Cao Ziyi, Harmon Dustin M, Yang Ruochen, Razumtcev Aleksandr, Li Minghe, Carlsen Mark S, Geiger Andreas C, Zemlyanov Dmitry, Sherman Alex M, Takanti Nita, Rong Jiayue, Hwang Yechan, Taylor Lynne S, Simpson Garth J
Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, Indiana47907, United States.
Department of Industrial and Physical Pharmacy, Purdue University, 575 Stadium Mall Drive, West Lafayette, Indiana47907, United States.
Anal Chem. 2023 Jan 31;95(4):2192-2202. doi: 10.1021/acs.analchem.2c02950. Epub 2023 Jan 19.
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.
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）相结合的方法，已被证明可用于在任意形状的分割区域内进行扩散率映射。光漂白过程中激发光束的周期性“梳状漂白”图案，将扩散的空间图谱编码到空间傅里叶变换中的谐波峰值上。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，被证明可用于对预期在一个区域内具有相似扩散率的任意形状区域进行信号池化，以增强信噪比。在基于具有已知真实图谱的模拟进行概念验证分析之后，FT - FRAP扩散成像被用于绘制模型非晶态固体分散体旋铸薄膜相分离域内空间分辨的扩散差异。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以及向更高迁移率界面层的交换在模拟非晶态/非晶态相分离薄膜内恢复动力学中的作用，其中界面扩散在恢复过程中起着关键作用。这些结果对用于治疗分子稳定储存和有效递送的非晶态系统设计具有直接影响。
