics, Friedrich-Schiller-University Jena, Jena, Germany.
出版信息
Biomed Opt Express. 2019 Nov 25;10(12):6462-6475. doi: 10.1364/BOE.10.006462. eCollection 2019 Dec 1.
Single molecule localization microscopy (SMLM) has been established to acquire images with unprecedented resolution down to several nanometers. A typical time scale for image acquisition is several minutes to hours. Yet it is difficult to avoid completely sample drift for long time measurements. To estimate drift, we present a method based on the evaluation of speckle patterns formed by backscattered laser light from the cells using a single molecule localization microscope setup. A z-stack of unique speckle patterns is recorded prior to the measurements as a three-dimensional position reference. During the experiment, images of scattered laser light were acquired, and correlated individually with each of the images of the speckle reference stack to estimate x, y and z drift. Our method shows highly comparable results with a fiducial marker approach, achieving a precision of several nanometers. This method allows for high precision three dimensional drift correction of microscope systems without any additional sample preparation.
单分子定位显微镜（SMLM）已被用于获取分辨率低至几纳米的前所未有的图像。图像采集的典型时间尺度是几分钟到几小时。然而，长时间测量很难完全避免样本漂移。为了估计漂移，我们提出了一种基于使用单分子定位显微镜装置评估细胞反向散射激光形成的散斑图案的方法。在测量之前记录一组独特散斑图案的z轴堆栈作为三维位置参考。在实验过程中，采集散射激光的图像，并将其分别与散斑参考堆栈的每个图像进行关联，以估计x、y和z方向的漂移。我们的方法与基准标记方法显示出高度可比的结果，实现了几纳米的精度。该方法无需任何额外的样品制备，即可对显微镜系统进行高精度的三维漂移校正。
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