Multicolor 3D Orbital Tracking.

Feedback-based single-particle tracking (SPT) is a powerful technique for investigating particle behavior with very high spatiotemporal resolution. The ability to follow different species and their interactions independently adds a new dimension to the information available from SPT. However, only a few approaches have been expanded to multiple colors and no method is currently available that can follow two differently labeled biomolecules in 4 dimensions independently. In this proof-of-concept paper, the new modalities available when performing 3D orbital tracking with a second detection channel are demonstrated. First, dual-color tracking experiments are described studying independently diffusing particles of different types. For interacting particles where their motion is correlated, a second modality is implemented where a particle is tracked in one channel and the position of the second fluorescence species is monitored in the other channel. As a third modality, 3D orbital tracking is performed in one channel while monitoring its spectral signature in a second channel. This last modality is used to successfully readout accurate Förster Resonance Energy Transfer (FRET) values over time while tracking a mobile particle.