Since its initial development in 1976, fluorescence recovery after photobleaching (FRAP) has been one of the most popular tools for studying diffusion and protein dynamics in living cells. Its popularity is derived from the widespread availability of confocal microscopes and the relative ease of the experiment and analysis. FRAP, however, is limited in its ability to resolve spatial heterogeneity. Here, we combine selective plane illumination microscopy (SPIM) and FRAP to create SPIM-FRAP, wherein we use a sheet of light to bleach a 2D plane and subsequently image the recovery of the same image plane. This provides simultaneous quantification of diffusion or protein recovery for every pixel in a given 2D slice, thus moving FRAP measurements beyond these previous limitations. We demonstrate this technique by mapping intranuclear diffusion of NLS-GFP in live MDA-MB-231 cells; SPIM-FRAP proves to be an order of magnitude faster than fluorescence correlation spectroscopy (FCS) based techniques for such measurements. We observe large length-scale (> ~500 nm) heterogeneity in the recovery times of NLS-GFP, which is validated against simulated data sets. 2D maps of recovery times were correlated with fluorescence images of H2B to address conflicting literature on the role of chromatin in diffusion of small molecules. We observed no correlation between histone density and diffusion. We developed a diffusion simulation for our SPIM-FRAP experiments to compare across techniques; our measured diffusion coefficients are on the order of previously reported results, thus validating the quantitative accuracy of SPIM-FRAP relative to well-established methods. With the recent rise of accessibility of SPIM systems, SPIM-FRAP is set to provide a simple and quick means of quantifying the spatial distribution of protein recovery or diffusion in living cells.

中文翻译：

---

组合选择性平面照明显微镜和FRAP绘制NLS-GFP的核内扩散图

自1976年首次开发以来，光漂白后的荧光恢复（FRAP）一直是研究活细胞中扩散和蛋白质动力学的最流行工具之一。它的流行是由于共聚焦显微镜的广泛应用以及实验和分析的相对简便性。但是，FRAP解决空间异质性的能力有限。在这里，我们结合选择性平面照明显微镜（SPIM）和FRAP来创建SPIM-FRAP，其中我们使用一片光对2D平面进行漂白，然后对同一图像平面的恢复进行成像。这样可以同时量化给定2D切片中每个像素的扩散或蛋白质回收率，从而使FRAP测量超出了先前的限制。我们通过在活的MDA-MB-231细胞中绘制NLS-GFP的核内扩散来证明该技术；事实证明，SPIM-FRAP比基于荧光相关光谱（FCS）的技术快一个数量级。我们在NLS-GFP的恢复时间中观察到了大尺度（> 500 nm）的异质性，这已针对模拟数据集进行了验证。恢复时间的二维图与H2B的荧光图像相关，以解决关于染色质在小分子扩散中作用的矛盾文献。我们观察到组蛋白密度和扩散之间没有相关性。我们为SPIM-FRAP实验开发了扩散模拟，以比较各种技术。我们测得的扩散系数约为先前报告的结果，因此，相对于公认的方法，可以验证SPIM-FRAP的定量准确性。随着SPIM系统可访问性的最新发展，SPIM-FRAP旨在提供一种简单快速的方法来量化活细胞中蛋白质回收或扩散的空间分布。