title: Emission ratiometry for simultaneous calcium and action potential measurements with coloaded dyes in rabbit hearts: reduction of motion and drift.
abstract: BACKGROUND
  Optical measurements of the cardiac calcium transient (Ca) and transmembrane action potential (AP) may be performed simultaneously with emission ratiometry to lessen motion artifacts and photobleaching effects. We examined changes in emission spectrum in perfused rabbit hearts coloaded with Rh237 and a green-emitting Ca dye (Fluo-4 or Oregon Green BAPTA 1) to determine wavelength bands for emission ratiometry and to test whether ratiometry reduces motion artifacts and drift.
  
  
  RESULTS
  A 488-nm laser illuminated hearts while a spectrofluorometer collected fluorescence from 489 to 838 nm at 1 kHz. Ratiometry with the Ca- and AP-insensitive emission band 663 to 685 nm (IS) as denominator and the Ca-sensitive band 510 to 532 nm as numerator lessened motion artifacts, which was quantified as a 1.4-fold increase in relative amplitude of the Ca (P < 0.05). Ratiometry with the AP-sensitive band 772 to 794 nm as denominator and the IS as numerator produced a 1.7-fold increase in relative amplitude of the AP (P < 0.05). The ratiometry decreased photobleaching-dependent drift by a factor of 0.6 (P < 0.05) for Ca and 0.45 (P < 0.05) for AP.
  
  
  CONCLUSIONS
  Simultaneous Ca and AP emission ratiometry reduces motion artifacts and drift in hearts with coloaded dyes.
authors.affiliation: Department of Biomedical Engineering of the School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7575, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Kong
authors.forename: Gregory P
authors.initials: GP
authors.lastname: Walcott
authors.forename: William M
authors.initials: WM
authors.lastname: Smith
authors.forename: Philip L
authors.initials: PL
authors.lastname: Johnson
authors.forename: Stephen B
authors.initials: SB
authors.lastname: Knisley
mesh_terms: D000200:Action Potentials / Q000502:physiology*; D000818:Animals; D016477:Artifacts; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology; D004396:Coloring Agents; D003971:Diastole / Q000502:physiology; D006321:Heart / Q000502:physiology*; D008564:Membrane Potentials / Q000502:physiology; D023421:Models, Animal; D008955:Models, Cardiovascular; D011817:Rabbits; D013057:Spectrum Analysis; D013599:Systole / Q000502:physiology; D013997:Time Factors
chemical_list: D004396:Coloring Agents; D002118:Calcium