Li Minghe, Razumtcev Aleksandr, Turner Gwendylan A, Hwang Yechan, Simpson Garth J
Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, Indiana 47907, United States.
Anal Chem. 2023 Sep 26;95(38):14331-14340. doi: 10.1021/acs.analchem.3c02638. Epub 2023 Sep 12.
Multiphoton-excited fluorescence recovery while photobleaching (FRWP) is demonstrated as a method for quantitative measurements of rapid molecular diffusion over microsecond to millisecond timescales. Diffusion measurements are crucial in assessing molecular mobility in cell biology, materials science, and pharmacology. Optical and fluorescence microscopy techniques enable non-invasive rapid analysis of molecular diffusion but can be challenging for systems with diffusion coefficients exceeding ∼100 μm/s. As an example, fluorescence recovery after photobleaching (FRAP) operates on the implicit assumption of a comparatively fast photobleaching step prior to a relatively slow recovery and is not generally applicable for systems exhibiting substantial recovery during photobleaching. These challenges are exacerbated in multiphoton excitation by the lower excitation efficiency and competing effects from local heating. Herein, beam-scanning FRWP with patterned line-bleach illumination is introduced as a technique that addresses FRAP limitations and further extends its application range by measuring faster diffusion events. In FRWP, the recovery of fluorescence is continuously probed after each pass of a fast-scanning mirror, and the upper bound of measurable diffusion rates is, therefore, only limited by the mirror scanning frequency. A theoretical model describing transient fluctuations in fluorescence intensity arising as a result of combined contributions from photobleaching and localized photothermal effect is introduced along with a mathematical framework for quantifying fluorescence intensity temporal curves and recovering room-temperature diffusion coefficients. FRWP is then tested by characterization of normal diffusion of rhodamine-labeled bovine serum albumin, green fluorescence protein, and immunoglobulin G molecules in aqueous solutions of varying viscosity.
多光子激发光漂白荧光恢复（FRWP）被证明是一种用于在微秒至毫秒时间尺度上定量测量快速分子扩散的方法。扩散测量在评估细胞生物学、材料科学和药理学中的分子流动性方面至关重要。光学和荧光显微镜技术能够对分子扩散进行非侵入性快速分析，但对于扩散系数超过约100μm/s的系统可能具有挑战性。例如，光漂白后荧光恢复（FRAP）基于在相对缓慢的恢复之前有一个相对快速的光漂白步骤这一隐含假设运行，并且通常不适用于在光漂白期间表现出大量恢复的系统。在多光子激发中，由于较低的激发效率和局部加热的竞争效应，这些挑战会加剧。在此，引入具有图案化线漂白照明的光束扫描FRWP作为一种解决FRAP局限性并通过测量更快的扩散事件进一步扩展其应用范围的技术。在FRWP中，在快速扫描镜每次扫描后连续探测荧光的恢复，因此，可测量扩散速率的上限仅受镜扫描频率限制。引入了一个理论模型，该模型描述了由于光漂白和局部光热效应的综合贡献而产生的荧光强度的瞬态波动，以及一个用于量化荧光强度时间曲线和恢复室温扩散系数的数学框架。然后通过表征罗丹明标记的牛血清白蛋白、绿色荧光蛋白和免疫球蛋白G分子在不同粘度水溶液中的正常扩散来测试FRWP。
