Parallax microscopy for 3D single-molecule tracking in bacteria

Session

Stream 6 (Frontiers): Quantifying Dynamic Movement in Living Cells

Authors

Dr Helen Miller (1), Mr Rohan Rao (1), Dr Sam Hickman (1), Mr Andreas Kjaer (1), Professor Ben Berks (1), Professor Richard Berry (1)

Affiliations

1. University of Oxford

Keywords

single-molecule; 3D tracking; parallax microscopy; fluorescence

Abstract text

Parallax microscopy¹ uses back focal plane splitting achieve to achieve 3D single molecule optical microscopy. The axial position is calculated from the distance between two localisations of the same particle, obtained from each half of the back-focal plane. The accuracy of this method depends on finding the centres of the two localisations. Most other 3D particle localization methods use shape fitting of a single point-spread function to determine the axial position, which is extremely sensitive to noise and to motion blur from movement of the particle. This has limited single molecule tracking experiments to slow-moving molecules.

We present the results of simulations demonstrating the movement regimes in which parallax microscopy can outperform astigmatism microscopy for localisation of moving particles. Combining parallax microscopy with Halo-Tag liganded Janelia Fluor dyes we demonstrate tracking of fast-moving bacterial proteins in vivo over timescales of seconds. We additionally demonstrate dual-colour tracking using parallax and introduce our suite of parallax microscopy analysis software.

References

1.        Sun, Y., McKenna, J. D., Murray, J. M., Ostap, E. M. & Goldman, Y. E. Parallax: high accuracy three-dimensional single molecule tracking using split images. Nano Lett. 9, 2676–82 (2009).