Ratiometric fluorescence measurements and imaging of the dipole potential in cell plasma membranes

摘要

Development of fluorescence microscopic methods is limited by the application of new dyes, the response of which could be sensitive to different functional states in the living cells, and, in particular, to electrostatic potentials on their plasma membranes. Recently, we showed that newly designed 3-hydroxyflavone fluorescence dyes are highly electrochromic and show a strong two-band ratiometric response to electric dipole potential in lipid membranes. In the present report we extend these observations and describe a new generation of these dyes as electrochromic probes in biomembrane research. Modification of the membrane dipole potential was achieved by addition of 6-ketocholestanol (6-KC), cholesterol and phloretin. The dipole potential was also estimated by the reference probe di-8-ANEPPS. As an example, we show that on addition of 6-KC there occurs a dramatic change of the intensity ratio of the two emission bands, which is easily detected as a change of color. We describe in detail the applications of one of these dyes, PPZ8, to the studies of cells in suspension or attached to the glass surface. Confocal microscopy demonstrates strong preference of the probe for the cell plasma membrane, which allows us to apply this dye for studying electrostatic and other biomembrane properties. We demonstrate that the two-color response provides a direct and convenient way to measure the dipole potential in the plasma membrane. Applying PPZ8 in confocal microcopy and two-photon microspectroscopy allowed us to provide two-color imaging of the membrane dipole potential on the level of a single cell.