Addressing the Requirements of High-Sensitivity Single-Molecule Imaging of Low-Copy-Number Proteins in Bacteria.
Addressing the Requirements of High-Sensitivity Single-Molecule Imaging of Low-Copy-Number Proteins in Bacteria.
复制标题
DOI：
10.1002/cphc.201600035
复制
发表时间：
2016-05-18
期刊：
影响因子：
--
通讯作者：
Biteen JS
中科院分区：
文献类型：
--
作者：
Tuson HH;Aliaj A;Brandes ER;Simmons LA;Biteen JS
单分子荧光超分辨率成像和跟踪提供了有关亚细胞蛋白质位置和动力学的纳米级信息。这些单分子成像实验可能非常强大，但它们最适合高拷贝数蛋白质，其中可以在每个细胞中顺序进行许多测量。我们描述的文物与成像的蛋白质表达在每个细胞只有几个副本的挑战。我们的形象活枯草芽孢杆菌在荧光显微镜下，并证明在标准的单分子成像条件下，未标记的B。subtilis细胞显示出与研究中的少数PAmCherry荧光蛋白单分子难以区分的点状红色荧光点。所有调查的芽孢杆菌属物种都受到这种人为因素的强烈影响，而我们在我们调查的另外两个物种，粪肠球菌和大肠杆菌中没有发现大量的这些背景来源。与单分子分辨率，我们表征的数量，空间分布，和这些杂质斑点的强度。在枯草芽孢杆菌细胞中报道了单分子样荧光背景信号，并且在几种芽孢杆菌属物种和其他革兰氏阴性和革兰氏阳性生物体中定量了这些斑点的密度和荧光强度。
Single-molecule fluorescence super-resolution imaging and tracking provide nanometer-scale information about subcellular protein positions and dynamics. These single-molecule imaging experiments can be very powerful, but they are best suited to high-copy number proteins where many measurements can be made sequentially in each cell. We describe artifacts associated with the challenge of imaging a protein expressed in only a few copies per cell. We image live Bacillus subtilis in a fluorescence microscope, and demonstrate that under standard single-molecule imaging conditions, unlabeled B. subtilis cells display punctate red fluorescent spots indistinguishable from the few PAmCherry fluorescent protein single molecules under investigation. All Bacillus species investigated were strongly affected by this artifact, whereas we did not find a significant number of these background sources in two other species we investigated, Enterococcus faecalis and Escherichia coli. With single-molecule resolution, we characterize the number, spatial distribution, and intensities of these impurity spots. Single-molecule-like fluorescent background signal is reported in Bacillus subtilis cells, and the density and fluorescence intensity of these spots are quantified in several Bacillus species and other Gram-negative and Gram-positive organisms.