title: 2-Photon excitation fluorescence microscopy enables deeper high-resolution imaging of voltage and Ca(2+) in intact mice, rat, and rabbit hearts.
abstract: We describe a novel two-photon (2P) laser scanning microscopy (2PLSM) protocol that provides ratiometric transmural measurements of membrane voltage (Vm ) via Di-4-ANEPPS in intact mouse, rat and rabbit hearts with subcellular resolution. The same cells were then imaged with Fura-2/AM for intracellular Ca(2+) recordings. Action potentials (APs) were accurately characterized by 2PLSM vs. microelectrodes, albeit fast events (<1 ms) were sub-optimally acquired by 2PLSM due to limited sampling frequencies (2.6 kHz). The slower Ca(2+) transient (CaT) time course (>1ms) could be accurately described by 2PLSM. In conclusion, Vm - and Ca(2+) -sensitive dyes can be 2P excited within the cardiac muscle wall to provide AP and Ca(2+) signals to ∼400 µm.
authors.affiliation: Institute of Cardiovascular and Medical Sciences; College of Medical, Veterinary, and Life Sciences, University of Glasgow, Glasgow, G12 8QQ, UK.
authors.forename: Iffath A
authors.initials: IA
authors.lastname: Ghouri
authors.forename: Allen
authors.initials: A
authors.lastname: Kelly
authors.forename: Francis L
authors.initials: FL
authors.lastname: Burton
authors.forename: Godfrey L
authors.initials: GL
authors.lastname: Smith
authors.forename: Ole Johan
authors.initials: OJ
authors.lastname: Kemi
mesh_terms: D000200:Action Potentials; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D004396:Coloring Agents / Q000737:chemistry; D006321:Heart / Q000502:physiology*; D042541:Intracellular Space / Q000378:metabolism; D051379:Mice; D008839:Microelectrodes; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation / Q000379:methods*; D009206:Myocardium / Q000166:cytology; D011817:Rabbits; D051381:Rats
chemical_list: D004396:Coloring Agents; D002118:Calcium
keywords: action potential