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.
KEYWORDS: Microscopes, 3D image processing, Stereoscopy, Particles, Viruses, Two photon imaging, Temporal resolution, Microscopy, 3D vision, 3D acquisition
The structure and dynamics of intracellular water constitute the cornerstone for understanding all aspects of cellular function. However, direct visualization of subcellular solvation heterogeneity has remained elusive. To explore this question, we have demonstrated a vibrational-shift imaging approach to probe solvation at the microscopic level by combining spectral-focusing hyperspectral stimulated Raman scattering (hsSRS) with an environmentally-sensitive nitrile probe. When applied to quantitatively measure the spatial variation of solvation in live cells, this new method reveals significantly reduced solvation in the cytoplasm compared to the nuclear compartment and bulk water! This work sheds light on heterogenous solvation at the subcellular level and opens up new avenues to explore solvation variance in complex systems.
Measuring the behavior of single molecules enables the discovery of states and dynamics obscured by bulk measurements. However, molecules in solution rapidly diffuse in three dimensions, precluding long-duration and high-temporal resolution measurement. To overcome this hurdle, we have developed 3D single-molecule active real-time tracking (3D-SMART) which enables active feedback tracking of rapidly diffusing (exceeding 10 μm2/sec) and lowly emitting fluorescent (rates of 10 kHz or less) particles. Here we demonstrate the application of 3D-SMART to a range targets, from single virus-like particles and quantum dots in water, all the way down to single fluorophores in viscous solution. This new single molecule tracking can be applied to continuously monitor single proteins and nucleic acids, including real-time measurement of transcription on a freely diffusing, single-dye labelled DNA strand. 3D-SMART represents a critical step towards the untethering of single molecule spectroscopy.
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