PHi and pHo at different depths in perfused myocardium measured by confocal fluorescence microscopy

Barbara J. Muller-Borer, Hua Yang, Sayed A.M. Marzouk, John J. Lemasters, Wayne E. Cascio

Research output: Contribution to journalArticlepeer-review

16 Citations (Scopus)


Confocal microscopy and the H+-sensitive fluorophore carboxyseminaphthorhodafluor-1 (SNARF-1) were used to measure either intracellular pH (pHj) or extracellular pH (pH0) in isolated, arterially perfused rabbit papillary muscles. Single-excitation, dual-emission fluorescent images of the endocardial surface and underlying myocardium to a depth of 300 ; /m were simultaneously recorded from perfused cylindrical muscles suspended in a controlled atmosphere oriented oblique to the focal plane. Contraction was inhibited by the addition of butanedione monoxime. In separate muscles, pHo was measured during continuous perfusion of SNARF-1 free acid. pH; measurements were made after the muscle was loaded with SNARF-1/AM and the extracellular space was cleared of residual fluorophore. Initial experiments demonstrated the uniformity of ratiometric measurements as a function of pH, image depth, and fluorophore concentration, thereby establishing the potential feasibility of this method for quantitative intramural pH measurements. In subsequent experiments, the method was validated in isolated, arterially perfused rabbit papillary muscle during normal arterial perfusion and as pHi and pH0 were altered by applying CC> 2 externally, exchanging HEPES and bicarbonate buffers, and changing pH; with NH4C1 washout. We conclude that in situ confocal fluorescent microscopy can measure pH; and pH0 changes at the endocardial surface and deeper endocardial layers in arterially perfused ventricular myocardium. This method has the potential to study pH; regulation in perfused myocardium at boundaries where diffusion of gases, metabolites, and peptides are expected to modify processes that regulate pHi.

Original languageEnglish
Pages (from-to)H1937-H1947
JournalAmerican Journal of Physiology
Issue number6 PART 2
Publication statusPublished - 1998
Externally publishedYes


  • Acidosis
  • Alkalosis
  • Carbon dioxide
  • Carboxyseminaphthorhodafluor-1
  • Ventricular myocardium

ASJC Scopus subject areas

  • Physiology (medical)


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