Accuracy and Precision in Camera-Based Fluorescence Correlation Spectroscopy Measurements
Accuracy and Precision in Camera-Based Fluorescence Correlation Spectroscopy Measurements
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DOI：
10.1021/ac303485t
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发表时间：
2013-04-16
影响因子：
7.4
通讯作者：
Wohland, Thorsten
中科院分区：
文献类型：
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作者：
Sankaran, Jagadish;Bag, Nirmalya;Wohland, Thorsten
使用阵列检测器进行的成像荧光相关光谱（FCS）已成功地用于量化细胞和生物体中生物分子的数量、迁移率和组织。然而，还没有任何系统的研究，在这些估计中的错误，由于仪器和实验因素。最先进的阵列检测器仍然受到每单位时间可以记录的帧数、灵敏度和噪声特性以及可以实际记录的帧数的限制。这些限制分别限制了时间分辨率、信噪比和总测量时间。这项工作解决了这些问题，通过使用模拟和实验相结合的脂质双层提供特性的性能参数和指导方针，管理的准确性和精度的扩散系数和浓度测量在基于相机的FCS。然后，我们继续证明这些参数的影响，基于相机的FCS的能力，以确定膜的异质性，通过FCS的扩散定律，表明有一个较低的长度尺度限制超过膜组织无法检测，可以通过选择合适的实验参数克服。在这些结果的基础上，我们提供了一个有效的实验设计，基于相机的FCS提取信息的流动性，浓度和异质性的指导方针。
Imaging fluorescence correlation spectroscopy (FCS) performed using array detectors has been successfully used to quantify the number, mobility, and organization of biomolecules in cells and organisms. However, there have not been any systematic studies on the errors in these estimates that are introduced due to instrumental and experimental factors. State-of-the-art array detectors are still restricted in the number of frames that can be recorded per unit time, sensitivity and noise characteristics, and the total number of frames that can be realistically recorded. These limitations place constraints on the time resolution, the signal-to-noise ratio, and the total measurement time, respectively. This work addresses these problems by using a combination of simulations and experiments on lipid bilayers to provide characteristic performance parameters and guidelines that govern accuracy and precision of diffusion coefficient and concentration measurements in camera-based FCS. We then proceed to demonstrate the effects of these parameters on the capability of camera-based FCS to determine membrane heterogeneity via the FCS diffusion laws, showing that there is a lower length scale limit beyond which membrane organization cannot be detected and which can be overcome by choosing suitable experimental parameters. On the basis of these results, we provide guidelines for an efficient experimental design for camera-based FCS to extract information on mobility, concentration, and heterogeneity.