Fluorescence colocalization microscopy analysis can be improved by combining object-recognition with pixel-intensity-correlation.
Fluorescence colocalization microscopy analysis can be improved by combining object-recognition with pixel-intensity-correlation.
复制标题
通过将对象识别与像素强度相关相结合可以改进荧光共定位显微镜分析。
DOI：
10.1002/biot.201600332
复制
发表时间：
2017-01
影响因子：
4.7
通讯作者：
Schmid JA
中科院分区：
文献类型：
--
作者：
Moser B;Hochreiter B;Herbst R;Schmid JA
两种蛋白质是否相互作用或者蛋白质是否定位于细胞的某个区域的问题通常通过荧光显微镜和荧光标记的潜在共定位分析来解决。由于单纯的视觉估计无法量化共定位程度，因此通常使用不同的像素强度相关性统计方法对其进行评分。我们观察到，这些相关系数很容易出现假阳性结果，并且即使对于驻留在不同细胞器中的分子也往往显示出较高的值。我们的目标是改进此类分析，并开发了一种新颖的方法，将基于对象识别的共定位分析与像素强度相关性相结合，以计算对象校正的皮尔逊系数。我们为 ImageJ 软件的斐济版设计了一个宏，并使用各种细胞器标记系统地测试了性能，表明我们的方法比经典方法具有更高的稳健性。为了证明共定位并不一定意味着物理相互作用，我们进行了 FRET（荧光共振能量转移）显微镜检查。这证实了非相互作用分子可以表现出几乎完全的共定位，但与彼此结合的蛋白质相比，它们没有表现出任何显着的 FRET 信号。
The question whether two proteins interact with each other or whether a protein localizes to a certain region of the cell is often addressed with fluorescence microscopy and analysis of a potential colocalization of fluorescence markers. Since a mere visual estimation does not allow quantification of the degree of colocalization, different statistical methods of pixel‐intensity correlation are commonly used to score it. We observed that these correlation coefficients are prone to false positive results and tend to show high values even for molecules that reside in different organelles. Our aim was to improve this type of analysis and we developed a novel method combining object‐recognition based colocalization analysis with pixel‐intensity correlation to calculate an object‐corrected Pearson coefficient. We designed a macro for the Fiji‐version of the software ImageJ and tested the performance systematically with various organelle markers revealing an improved robustness of our approach over classical methods. In order to prove that colocalization does not necessarily mean a physical interaction, we performed FRET (fluorescence resonance energy transfer) microscopy. This confirmed that non‐interacting molecules can exhibit a nearly complete colocalization, but that they do not show any significant FRET signal in contrast to proteins that are bound to each other.