基于多维TCSPC技术的心肌细胞Ca^(2+)动力学研究

Investigation of intracellular calcium dynamics during cardiac cell contraction by multi-dimensional TCSPC

采用Fluo-4和Ca-orange标记细胞质及线粒体中的Ca2+,通过多维时间相关单光子计数技术研究心肌收缩时Ca2+动力学.结果表明,在心肌收缩后10～110 ms的Fluo-4荧光强度明显强于心肌收缩后1 s的荧光强度,归一化光谱和细胞自发荧光一致;静止状态下,成分荧光寿命时间和相关振幅在峰值540nm波长处τ1=(0.42±0.01)ns(73±5)%,τ2=(2.74±0.67)ns(25±5)%,平均荧光衰减时间τmean=0.83 ns;Ca-orange荧光峰值560 nm,加入线粒体呼吸链阻断剂Rotenone后,10～110 ms的Ca-orange荧光强度显著降低.综合评估了心肌细胞收缩时细胞质和线粒体中荧光团的光谱及时间分辨荧光特性,该方法可为心肌收缩过程提供满意的光谱和时间分辨荧光记录,有助于理解细胞综合行为.

Spectrally-resolved time correlated single photon counting(TCSPC) technique was applied to investigate the changes of mitochondrial vs cytosolic calcium.Calcium-sensitive fluorescence probes Fluo-4 and Ca-orange were used to test cytosolic and/or mitochondrial calcium dynamics.The results show that upon comparison with steady state conditions(measured 1 s after the beginning of contraction),Fluo-4 fluorescence intensity is significantly higher when measured at 10～110 ms after the start of contraction,as cal...