Properties of New,Long-Wavelength,Voltage-sensitive Dyes in the Heart |
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Authors: | Email author" target="_blank">G?SalamaEmail author B-R?Choi G?Azour M?Lavasani V?Tumbev BM?Salzberg MJ?Patrick LA?Ernst AS?Waggoner |
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Institution: | (1) Department of Cell Biology and Physiology, University of Pittsburgh, School of Medicine, S314 Biomedical Science Tower, 3500 Terrace Street, Pittsburgh, PA 15261, USA;(2) Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA 15261, USA;(3) Departments of Neuroscience and Physiology, University of Pennsylvania, Philadelphia, PA 19104, USA;(4) Molecular Biosensor and Imaging Center, Carnegie Mellon University, Pittsburgh, PA 15213, USA |
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Abstract: | Membrane potential measurements using voltage-sensitive dyes (VSDs) have made important contributions to our understanding
of electrophysiological properties of multi-cellular systems. Here, we report the development of long wavelength VSDs designed
to record cardiac action potentials (APs) from deeper layers in the heart. The emission spectrum of styryl VSDs was red-shifted
by incorporating a thienyl group in the polymethine bridge to lengthen and retain the rigidity of the chromophore. Seven dyes,
Pittsburgh I to IV and VI to VIII (PGH I-VIII) were synthesized and characterized with respect to their spectral properties in organic solvents and heart muscles. PGH VSDs
exhibited 2 absorption, 2 excitation and 2 voltage-sensitive emission peaks, with large Stokes shifts (> 100 nm). Hearts (rabbit,
guinea pig and Rana pipiens) and neurohypophyses (CD-1 mice) were effectively stained by injecting a bolus (10–50 μl) of stock solution of VSD (2–5 mM) dissolved in in dimethylsulfoxide plus low molecular weight Pluronic (16% of L64). Other preparations were better stained
with a bolus of VSD (2–5 mM) Tyrode’s solution at pH 6.0. Action spectra measured with a fast CCD camera showed that PGH I exhibited an increase in fractional
fluorescence, ΔF/F = 17.5 % per AP at 720 nm with 550 nm excitation and ΔF/F = − 6% per AP at 830 nm with 670 nm excitation. In frog hearts, PGH1 was stable with ∼30% decrease in fluorescence and AP
amplitude during 3 h of intermittent excitation or 1 h of continuous high intensity excitation (300 W Xe-Hg Arc lamp), which
was attributed to a combination of dye wash out > photobleaching > dynamic damage > run down of the preparation. The long
wavelengths, large Stokes shifts, high ΔF/F and low baseline fluorescence make PGH dyes a valuable tool in optical mapping and for simultaneous mapping of APs and intracellular
Ca2+. |
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Keywords: | Voltage-dependent optical signals Action potential Action spectral Surfactants |
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