Confocal imaging of [Ca2+] in cellular organelles by SEER, shifted excitation and emission ratioing of fluorescence
Confocal imaging of [Ca2+] in cellular organelles by SEER, shifted excitation and emission ratioing of fluorescence
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DOI：
10.1113/jphysiol.2005.087973
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发表时间：
2005-09-01
影响因子：
5.5
通讯作者：
Ríos, E
中科院分区：
文献类型：
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作者：
Launikonis, BS;Zhou, JS;Ríos, E
细胞内钙信号调节多种细胞功能。它们依赖于细胞内钙离子从细胞内释放到胞浆中，这一过程似乎受到细胞内[Ca2+]变化的严格控制。设计了一种对细胞器内游离[Ca~(2+)]进行成像的方法，首次提供了骨骼肌肌浆网(SR)内[Ca~(2+)]的定量共聚焦图像。为了获得更高的灵敏度，该方法利用了一些荧光染料在结合钙离子时发生的双重光谱位移。用捕获在青蛙骨骼肌细胞内细胞器中的MAG-INDO-1实现了该方法，并得到了验证，表明它主要监测具有SR池结构的咖啡因敏感区域中的[Ca+]。试探性的现场校准表明，染料的离解常数增加了，这与在细胞环境中观察到的其他染料的情况相似。这一增加，加上比率方法的其他特征，使半信号[钙]接近1 mm，这是一个适合细胞存储的值。这项技术显示的优势包括准确性(校准比率法)、动态范围和灵敏度(来自两个光谱位移的组合)、空间和时间分辨率，以及与大量可见染料的兼容性，以监测细胞功能的不同方面。SEER(移位激发和发射比)也提供了细胞内染料浓度的不依赖于[Ca2+]的测量方法。利用SEER的高空间分辨率，可以分别测量细胞内和线粒体的[Ca~(2+)]([Ca~(2+)](SR)和[Ca~(2+)](MITO))。[Ca~(2+)](SR)随细胞内[Ca~(2+)]([Ca~(2+)](Cyto))的变化而变化。在[Ca~(2+)](Cyto)=100 nm时，[Ca~(2+)](MITO)保持在检测下限附近，[Ca~(21+)]SR稳定在我们初步校准的亚毫米尔值。稳定的[Ca~(2+)](SR)在800 nm处仅略高于[Ca~(2+)](Cyto)，除非[Ca~(2+)](Cyto)降至10 nm以下，否则基本不会下降。[Ca~(2+)](SR)的增加受限于通过钙释放通道的损失，而低[Ca~(2+)](Cyto)的减少则主要依赖于通过SR钙泵的泄漏。
Intracellular calcium signals regulate multiple cellular functions. They depend on release of Ca2+ from cellular stores into the cytosol, a process that appears to be tightly controlled by changes in [Ca2+] within the store. A method to image free [Ca2+] within cellular organelles was devised, which provided the first quantitative confocal images of [Ca2+] inside the sarcoplasmic reticulum (SR) of skeletal muscle. The method exploits, for greater sensitivity, the dual spectral shifts that some fluorescent dyes undergo upon binding Ca2+. It was implemented with mag-indo-1 trapped in the intracellular organelles of frog skeletal muscle and validated showing that it largely monitors [Ca2+] in a caffeine-sensitive compartment with the structure of the SR cisternae. A tentative calibration in situ demonstrated an increase in the dye's dissociation constant, not unlike that observed for other dyes in cellular environments. This increase, together with other characteristics of the ratioing method, placed the half-signal [Ca2+] near 1 mm, a value suitable for cellular stores. Demonstrated advantages of the technique include accuracy (that of a calibrated ratiometric method), dynamic range and sensitivity (from the combination of two spectral shifts), spatial and temporal resolution, and compatibility with a vast array of visible dyes to monitor diverse aspects of cellular function. SEER (shifted excitation and emission ratioing) also provides a [Ca2+] -independent measure of dye concentration in the cell. Store and mitochondrial [Ca2+] ([Ca2+](SR) and [Ca2+](mito)) could be measured separately using the high spatial resolution of SEER. Evolution of [Ca2+](SR) was followed upon changes in cytosolic [Ca2+]([Ca2+](cyto)). At [Ca2+](cyto) = 100nm, [Ca2+](mito) remained near the lower limit of detection and [Ca 21+] SR stabilized at values that were submillimolar according to our tentative calibration. Steady [Ca2+](SR) was only slightly higher in 800 nm [Ca2+](cyto), and essentially did not decrease unless [Ca2+](cyto) was reduced below 10 nm. While the increase of [Ca2+](SR) was limited by loss through Ca2+ release channels, its decrease in low [Ca2+](cyto) was largely dependent on leaks through the SR Ca2+ pump.