title: Dual excitation wavelength epifluorescence imaging of transmural electrophysiological properties in intact hearts.
abstract: BACKGROUND
  Epifluorescence imaging using voltage-sensitive dyes has provided unique insights into cardiac electrical activity and arrhythmias. However, conventional dyes use blue-green excitation light, which has limited depth penetration.
  
  
  OBJECTIVE
  The aim of this study was to demonstrate that combining a short and a long excitation wavelength using near-infrared (NIR) dyes allows for epifluorescence imaging of transmural electrophysiological properties in intact hearts.
  
  
  METHODS
  Epifluorescence imaging was performed in rat hearts (N = 11) using DI-4-ANEPPS and the NIR dye DI-4-ANBDQBS. Activation and action potential duration (APD) patterns were investigated at 2 excitation wavelengths (530 and 660 nm) after epicardial stimulation at various cycle lengths (160 to 70 ms).
  
  
  RESULTS
  Optical action potential upstrokes acquired with 660-nm excitation of DI-4-ANBDQBS were significantly longer than upstrokes obtained with 530-nm excitation of DI-4-ANEPPS (P < .001). Comparison of activation maps showed counterclockwise rotation of isochrones consistent with a transmural rotation of myofibers. Pronounced APD modulation by the activation sequence was observed at both excitation wavelengths. Significantly prolonged APDs (P = .016) and steeper APD restitution curves were found with DI-4-ANBDQBS (660-nm excitation) when compared with DI-4-ANEPPS (530-nm excitation). Dual excitation wavelength experiments using solely DI-4-ANBDQBS yielded similar results. Monophasic action potential recordings showed prolonged APD and steeper APD restitution curves in the endocardium, indicating that 660-nm excitation provides a significant endocardial contribution to the signal. Three-dimensional computer simulations confirmed our findings.
  
  
  CONCLUSIONS
  Dual excitation wavelength epifluorescence allows detecting transmural heterogeneity in intact hearts. It therefore has the potential to become an important tool in experimental cardiac electrophysiology.
authors.affiliation: Institute of Membrane and Systems Biology, Faculty of Biological Sciences, Multidisciplinary Cardiovascular Research Centre, University of Leeds, Leeds LS2 9JT, UK.
authors.forename: Richard D
authors.initials: RD
authors.lastname: Walton
authors.forename: David
authors.initials: D
authors.lastname: Benoist
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Hyatt
authors.forename: Stephen H
authors.initials: SH
authors.lastname: Gilbert
authors.forename: Ed
authors.initials: E
authors.lastname: White
authors.forename: Olivier
authors.initials: O
authors.lastname: Bernus
mesh_terms: D015081:2-Naphthylamine / Q000031:analogs & derivatives; D000818:Animals; D003198:Computer Simulation; D022062:Electrophysiologic Techniques, Cardiac / Q000379:methods*; D005456:Fluorescent Dyes; D006321:Heart / Q000294:innervation / Q000502:physiology*; D006854:Hydrocortisone / Q000031:analogs & derivatives; D066298:In Vitro Techniques; D008297:Male; D011726:Pyridinium Compounds; D011806:Quinolinium Compounds; D051381:Rats; D056969:Voltage-Sensitive Dye Imaging / Q000379:methods*
chemical_list: C553754:1-(4-sulfanatobutyl)-4-(beta-(2-(di-n-butylamino)-6-naphthyl)butadienyl)quinolinium betaine; D005456:Fluorescent Dyes; D011726:Pyridinium Compounds; D011806:Quinolinium Compounds; C021650:hydrocortisone acetate; C050019:1-(3-sulfonatopropyl)-4-(beta)(2-(di-n-butylamino)-6-naphthylvinyl)pyridinium betaine; D015081:2-Naphthylamine; D006854:Hydrocortisone