This application consists of 2 Aims that propose to develop new membrane potential imaging methodologies and 1 Aim that applies membrane potential imaging to the discovery and elucidation of novel cell physiological phenomena. 1) We will continue the development of potential sensitive dyes by synthesizing and characterizing chromophores with absorbances in the 700nm-900nm range. These as well as some of the older chromophores will be functionalized variably to optimize membrane binding or solubility, for covalent labeling applications, and to append chiral moieties for enhanced second harmonic generation. 2) The adaptation of second harmonic generation (SHG), a non-linear optical phenomenon, to high resolution microscopy has been achieved. SHG from cells stained with potentiometric styryl dyes will be thoroughly characterized. We will optimize both the dyes and the measurements so as to exploit the high sensitivity of SHG signals to membrane potential. Our goal is to determine if this can be an advantageous modality for optical recording of electrical activity. 3) We have shown that intramembrane electric fields (IEF), originating from dipole potentials or differences in surface potential, can be measured by ratiometric imaging of potentiometric dyes. The IEF changes significantly during receptor mediated signaling via the phosphoinositide pathway. We will use both biochemical analysis and fluorescent probe imaging to determine how the inositide lipid composition affects the IEF. We will determine if direct manipulation of the IEF, particularly with reagents that alter dipole potentials, can modulate the activity of ion channels and the intracellular signals that they control. The goal of this aim is to determine if the apparently large receptor mediated changes in IEF can directly modulate membrane physiology ? ?
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