Using COBWEBS for 3D active-feedback tracking of freely diffusing particles in tricky environments

Abstract

Active-feedback 3D single-particle tracking in complex environments is limited to bright slow-moving subjects. Extending this technique to include complex background environments requires the development of particle localization strategies that can account for changing particle and background intensities. In previously reported simulations, 2D tracking utilizing combined online Bayesian with estimation of background and signal (COBWEBS) position estimation yielded improved stability in complex background environments for a variety of particle intensities, diffusive speeds, and patterns. Here, COBWEBS’ improved stability in spatially dependent backgrounds is demonstrated to extend to 3D tracking in numerical simulations. In even background tracking, 3D implementations including COBWEBS estimation show slightly reduced position estimation errors and similar tracking accuracy to traditional Kalman estimation. Uneven background tracking shows improved tracking stability for COBWEBS estimation along XY paired with conventional Kalman estimation. Pairing COBWEBSXY with COBWEBS adapted to the Z axis further improves performance in limited cases. In 3D, the windowed signal and background estimation approach remains proportionally responsive to changing particle and background intensities but systematically underestimates true signal values. Overall, COBWEBS’ improved stability in 3D complex environments should expand the application scope of active-feedback single-particle tracking approaches.