Microscope calibration protocol for single-molecule microscopy.
Microscope calibration protocol for single-molecule microscopy.
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DOI：
10.1364/oe.408361
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发表时间：
2021-01-04
期刊：
影响因子：
3.8
通讯作者：
Ober RJ
中科院分区：
文献类型：
--
作者：
You S;Chao J;Cohen EAK;Ward ES;Ober RJ
单分子显微镜可以在高空间分辨率下研究单个分子的动力学和亚细胞结构的可视化。对于单分子成像实验，特别是那些需要获取多色数据的实验，因此需要以高精度对显微镜及其光学部件进行校准。在这里，我们提出了一种在纳米尺度上校准显微镜的方法，其意义是通过纳米量级的点源定位误差来确定光学像差。该方法基于标准样品的成像来检测和评估光学光路中引入的几何像差量。为了支持多色成像，它还包括评估由二向色滤光片引起的几何像差和由物镜引入的轴向色差的程序。
Single-molecule microscopy allows for the investigation of the dynamics of individual molecules and the visualization of subcellular structures at high spatial resolution. For single-molecule imaging experiments, and particularly those that entail the acquisition of multicolor data, calibration of the microscope and its optical components therefore needs to be carried out at a high level of accuracy. We propose here a method for calibrating a microscope at the nanometer scale, in the sense of determining optical aberrations as revealed by point source localization errors on the order of nanometers. The method is based on the imaging of a standard sample to detect and evaluate the amount of geometric aberration introduced in the optical light path. To provide support for multicolor imaging, it also includes procedures for evaluating the geometric aberration caused by a dichroic filter and the axial chromatic aberration introduced by an objective lens.