Advances in molecularly specific fluorescent imaging have revolutionized the tracking of biochemical processes in cells. A continuing problem, however, has been the relatively poor signal-to-noise ratio in the presence of background fluorescence from endogenous or exogenous fluorophores. We propose to employ a signal processing technique called optical modulation to improve that ratio. We will design and synthesize proteins based upon a blue fluorescent protein which will undergo fast conversion to a green-absorbing form. This form can be converted back to the fluorescent form by long- wavelength irradiation, producing a tracking signal which can be used for optical modulation and lock-in amplification. By mutating known blue fluorescent proteins or photoswitchable proteins toward the right absorption/isomerizations characteristics, we will develop optically modulatable fluorescent proteins capable of true intracellular single molecule imaging.
Public health statement Optically Modulated Fluorescent Proteins Fluorescent proteins have revolutionized the tracking of biochemical processes in cells, as recognized by the 2008 Nobel Prize in the field and by the presence of over 100,000 references to the use of fluorescent proteins in the scientific literature. Using new proteins derived from known fluorescent proteins, we propose to develop a new set of proteins, coupling them to a signal processing technique called optical modulation, to greatly improve sensitivity. The ability to follow the dynamics of cellular pathways at the near molecular level will allow significant progress to be made in disease states that are dependent upon defects in cell transport, including cancer and autoimmune diseases.
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