Abstract：
Here we provide demonstration that fast fluorescence fluctuation spectroscopy is a fast and robust approach to extract information on the dynamics of molecules enclosed within subcellular nanostructures (e.g., organelles or vesicles) which are also moving in the complex cellular environment. In more detail, Raster Image Correlation Spectroscopy (RICS) performed at fast timescales (i.e., microseconds) reveals the fast motion of fluorescently labeled molecules within two exemplary dynamic subcellular nanostructures of biomedical interest, the lysosome and the insulin secretory granule (ISG). The measurement of molecular diffusion is then used to extract information on the average properties of subcellular nanostructures, such as macromolecular crowding or molecular aggregation. Concerning the lysosome, fast RICS on a fluorescent tracer allowed us to quantitatively assess the increase in organelle viscosity in the pathological condition of Krabbe disease. In the case of ISGs, fast RICS on two ISG-specific secreting peptides unveiled their differential aggregation propensity depending on intragranular concentration. Finally, a combination of fast RICS and feedback-based 3D orbital tracking was used to subtract the slow movement of subcellular nanostructures from the fast diffusion of molecules contained within them and independently validate the results. Results presented here not only demonstrate the acquired ability to address the dynamic behavior of molecules in moving, nanoscopic reference systems, but prove the relevance of this approach to advance our knowledge on cell function at the subcellular scale.
摘要：
在这里，我们提供了快速荧光波动光谱是一种快速而稳健的方法来提取有关亚细胞纳米结构内封闭的分子动力学的信息（例如，细胞器或囊泡）也在复杂的细胞环境中移动。更详细地说,在快速时间尺度上执行的光栅图像相关光谱学(RICS)(即，微秒）揭示了生物医学兴趣的两个示例性动态亚细胞纳米结构中荧光标记的分子的快速运动，溶酶体和胰岛素分泌颗粒(ISG)。然后使用分子扩散的测量来提取有关亚细胞纳米结构的平均性质的信息，如大分子拥挤或分子聚集。关于溶酶体，荧光示踪剂上的快速RICS使我们能够定量评估Krabbe病病理状态下细胞器粘度的增加。在ISG的情况下，对两种ISG特异性分泌肽的快速RICS揭示了它们依赖于颗粒内浓度的差异聚集倾向。最后,使用快速RICS和基于反馈的3D轨道跟踪相结合的方法,从亚细胞纳米结构中所含分子的快速扩散中减去亚细胞纳米结构的缓慢移动,并独立验证结果.这里提出的结果不仅证明了获得的解决分子在运动中的动态行为的能力，纳米参考系统，但证明了这种方法在亚细胞尺度上提高我们对细胞功能的认识的相关性。
