Churchman L Stirling, Spudich James A
Cold Spring Harb Protoc. 2012 Feb 1;2012(2):141-9. doi: 10.1101/pdb.top067918.
Colocalization of fluorescent probes is commonly used in cell biology to discern the proximity of two proteins in the cell. Considering that the resolution limit of optical microscopy is on the order of 250 nm, there has not been a need for high-resolution colocalization techniques. However, with the advent of higher resolution techniques for cell biology and single-molecule biophysics, colocalization must also improve. For diffraction-limited applications, a geometric transformation (i.e., translation, scaling, and rotation) is typically applied to one color channel to align it with the other; however, to achieve high-resolution colocalization, this is not sufficient. Single-molecule high-resolution colocalization (SHREC) of single probes uses the local weighted mean transformation to achieve a colocalization resolution of at least 10 nm. This article describes the process of collecting a calibration data set of fiducials and the appropriate analysis to determine the transformation for colocalization.
荧光探针的共定位在细胞生物学中常用于识别细胞内两种蛋白质的接近程度。鉴于光学显微镜的分辨率极限约为250纳米，一直以来都不需要高分辨率的共定位技术。然而，随着细胞生物学和单分子生物物理学中更高分辨率技术的出现，共定位也必须得到改进。对于受衍射限制的应用，通常会对一个颜色通道应用几何变换（即平移、缩放和旋转），使其与另一个通道对齐；然而，要实现高分辨率共定位，这还不够。单探针的单分子高分辨率共定位（SHREC）使用局部加权平均变换来实现至少10纳米的共定位分辨率。本文描述了收集基准校准数据集的过程以及用于确定共定位变换的适当分析方法。
