Theoretical comparison of real-time feedback-driven single-particle tracking techniques

Abstract

Real-time feedback-driven single-particle tracking is a technique that uses feedback control to enable single-molecule spectroscopy of freely diffusing particles in native or near-native environments. A number of different real-time feedback-driven single-particle tracking (RT-FD-SPT) approaches exist, and comparisons between methods based on experimental results are of limited use due to differences in samples and setups. In this study, we used statistical calculations and dynamical simulations to directly compare the performance of different methods. The methods considered were the orbital method, the knight‘s tour (grid scan) method, and MINFLUX, and we considered both fluorescence-based and interferometric scattering (iSCAT) approaches. There is a fundamental trade-off between precision and speed, with the knight’s tour method being able to track the fastest diffusion but with low precision, and MINFLUX being the most precise but only tracking slow diffusion. To compare iSCAT and fluorescence, different biological samples were considered, including labeled and intrinsically fluorescent samples. The success of iSCAT as compared to fluorescence is strongly dependent on the particle size and the density and photophysical properties of the fluorescent particles. Using a wavelength for iSCAT that is negligibly absorbed by the tracked particle allows for an increased illumination intensity, which results in iSCAT providing better tracking for most samples. This work highlights the fundamental aspects of performance in RT-FD-SPT and should assist with the selection of an appropriate method for a particular application. The approach used can easily be extended to other RT-FD-SPT methods.

Combined online Bayesian and windowed estimation of background and signal localization facilitates active-feedback particle tracking in complex environments

“…In homogenous background environments, COBWEBS tracking of dim particles occasionally fails at background levels as low as the approximate OCR of the particle (Figure S11A). Despite these occasional failures in the even background environment, introduction of a background interface shows superior COBWEBS tracking for cases (3)(4)(5) containing background levels and equivalent tracking for cases (1-2) (Figure S11C).…”

“…Active-feedback particle tracking microscopy applies to an increasing range of critical biological and chemical research questions. [1][2][3][4][5][6][7][8][9] Despite successes in tracking single dye labeled molecules in vitro and brighter particles in live cells, the extension of these powerful methods to tracking rapidly diffusing and unconfined proteins in live cells has not been realized. Tetrahedral approaches have been applied to single fluorescent dyes and fluorescent proteins in glycerol/water solutions, as well as to membrane bound or vesicle confined quantum dot (bright) labeled proteins in live cells.…”

Active-Feedback 3D Single-Molecule Tracking Using a Fast-Responding Galvo Scanning Mirror

“…Limiting the displacement of the molecule within the response time of the control actuator leads to an increased tracking performance. We note here that even an infinitely fast response element will have degraded tracking performance with increased diffusion coefficient . The effect of the increased diffusion speed could be mitigated by implementing a more sophisticated feedback controller.…”