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 two-dimensional (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 both intranuclear diffusion of NLS-GFP and recovery of 53BP1-mCherry, a marker for DNA damage, in live MDA-MB-231 cells. SPIM-FRAP proves to be an order of magnitude faster than fluorescence-correlation-spectroscopy-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 NLS-GFP recovery times showed no pixel-by-pixel correlation with histone density, although slower diffusion was observed in nucleoli. Additionally, recovery of 53BP1-mCherry was observed to be slowed at sites of DNA damage. We finally 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 straightforward means of quantifying the spatial distribution of protein recovery or diffusion in living cells.

中文翻译：

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结合选择性平面照明显微镜和 FRAP 映射 NLS-GFP 的核内扩散

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。它的流行源于共聚焦显微镜的广泛可用性以及实验和分析的相对容易性。然而，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来漂白二维 (2D) 平面，然后对同一图像平面的恢复进行成像。这为给定 2D 切片中的每个像素提供了扩散或蛋白质恢复的同步量化，从而使 FRAP 测量超出了这些先前的限制。我们通过在活的 MDA-MB-231 细胞中绘制 NLS-GFP 的核内扩散和 53BP1-mCherry（一种 DNA 损伤标记物）的恢复来证明这项技术。SPIM-FRAP 被证明比基于荧光相关光谱的技术快一个数量级，用于此类测量。我们观察到 NLS-GFP 恢复时间的大长度尺度（>~500 nm）异质性，这已针对模拟数据集进行了验证。NLS-GFP 恢复时间的二维图显示与组蛋白密度没有逐像素相关性，尽管在核仁中观察到较慢的扩散。此外，观察到 53BP1-mCherry 的恢复在 DNA 损伤部位减慢。我们最终为我们的 SPIM-FRAP 实验开发了一个扩散模拟，以比较不同的技术。我们测量的扩散系数与先前报告的结果一致，从而验证了 SPIM-FRAP 相对于成熟方法的定量准确性。随着最近 SPIM 系统可访问性的兴起，SPIM-FRAP 将提供一种直接的方法来量化活细胞中蛋白质回收或扩散的空间分布。