Lorén Niklas, Hagman Joel, Jonasson Jenny K, Deschout Hendrik, Bernin Diana, Cella-Zanacchi Francesca, Diaspro Alberto, McNally James G, Ameloot Marcel, Smisdom Nick, Nydén Magnus, Hermansson Anne-Marie, Rudemo Mats, Braeckmans Kevin
SP Food and Bioscience,PO 5401, SE-402 29, Göteborg,Sweden.
Department of Mathematical Sciences,Chalmers University of Technology,SE-412 96 Göteborg,Sweden.
Q Rev Biophys. 2015 Aug;48(3):323-87. doi: 10.1017/S0033583515000013.
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.
光漂白后荧光恢复（FRAP）是一种用于确定生物和材料科学中扩散以及相互作用/结合特性的多功能工具。要理解控制扩散的机制，就需要深入了解结构 - 相互作用 - 扩散关系。例如，在细胞生物学中，这适用于蛋白质和脂质在质膜、细胞质和细胞核中的移动。在与制药、食品、纺织品、卫生用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质和功能有很大贡献。所有这些系统都是非均匀的，因此在局部水平上准确量化质量传输过程对于理解软（生物）材料的性质至关重要。FRAP是一种常用的基于荧光显微镜的技术，用于确定微米尺度上的局部分子传输。向样品局部施加一个短暂的高强度激光脉冲，使照射区域内的荧光分子发生大量光漂白。这会导致荧光分子的局部浓度梯度，从而使完整的荧光团从局部周围环境扩散流入漂白区域。使用合适的模型，可以从漂白区域荧光恢复的时间演变中提取有关分子传输的定量信息。多年来已经开发了多种FRAP模型，每个模型都基于特定的假设。这使得非专业人员难以决定哪种模型最适合特定应用。此外，进行准确的FRAP实验存在许多微妙之处。出于这些原因，本综述旨在提供一个广泛的教程，涵盖基本的理论和实践方面，以便能够在软（生物）材料中进行准确的定量FRAP实验以测量分子传输。
