Complex cellular processes can be characterised using single particle tracking techniques (SPT). ‘Labels’ such as metal nanoparticles are introduced into cells and tracked to reveal molecular...Show moreComplex cellular processes can be characterised using single particle tracking techniques (SPT). ‘Labels’ such as metal nanoparticles are introduced into cells and tracked to reveal molecular trajectories. Many current techniques are based on fluorescence microscopy, and have nanometre-resolution. To accurately probe cellular processes, a technique must also have long-term 3D in-vivo observing capability with a minimal toxic effect. Gold nanorods (GNRs) in two-photon microscopy is a promising technique. GNRs are non-toxic, easily functionalisable, and exhibit a bright two-photon fluorescence. However, the theoretical positional accuracy for this technique is not yet known. Furthermore, the detailed trajectory data present statistical challenges. We have addressed these issues here numerically, using simulated image data. We found the accuracy to be between 5.2nm and 8.9nm for stationary GNRS, dependent on the separation between slices. We also found that diffusive movement of a nanorod lowers the accuracy, at worst case to 31.8nm. We have also investigated optimising the extraction of behaviour properties from MSD plots, and have used Welsh’s test to detect transitions. GNRs in two-photon microscopy has been shown to be a very accurate technique, and its trajectory data can yield accurate behavioural information. The fit of the PSF may be improved, but the techniques already compares well against others.Show less
