Periodic Photobleaching with Structured Illumination for Diffusion Imaging
Periodic Photobleaching with Structured Illumination for Diffusion Imaging
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DOI：
10.1021/acs.analchem.2c02950
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发表时间：
2023-01-19
影响因子：
7.4
通讯作者：
Simpson，Garth J.
中科院分区：
文献类型：
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作者：
Cao，Ziyi;Harmon，Dustin M.;Simpson，Garth J.
周期性结构照明与光漂白后空间傅里叶变换荧光恢复 (FT-FRAP) 的结合被证明可以支持任意形状的分段域内的扩散率映射。光漂白过程中激发光束的周期性“梳状漂白”图案将扩散的空间图编码到空间傅立叶变换中的谐波峰上。扩散表现为给定谐波的简单指数衰减，提高了信噪比并简化了数学分析。傅里叶变换之前的图像分割被证明支持对预期在域内表现出类似扩散率的任意形状的区域进行信噪比增强的池化。基于已知地面实况图的模拟进行概念验证分析后，使用 FT-FRAP 扩散成像来绘制模型非晶固体分散体旋铸薄膜的相分离域内的空间分辨扩散差异。值得注意的是，FT-FRAP 的多谐波分析能够明确区分和量化内部扩散和交换到较高迁移率界面层的作用，以模拟薄非晶/非晶相分离域内的恢复动力学，其中界面扩散在恢复中发挥着关键作用。这些结果对稳定储存和有效递送治疗分子的无定形系统的设计具有直接影响。
The use of periodically structured illumination coupled with spatial Fourier-transform fluorescence recovery after photobleaching (FT-FRAP) was shown to support diffusivity mapping within segmented domains of arbitrary shape. Periodic “comb-bleach” patterning of the excitation beam during photobleaching encoded spatial maps of diffusion onto harmonic peaks in the spatial Fourier transform. Diffusion manifests as a simple exponential decay of a given harmonic, improving the signal to noise ratio and simplifying mathematical analysis. Image segmentation prior to Fourier transformation was shown to support pooling for signal to noise enhancement for regions of arbitrary shape expected to exhibit similar diffusivity within a domain. Following proof-of-concept analyses based on simulations with known ground-truth maps, diffusion imaging by FT-FRAP was used to map spatially-resolved diffusion differences within phase-separated domains of model amorphous solid dispersion spin-cast thin films. Notably, multi-harmonic analysis by FT-FRAP was able to definitively discriminate and quantify the roles of internal diffusion and exchange to higher mobility interfacial layers in modeling the recovery kinetics within thin amorphous/amorphous phase-separated domains, with interfacial diffusion playing a critical role in recovery. These results have direct implications for the design of amorphous systems for stable storage and efficacious delivery of therapeutic molecules.