Tracking the single molecule dynamics of transcription factors in a living cell

Stroboscopic photo-activation (SPA) coupled with single particle tracking (SPT) constitutes a powerful method to probe the intracellular journey of proteins in a living cell with single molecule resolution. This technology, which is based on: (1) pulsing the laser light to eliminate motion blur via use of an acousto-optic tunable filter and (2) a total internal reflection fluorescence (TIRF) microscope equipped with a perfect focusing system to correct for axial drift and motorised laser illumination to allow for a highly inclined and laminated optical (HILO) sheet; has enabled the DNA target search strategy of transcription factors to be elucidated with unprecedented spatiotemporal resolution. Here we request this modified TIRF microscope set-up that is capable of performing spaSPT coupled to a time resolved camera that would enable concomitant fluorescence lifetime imaging microscopy (FLIM) of Förster resonance energy transfer (FRET) interaction - a phenomenon that can report transcription factor interaction with a fluorescently labelled target DNA sequence. This one of a kind research infrastructure would enable scientists in Australia to image for the first time the biophysical mechanism by which a transcription factor navigates the nuclear architecture of a living cell and the kinetics of transcription factor interaction with a DNA target sequence that lead to a specific gene expression program.