Fast Diffusion Characterization by Multiphoton Excited Fluorescence Recovery while Photobleaching
Fast Diffusion Characterization by Multiphoton Excited Fluorescence Recovery while Photobleaching
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DOI：
10.1021/acs.analchem.3c02638
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发表时间：
2023-09-12
影响因子：
7.4
通讯作者：
Simpson，Garth J.
中科院分区：
文献类型：
--
作者：
Li，Minghe;Razumtcev，Aleksandr;Simpson，Garth J.
多光子激发的荧光恢复，而光漂白（FRWP）被证明是一种方法，用于定量测量的快速分子扩散超过微秒到毫秒的时间尺度。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100 μm2/s的系统可能具有挑战性。例如，光漂白后的荧光恢复（FRAP）是基于在相对缓慢的恢复之前相对较快的光漂白步骤的隐含假设进行的，并且通常不适用于在光漂白期间表现出实质性恢复的系统。这些挑战在多光子激发中由于较低的激发效率和来自局部加热的竞争效应而加剧。本文中，引入具有图案化线漂白照明的光束扫描FRWP作为解决FRAP限制的技术，并通过测量更快的扩散事件来进一步扩展其应用范围。在FRWP中，每次通过快速扫描镜后都会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的限制。介绍了一个描述荧光强度瞬态波动的理论模型，该模型是由于光漂白和局部光热效应的综合贡献而产生的，沿着引入了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分子在不同粘度的水溶液中的正常扩散来测试FRWP。
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 μm2/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.