We aim to develop new probes and methods that will allow direct measurements of the spatio- temporal behavior of proteins in living cells. Using multifunctional dendrimers decorated with two dyes, a fluorescent donor array and a fluorogenic quencher dye, we will develop very high brightness probes that are activated by small fusion protein tags. Because the binding of these modules produces an intermittent signal, we will integrate these probes into a new superresolution imaging method that enables molecular scale localization and tracking of all tagged proteins in a sample region by stochastic activation at equilibrium. We will validate these new probes and methods in studies of ?2 adrenergic receptor and cytoskeletal dynamics. Collectively, this program will develop, validate, and deliver new imaging tools and methods to track positions and dynamics of tagged proteins at nanometer length scales. Collectively, these methods are anticipated to deliver at least an order of magnitude improvement in the single molecule brightness, the localization accuracy, and the timescale of dynamic single molecule measurements in living cells.
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