Fluorescence recovery after photobleaching (FRAP) is a versatile tool for determining diffusion and interaction/binding properties in biological and material sciences. An understanding of the mechanisms controlling the diffusion requires a deep understanding of structure–interaction–diffusion relationships. In cell biology, for instance, this applies to the movement of proteins and lipids in the plasma membrane, cytoplasm and nucleus. In industrial applications related to pharmaceutics, foods, textiles, hygiene products and cosmetics, the diffusion of solutes and solvent molecules contributes strongly to the properties and functionality of the final product. All these systems are heterogeneous, and accurate quantification of the mass transport processes at the local level is therefore essential to the understanding of the properties of soft (bio)materials. FRAP is a commonly used fluorescence microscopy-based technique to determine local molecular transport at the micrometer scale. A brief high-intensity laser pulse is locally applied to the sample, causing substantial photobleaching of the fluorescent molecules within the illuminated area. This causes a local concentration gradient of fluorescent molecules, leading to diffusional influx of intact fluorophores from the local surroundings into the bleached area. Quantitative information on the molecular transport can be extracted from the time evolution of the fluorescence recovery in the bleached area using a suitable model. A multitude of FRAP models has been developed over the years, each based on specific assumptions. This makes it challenging for the non-specialist to decide which model is best suited for a particular application. Furthermore, there are many subtleties in performing accurate FRAP experiments. For these reasons, this review aims to provide an extensive tutorial covering the essential theoretical and practical aspects so as to enable accurate quantitative FRAP experiments for molecular transport measurements in soft (bio)materials.

中文翻译：

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材料和生命科学中光漂白后的荧光恢复：将理论付诸实践

光漂白后的荧光恢复 (FRAP) 是一种多功能工具，用于确定生物和材料科学中的扩散和相互作用/结合特性。了解控制扩散的机制需要深入了解结构-相互作用-扩散关系。例如，在细胞生物学中，这适用于质膜、细胞质和细胞核中蛋白质和脂质的运动。在与制药、食品、纺织品、卫生产品和化妆品相关的工业应用中，溶质和溶剂分子的扩散对最终产品的性能和功能有很大影响。所有这些系统都是异构的，因此，在地方层面准确量化质量传输过程对于理解软（生物）材料的特性至关重要。FRAP 是一种常用的基于荧光显微镜的技术，用于确定微米级的局部分子传输。将短暂的高强度激光脉冲局部应用于样品，导致照明区域内的荧光分子大量光漂白。这会导致荧光分子的局部浓度梯度，导致完整的荧光团从局部环境扩散流入漂白区域。可以使用合适的模型从漂白区域中荧光恢复的时间演变中提取分子传输的定量信息。多年来已经开发了多种 FRAP 模型，每个都基于特定的假设。这使得非专业人士很难决定哪种模型最适合特定应用。此外，在进行准确的 FRAP 实验时有许多微妙之处。出于这些原因，本综述旨在提供一个涵盖基本理论和实践方面的广泛教程，以便为软（生物）材料中的分子传输测量提供准确的定量 FRAP 实验。