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首页> 外文期刊>American Journal of Physiology >pHi and pHo at different depths in perfused myocardium measured by confocal fluorescence microscopy.
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pHi and pHo at different depths in perfused myocardium measured by confocal fluorescence microscopy.

机译：通过共聚焦荧光显微镜测量灌注心肌中不同深度的pHi和pHo。

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Confocal microscopy and the H+-sensitive fluorophore carboxyseminaphthorhodafluor-1 (SNARF-1) were used to measure either intracellular pH (pHi) or extracellular pH (pHo) 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 micron 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. pHi 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 pHo were altered by applying CO2 externally, exchanging HEPES and bicarbonate buffers, and changing pHi with NH4Cl washout. We conclude that in situ confocal fluorescent microscopy can measure pHi and pHo changes at the endocardial surface and deeper endocardial layers in arterially perfused ventricular myocardium. This method has the potential to study pHi regulation in perfused myocardium at boundaries where diffusion of gases, metabolites, and peptides are expected to modify processes that regulate pHi.
机译：共聚焦显微镜和H +敏感的荧光团羧基半胱氨酸-hodafluor-1（SNARF-1）用于测量分离的动脉灌注兔乳头肌中的细胞内pH（pHi）或细胞外pH（pHo）。从悬浮在倾斜于焦平面的受控气氛中的灌注圆柱肌同时记录心内膜表面和下方心肌的单次激发，两次发射荧光图像，深度为300微米。通过添加丁二酮一肟抑制收缩。在单独的肌肉中，在SNARF-1游离酸的连续灌注过程中测量了pHo。在肌肉中加载SNARF-1 / AM并清除细胞外空间的残留荧光团后，进行pHi测量。最初的实验证明了比例测量的均匀性是pH，图像深度和荧光团浓度的函数，从而确立了该方法用于定量壁内pH测量的潜在可行性。在随后的实验中，该方法在正常动脉灌注过程中在分离的动脉灌注兔乳头肌中得到了验证，并且pHi和pHo通过外部施加CO2，交换HEPES和碳酸氢盐缓冲液并用NH4Cl冲洗液改变pHi来改变。我们得出的结论是，原位共聚焦荧光显微镜可以测量动脉灌注心肌室的心内膜表面和更深的心内膜层的pHi和pHo变化。该方法有可能研究灌注心肌中边界处的pHi调节，在该边界处，气体，代谢物和肽的扩散会改变调节pHi的过程。

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