High Density Single Particle Tracking with Various Probes

Abstract

Single Particle Tracking (SPT) is a set of single molecule techniques that identify and characterize the motion of individual particles in a medium. High Density Single Particle Tracking (HDSPT) is a subset of advanced SPT techniques designed to overcome some of the limitations of current SPT methods, primarily to compensate for poor statistical information due to low trajectory counts per observed area as seen in conventional SPT. Quantum Dots (QDs), Fluorogen Activating Peptides (FAPs), and Organic Dyes are recognized probes that have been used in SPT experiments. QDs are very bright, photo stable probes with blinking dictated by power law behaviors. A FAP will bind to a receptor and activate, emitting photons at a constant rate and intensity until it bleaches. ODs have emission behaviors characterized by Jablonski diagrams; depending on the particular OD and buffer combination, they may blink or bleach. Variable emission dynamics provide a challenge in SPT analysis because it requires localization techniques that can properly account for varying probe behaviors. We have modified our multi-emitter fitting algorithm [1] with a Bayesian approach in order to localize overlapping probes with variable intensity profiles. This advancement in localization capturing allows us to overcome one of the more difficult challenges present in high density SPT- reliable probe identification in high density regions. We have tested our modified SPT software on simulated data as well as on live Rat Basophil Leukemia (RBL) cells with various probes to demonstrate our approach to high density SPT.[1] Fang Huang, Samantha L. Schwartz, Jason M. Byars, and Keith A. Lidke, Biomedical Optics Express, Vol. 2, Issue 5, pp. 1377-1393 (2011).