Accuracy of the detection of binding events using 3D single particle tracking.
BACKGROUND
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 coefficientD. However, the precision ofDsets 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.


RESULTS
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.


CONCLUSIONS
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.
Huygens-Kamerlingh Onnes Laboratory, Leiden University, Postbus 9504, Leiden, 2300RA Netherlands.
Sara
S
Carozza
Huygens-Kamerlingh Onnes Laboratory, Leiden University, Postbus 9504, Leiden, 2300RA Netherlands.
Jamie
J
Culkin
Huygens-Kamerlingh Onnes Laboratory, Leiden University, Postbus 9504, Leiden, 2300RA Netherlands.
John
0000-0002-9094-5598
J
van Noort
Binding event; Diffusion; Mean square displacement analysis; Single particle tracking