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.
Fluorescence microscopy has greatly enhanced our understanding of biological processes, and tools like green fluorescent protein have allowed us to investigate protein behavior in living cells. The properties of traditional imaging, however, are not matched with true biological processes, which occur on lengths that are 50-100-fold shorter than optical imaging traditionally permits. As biologists, it is as if we are looking down from the sky at a building, attempting to understand what is going on inside. New methods are needed to directly investigate the behavior of single proteins in living cells at molecular length scales. In this proposal, we develop a novel approach to labeling, detection, and analysis that will allow direct measurements of the location and behavior of tagged proteins within a living cell at biologically relevant length and timescales.
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