Anomalous Diffusion Characterization by Fourier Transform-FRAP with Patterned Illumination
Anomalous Diffusion Characterization by Fourier Transform-FRAP with Patterned Illumination
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DOI：
10.1016/j.bpj.2020.07.013
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发表时间：
2020-08-18
影响因子：
3.4
通讯作者：
Simpson, Garth J.
中科院分区：
文献类型：
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作者：
Geiger, Andreas C.;Smith, Casey J.;Simpson, Garth J.
傅立叶变换荧光恢复后的光漂白（FT-FRAP）与图案照明理论和证明了定量评估正常和异常扩散。扩散特性是在细胞生物学、药理学和食品科学中评估流动性的常规方法。传统的FRAP是非侵入性的，样品体积要求低，可以在几微米的距离内快速测量扩散。然而，传统的点漂白剂测量由于信号噪声限制、对光漂白剂光束轮廓的精确了解、样品非均质性造成的潜在偏差以及由于局部加热而与多光子激发的兼容性差而变得复杂。在有图案照明的FT-FRAP中，随时间变化的荧光恢复信号被集中到空间傅里叶域中的点，在信噪比、数学简单性、代表性采样和多光子兼容性方面有了实质性的改进。一个定制的非线性光束扫描显微镜使图案照明的光漂白通过双光子激发。在空间傅里叶域中的测量消除了对光漂白剖面的依赖，抑制了对点扩散函数的不精确知识的偏差。对于正常扩散，荧光恢复在空间傅里叶域中产生简单的单指数衰减，与理论预测非常吻合。通过分析光漂白图案的多个空间谐波，可以在多个长度尺度上同时测量扩散。FT-FRAP通过对荧光恢复的多个空间谐波的非线性拟合来表征异常扩散。约束拟合来描述在多个长度尺度上的扩散导致恢复的拟合参数具有更高的置信度。此外，FT-FRAP中的相位分析被证明可以为流程/样品转换提供信息。
Fourier transform fluorescence recovery after photobleaching (FT-FRAP) with patterned illumination is theorized and demonstrated for quantitatively evaluating normal and anomalous diffusion. Diffusion characterization is routinely performed to assess mobility in cell biology, pharmacology, and food science. Conventional FRAP is noninvasive, has low sample volume requirements, and can rapidly measure diffusion over distances of a few micrometers. However, conventional point-bleach measurements are complicated by signal-to-noise limitations, the need for precise knowledge of the photobleach beam profile, potential for bias due to sample heterogeneity, and poor compatibility with multiphoton excitation because of local heating. In FT-FRAP with patterned illumination, the time-dependent fluorescence recovery signal is concentrated to puncta in the spatial Fourier domain, with substantial improvements in signal-to-noise, mathematical simplicity, representative sampling, and multiphoton compatibility. A custom nonlinear optical beam-scanning microscope enabled patterned illumination for photobleaching through two-photon excitation. Measurements in the spatial Fourier domain removed dependence on the photobleach profile, suppressing bias from imprecise knowledge of the point spread function. For normal diffusion, the fluorescence recovery produced a simple single-exponential decay in the spatial Fourier domain, in excellent agreement with theoretical predictions. Simultaneous measurement of diffusion at multiple length scales was enabled through analysis of multiple spatial harmonics of the photobleaching pattern. Anomalous diffusion was characterized by FT-FRAP through a nonlinear fit to multiple spatial harmonics of the fluorescence recovery. Constraining the fit to describe diffusion over multiple length scales resulted in higher confidence in the recovered fitting parameters. Additionally, phase analysis in FT-FRAP was shown to inform on flow/sample translation.