Carozza Sara, Culkin Jamie, van Noort John
Huygens-Kamerlingh Onnes Laboratory, Leiden University, Postbus 9504, Leiden, 2300RA Netherlands.
BMC Biophys. 2017 Mar 23;10:3. doi: 10.1186/s13628-017-0035-8. eCollection 2017.
Nanoparticles can be used as markers to track the position of biomolecules, such as single proteins, inside living cells. The activity of a protein can sometimes be inferred from changes in the mobility of the attached particle. Mean Square Displacement analysis is the most common method to obtain mobility information from trajectories of tracked particles, such as the diffusion coefficient . However, the precision of sets a limit to discriminate changes in mobility caused by biological events from changes that reflect the stochasticity inherent to diffusion. This issue is of particular importance in an experiment aiming to quantify dynamic processes.
Here, we present simulations and 3D tracking experiments with Gold Nanorods freely diffusing in glycerol solution to establish the best analysis parameters to extract the diffusion coefficient. We applied this knowledge to the detection of a temporary change in diffusion, as it can occur due to the transient binding of a particle to an immobile structure within the cell, and tested its dependence on the magnitude of the change in diffusion and duration of this event.
The simulations show that the spatial accuracy of particle tracking generally does not limit the detection of short binding events. Careful analysis of the magnitude of the change in diffusion and the number of frames per binding event is required for accurate quantification of such events.
纳米颗粒可作为标记物来追踪生物分子（如单个蛋白质）在活细胞内的位置。有时可以从附着颗粒的迁移率变化推断蛋白质的活性。均方位移分析是从被追踪颗粒的轨迹获取迁移率信息（如扩散系数）的最常用方法。然而， 的精度限制了区分由生物事件引起的迁移率变化与反映扩散固有随机性的变化。在旨在量化动态过程的实验中，这个问题尤为重要。
在这里，我们展示了金纳米棒在甘油溶液中自由扩散的模拟和三维追踪实验，以确定提取扩散系数的最佳分析参数。我们将这一知识应用于检测扩散的临时变化，这种变化可能由于颗粒与细胞内固定结构的瞬时结合而发生，并测试了其对扩散变化幅度和该事件持续时间的依赖性。
模拟表明，颗粒追踪的空间精度通常不会限制短结合事件的检测。为了准确量化此类事件，需要仔细分析扩散变化的幅度和每个结合事件的帧数。
