title: Confocal imaging of transmembrane voltage by SEER of di-8-ANEPPS.
abstract: Imaging, optical mapping, and optical multisite recording of transmembrane potential (V(m)) are essential for studying excitable cells and systems. The naphthylstyryl voltage-sensitive dyes, including di-8-ANEPPS, shift both their fluorescence excitation and emission spectra upon changes in V(m). Accordingly, they have been used for monitoring V(m) in nonratioing and both emission and excitation ratioing modes. Their changes in fluorescence are usually much less than 10% per 100 mV. Conventional ratioing increases sensitivity to between 3 and 15% per 100 mV. Low sensitivity limits the value of these dyes, especially when imaged with low light systems like confocal scanners. Here we demonstrate the improvement afforded by shifted excitation and emission ratioing (SEER) as applied to imaging membrane potential in flexor digitorum brevis muscle fibers of adult mice. SEER--the ratioing of two images of fluorescence, obtained with different excitation wavelengths in different emission bands-was implemented in two commercial confocal systems. A conventional pinhole scanner, affording optimal setting of emission bands but less than ideal excitation wavelengths, achieved a sensitivity of up to 27% per 100 mV, nearly doubling the value found by conventional ratioing of the same data. A better pair of excitation lights should increase the sensitivity further, to 35% per 100 mV. The maximum acquisition rate with this system was 1 kHz. A fast "slit scanner" increased the effective rate to 8 kHz, but sensitivity was lower. In its high-sensitivity implementation, the technique demonstrated progressive deterioration of action potentials upon fatiguing tetani induced by stimulation patterns at >40 Hz, thereby identifying action potential decay as a contributor to fatigue onset. Using the fast implementation, we could image for the first time an action potential simultaneously at multiple locations along the t-tubule system. These images resolved the radially varying lag associated with propagation at a finite velocity.
authors: Section of Cellular Signaling, Department of Molecular Biophysics and Physiology, Rush University, Chicago, IL 60612, USA.
authors: Carlo
authors: C
authors: Manno
authors: Lourdes
authors: L
authors: Figueroa
authors: Robert
authors: R
authors: Fitts
authors: Eduardo
authors: E
authors: Ríos
mesh_terms: D000200:Action Potentials / Q000187:drug effects; D000818:Animals; D002478:Cells, Cultured; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects*; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods*; D018485:Muscle Fibers, Skeletal / Q000187:drug effects* / Q000378:metabolism; D011726:Pyridinium Compounds / Q000378:metabolism*
chemical_list: C089669:1-(3-sulfonatopropyl)-4-(beta-(2-(di-n-octylamino)-6-naphthyl)vinyl)pyridinium betaine; D005456:Fluorescent Dyes; D011726:Pyridinium Compounds