Fluorescence recovery after photobleaching (FRAP) and single particle tracking (SPT) techniques determine the diffusion coefficient from average diffusive motion of high-concentration molecules and from trajectories of low-concentration single molecules, respectively. Lateral diffusion coefficients measured by FRAP and SPT techniques for the same biomolecule on cell membrane have exhibited inconsistent values across laboratories and platforms with larger diffusion coefficient determined by FRAP, but the sources of the inconsistency have not been investigated thoroughly. Here, we designed an image-based FRAP-SPT system and made a direct comparison between FRAP and SPT for diffusion coefficient of submicron particles with known theoretical values derived from Stokes–Einstein equation in aqueous solution. The combined iFRAP-SPT technique allowed us to measure the diffusion coefficient of the same fluorescent particle by utilizing both techniques in a single platform and to scrutinize inherent errors and artifacts of FRAP. Our results reveal that diffusion coefficient overestimated by FRAP is caused by inaccurate estimation of the bleaching spot size and can be corrected by simple image analysis. Our iFRAP-SPT technique can be potentially used for not only cellular membrane dynamics but also for quantitative analysis of the spatiotemporal distribution of the solutes in small scale analytical devices.

中文翻译：

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光漂白后基于图像的荧光恢复测定扩散系数和在单个平台上实施的单粒子跟踪系统

光漂白 (FRAP) 和单粒子跟踪 (SPT) 技术后的荧光恢复分别从高浓度分子的平均扩散运动和低浓度单分子的轨迹确定扩散系数。FRAP 和 SPT 技术测量的细胞膜上相同生物分子的横向扩散系数在实验室和平台上表现出不一致的值，FRAP 确定的扩散系数较大，但不一致的来源尚未彻底调查。在这里，我们设计了一个基于图像的 FRAP-SPT 系统，并直接比较了 FRAP 和 SPT 之间的亚微米粒子扩散系数，已知理论值来自于水溶液中的 Stokes-Einstein 方程。结合的一世FRAP-SPT 技术使我们能够通过在单个平台上利用这两种技术来测量同一荧光粒子的扩散系数，并仔细检查 FRAP 的固有误差和伪影。我们的结果表明，FRAP 高估的扩散系数是由于对漂白点大小的估计不准确造成的，并且可以通过简单的图像分析来纠正。我们的一世FRAP-SPT 技术不仅可以潜在地用于细胞膜动力学，还可以用于定量分析小规模分析设备中溶质的时空分布。