Simultaneous optical imaging of membrane potential and intracellular calcium
Simultaneous optical imaging of membrane potential and intracellular calcium
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DOI：
10.1016/j.jelectrocard.2005.06.023
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发表时间：
2005-10-01
影响因子：
1.3
通讯作者：
Fast, VG
中科院分区：
文献类型：
--
作者：
Fast, VG
心肌细胞膜电位（V-m）和细胞内钙离子（Ca-i（2+））的成像对于研究心脏兴奋、心律失常和除颤的机制具有重要意义。我们发展了一种用荧光V-m-和Ca ~（2+）敏感染料同时测定培养细胞单层中V-m和Ca ~（2+）的光学技术。用染料RH-237（V-m）和Fluo-3或Rhod-2染料（Ca 2+）的类似物对新生大鼠心肌细胞培养物进行双重染色。这些染料具有重叠的激发光谱，允许在相同的波长范围内同时激发，并且具有分离的发射光谱，允许将荧光分成对V-m和Ca-1（2+）敏感的组分，其用两个16 × 16光电二极管阵列测量。发现Ca ~（2+）的测量强烈依赖于Ca ~（2+）敏感染料的性质。因此，高亲和力Ca 2+染料（例如Fluo-4和Rhod-2）报告的Ca-1（2+）瞬变时间大约是低亲和力染料Fluo-4FF和Rhod-F-F报告的时间的两倍。此外，具有不同亲和性的染料导致对电击的Ca-i（2+）响应的不同测量。当在动作电位的早期平台期施加电击时，低亲和力染料报告在负和正V-m变化的位点处的瞬时Ca-i（2+）降低。相比之下，高亲和力Ca 2+染料仅报道了平台Ca 1（2+）的可忽略的变化和舒张期Ca 1（2+）的大幅升高。高亲和力和低亲和力染料之间的这些差异由染料-离子相互作用的模型解释，这表明，用高亲和力染料测量的明显较长的Ca ~（2+）瞬变和Ca ~（2+）对电击的响应是由于它们的非-线性响应这些结果表明，光学测量Ca ~（2+）瞬态持续时间和冲击引起的Ca ~（2+）变化需要使用低亲和力Ca ~（2+）染料。(c)2005年爱思唯尔公司All rights reserved.
Imaging of membrane potential (V-m) and intracellular calcium (Ca-i(2+)) is important for studying mechanisms of cardiac excitation, arrhythmias and defibrillation. We have developed an optical technique for simultaneous mapping of V-m and Ca-i(2+) in Cultured cell monolayers using fluorescent V-m- and Ca2+ sensitive dyes. Cultures of neonatal rat myocytes were double-stained with dyes RH-237 (V-m) and an analog of Fluo-3 or Rhod-2 dyes (Ca2+). These dyes have overlapping excitation spectra, allowing simultaneous excitation at the same wavelength range, and separate emission spectra allowing division of the fluorescent light into components sensitive to V-m and Ca-i(2+) which were measured with two 16 x 16 photodiode arrays. It was found that Ca-i(2+) measurements were strongly dependent on the properties of Ca2+-sensitive dyes. Thus, high-affinity Ca2+ dyes such as Fluo-4 and Rhod-2 reported Ca-i(2+) transients approximately twice as long as those reported by low-affinity dyes Fluo-4FF and Rhod-F-F. In addition, dyes with different affinities resulted in different measurements of Ca-i(2+) responses to electrical shocks. When shocks were applied during the early plateau phase of the action potential, low-affinity dyes reported transient Ca-i(2+) decreases at sites of both negative and positive V-m changes. In contrast, high-affinity Ca2+ dyes reported only a negligible change of plateau Ca-i(2+) and a large elevation of diastolic Ca-i(2+) These discrepancies between high- and low-affinity dyes were explained by a model of dye-ion interaction, which indicated that apparently long Ca-i(2+) transients and Ca-i(2+) responses to electrical shocks measured with high-affinity dyes were due to their non-linear response. These results indicate that optical measurements of Ca-i(2+) transient duration and shock-induced Ca-i(2+) changes require the use of low-affinity Ca-i(2+) dyes. (c) 2005 Elsevier Inc. All rights reserved.