J E González1, R Y Tsien. 1. Howard Hughes Medical Institute and Department of Pharmacology, 310 Cellular and Molecular Medicine West, University of California, 9500 Gilman Drive, CA 92093-0647, USA.
Abstract
BACKGROUND: Fluorescence detection of cell membrane potentials is an important technique in neurobiology, cell physiology and pharmaceutical screening, but traditional one-fluorophore indicators either respond too slowly or have limited sensitivity. Recently, we introduced two-component sensors based on the transfer of fluorescence resonance energy from fluorescent lectins bound on one side of the plasma membrane to highly fluorescent oxonol acceptors that electrophorese from one face of the membrane to the other in response to membrane potential. RESULTS: We have found that fluorescent lectins can often be advantageously replaced in such sensors by fluorescently labeled phospholipids. A coumarinlabeled phosphatidylethanolamine donor and a bis(1,3-dihexyl-2-thiobarbiturate)trimethineoxonol acceptor gave the largest sensitivity of fluorescence ratio (>50% per 100 mV) ever reported. The response was also speeded several-fold by lengthening the mobile dye to the pentamethineoxonol analog, the <0.4 ms time constant of which was shorter than action potential durations. Photodynamic damage due to singlet oxygen was reduced by administering a natural carotenoid, astaxanthin. CONCLUSIONS: Voltage-sensitive fluorescence resonance energy transfer already gives record-setting performance on single cells and will continue to be rationally improvable.
BACKGROUND: Fluorescence detection of cell membrane potentials is an important technique in neurobiology, cell physiology and pharmaceutical screening, but traditional one-fluorophore indicators either respond too slowly or have limited sensitivity. Recently, we introduced two-component sensors based on the transfer of fluorescence resonance energy from fluorescent lectins bound on one side of the plasma membrane to highly fluorescent oxonol acceptors that electrophorese from one face of the membrane to the other in response to membrane potential. RESULTS: We have found that fluorescent lectins can often be advantageously replaced in such sensors by fluorescently labeled phospholipids. A coumarinlabeledphosphatidylethanolaminedonor and a bis(1,3-dihexyl-2-thiobarbiturate)trimethineoxonol acceptor gave the largest sensitivity of fluorescence ratio (>50% per 100 mV) ever reported. The response was also speeded several-fold by lengthening the mobile dye to the pentamethineoxonol analog, the <0.4 ms time constant of which was shorter than action potential durations. Photodynamic damage due to singlet oxygen was reduced by administering a natural carotenoid, astaxanthin. CONCLUSIONS: Voltage-sensitive fluorescence resonance energy transfer already gives record-setting performance on single cells and will continue to be rationally improvable.
Authors: Evan W Miller; John Y Lin; E Paxon Frady; Paul A Steinbach; William B Kristan; Roger Y Tsien Journal: Proc Natl Acad Sci U S A Date: 2012-01-24 Impact factor: 11.205