Harmon Dustin M, Cao Ziyi, Sherman Alex M, Takanti Nita, Murati Kevin, Wimsatt Maura M, Cousineau Michelle L, Hwang Yechan, Taylor Lynne S, Simpson Garth J
Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, United States.
Department of Industrial and Molecular Pharmaceutics, Purdue University, West Lafayette, Indiana 47907, United States.
Anal Chem. 2024 Jun 25;96(25):10161-10169. doi: 10.1021/acs.analchem.3c05728. Epub 2024 Jun 12.
Fourier transform-fluorescence recovery after photobleaching (FT-FRAP) using a diffractive optical element (DOE) is shown to support distance-dependent diffusion analysis in biologically relevant media. Integration of DOEs enables patterning of a dot array for parallel acquisition of point-bleach FRAP measurements at multiple locations across the field of view. In homogeneous media, the spatial harmonics of the dot array analyzed in the spatial Fourier transform domain yield diffusion recovery curves evaluated over specific well-defined distances. Relative distances for diffusive recovery in the spatial Fourier transform domain are directly connected to the 2D () Miller indices of the corresponding lattice lines. The distribution of the photobleach power across the entire field of view using a multidot array pattern greatly increases the overall signal power in the spatial FT-domain for signal-to-noise improvements. Derivations are presented for the mathematical underpinnings of FT-FRAP performed with 2D periodicity in the photobleach patterns. Retrofitting of FT-FRAP into instrumentation for high-throughput FRAP analysis (Formulatrix) supports automated analysis of robotically prepared 96-well plates for precise quantification of molecular mobility. Figures of merit are evaluated for FT-FRAP in analysis for both slow diffusion of fluorescent dyes in glassy polymer matrices spanning several days and model proteins and monoclonal antibodies within aqueous solutions recovering in matters of seconds.
使用衍射光学元件（DOE）的傅里叶变换-光漂白后荧光恢复（FT-FRAP）被证明可支持在生物相关介质中进行距离依赖性扩散分析。DOE的集成能够对点阵进行图案化，以便在整个视野的多个位置并行采集点漂白FRAP测量数据。在均匀介质中，在空间傅里叶变换域中分析的点阵空间谐波会产生在特定明确距离上评估的扩散恢复曲线。空间傅里叶变换域中扩散恢复的相对距离直接与相应晶格线的二维（）米勒指数相关。使用多点阵图案在整个视野上分布光漂白功率，极大地增加了空间傅里叶变换域中的整体信号功率，从而改善了信噪比。文中给出了在光漂白图案中具有二维周期性的FT-FRAP数学基础的推导。将FT-FRAP改装到用于高通量FRAP分析的仪器（Formulatrix）中，支持对机器人制备的96孔板进行自动分析，以精确量化分子迁移率。对FT-FRAP在分析荧光染料在玻璃态聚合物基质中持续数天的缓慢扩散以及水溶液中模型蛋白质和单克隆抗体在数秒内恢复的情况进行了品质因数评估。
