Optimization of sample preparation and green color imaging using the mNeonGreen fluorescent protein in bacterial cells for photoactivated localization microscopy.
Optimization of sample preparation and green color imaging using the mNeonGreen fluorescent protein in bacterial cells for photoactivated localization microscopy.
复制标题
DOI：
10.1038/s41598-018-28472-0
复制
发表时间：
2018-07-04
影响因子：
4.6
通讯作者：
Shin JY
中科院分区：
文献类型：
--
作者：
Stockmar I;Feddersen H;Cramer K;Gruber S;Jung K;Bramkamp M;Shin JY
mNeonGreen荧光蛋白能够光开关，因此原则上适用于超分辨率成像。然而，难以控制的闪烁动力学，导致多个附近的mNeonGreen分子的同时发射，阻碍了其用于PALM。在这里，我们确定了开关速率和照明功率对同时发射的影响。增加照明功率降低了同时发射的概率，但不足以生成高质量的PALM图像。因此，我们引入了一个简单的数据后处理步骤，该步骤使用分子定位的时间和空间信息来进一步减少由附近发射器的同时发射引起的伪影。我们还系统地评估了各种样品制备步骤，以建立一个优化的协议，以保持细胞形态和荧光信号。总之，我们提出了一个工作流程的超分辨率成像与mNeonGreen的基础上优化的样品制备，数据采集和简单的采集后数据处理。我们的方案的应用使我们能够解析细菌细胞分裂蛋白DivIVA的预期双带，并可视化染色体组织蛋白ParB组织成亚簇，而不是通常观察到的衍射限制焦点。我们希望我们的工作流程允许mNeonGreen广泛用于超分辨率显微镜，这是迄今为止难以实现的。
mNeonGreen fluorescent protein is capable of photo-switching, hence in principle applicable for super-resolution imaging. However, difficult-to-control blinking kinetics that lead to simultaneous emission of multiple nearby mNeonGreen molecules impedes its use for PALM. Here, we determined the on- and off- switching rate and the influence of illumination power on the simultaneous emission. Increasing illumination power reduces the probability of simultaneous emission, but not enough to generate high quality PALM images. Therefore, we introduce a simple data post-processing step that uses temporal and spatial information of molecule localizations to further reduce artifacts arising from simultaneous emission of nearby emitters. We also systematically evaluated various sample preparation steps to establish an optimized protocol to preserve cellular morphology and fluorescence signal. In summary, we propose a workflow for super-resolution imaging with mNeonGreen based on optimization of sample preparation, data acquisition and simple post-acquisition data processing. Application of our protocol enabled us to resolve the expected double band of bacterial cell division protein DivIVA, and to visualize that the chromosome organization protein ParB organized into sub-clusters instead of the typically observed diffraction-limited foci. We expect that our workflow allows a broad use of mNeonGreen for super-resolution microscopy, which is so far difficult to achieve.
登录
查看更多内容
影响因子：
9.2
作者：
Gruber, Stephan;Veening, Jan-Willem;Bach, Juri;Blettinger, Martin;Bramkamp, Marc;Errington, Jeff
通讯作者：
Errington, Jeff
影响因子：
48
作者：
Biteen, Julie S.;Thompson, Michael A.;Tselentis, Nicole K.;Bowman, Grant R.;Shapiro, Lucy;Moerner, W. E.
通讯作者：
Moerner, W. E.
影响因子：
3.2
作者：
REEVE, JN;MENDELSO.NH;COLE, RM
通讯作者：
COLE, RM
DOI：
10.1073/pnas.0500489102
发表时间：
2005-07-05
影响因子：
11.1
作者：
Habuchi, S;Ando, R;Hofkens, J
通讯作者：
Hofkens, J
一系列独特的可逆可切换荧光蛋白，具有适合各种应用的有益特性
DOI：
10.1073/pnas.1113770109
发表时间：
2012-03-20
影响因子：
11.1
作者：
Chang, Hao;Zhang, Mingshu;Xu, Tao
通讯作者：
Xu, Tao