Smith Carlas S, Stallinga Sjoerd, Lidke Keith A, Rieger Bernd, Grunwald David
RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01605.
Quantitative Imaging Group, Department of Imaging Science and Technology, Faculty of Applied Sciences, Delft University of Technology, 2628 CJ Delft, Netherlands.
Mol Biol Cell. 2015 Nov 5;26(22):4057-62. doi: 10.1091/mbc.E15-06-0448. Epub 2015 Sep 30.
Single-molecule detection in fluorescence nanoscopy has become a powerful tool in cell biology but can present vexing issues in image analysis, such as limited signal, unspecific background, empirically set thresholds, image filtering, and false-positive detection limiting overall detection efficiency. Here we present a framework in which expert knowledge and parameter tweaking are replaced with a probability-based hypothesis test. Our method delivers robust and threshold-free signal detection with a defined error estimate and improved detection of weaker signals. The probability value has consequences for downstream data analysis, such as weighing a series of detections and corresponding probabilities, Bayesian propagation of probability, or defining metrics in tracking applications. We show that the method outperforms all current approaches, yielding a detection efficiency of >70% and a false-positive detection rate of <5% under conditions down to 17 photons/pixel background and 180 photons/molecule signal, which is beneficial for any kind of photon-limited application. Examples include limited brightness and photostability, phototoxicity in live-cell single-molecule imaging, and use of new labels for nanoscopy. We present simulations, experimental data, and tracking of low-signal mRNAs in yeast cells.
荧光纳米显微镜中的单分子检测已成为细胞生物学中的一种强大工具，但在图像分析中可能会出现棘手的问题，例如信号有限、非特异性背景、凭经验设置阈值、图像滤波以及假阳性检测，这些都会限制整体检测效率。在此，我们提出了一个框架，其中基于概率的假设检验取代了专家知识和参数调整。我们的方法能够进行稳健的、无阈值的信号检测，并给出定义好的误差估计，同时提高对较弱信号的检测能力。概率值对下游数据分析有影响，例如权衡一系列检测结果及其相应概率、概率的贝叶斯传播，或者在跟踪应用中定义指标。我们表明，该方法优于所有当前方法，在背景低至17光子/像素、信号为180光子/分子的条件下，检测效率大于70%，假阳性检测率小于5%，这对任何光子受限的应用都是有益的。示例包括有限的亮度和光稳定性、活细胞单分子成像中的光毒性以及纳米显微镜新标记的使用。我们展示了模拟结果、实验数据以及酵母细胞中低信号mRNA的跟踪情况。
