Optimal microscopic systems for long-term imaging of intracellular calcium using a ratiometric genetically-encoded calcium indicator.
Optimal microscopic systems for long-term imaging of intracellular calcium using a ratiometric genetically-encoded calcium indicator.
复制标题
DOI：
10.1016/j.bbrc.2013.02.112
复制
发表时间：
2013-05
影响因子：
3.1
通讯作者：
Akitoshi Miyamoto;H. Bannai;T. Michikawa;K. Mikoshiba
中科院分区：
文献类型：
--
作者：
Akitoshi Miyamoto;H. Bannai;T. Michikawa;K. Mikoshiba
监测控制许多不同细胞过程的细胞内Ca2+信号的模式对于理解细胞功能的调节机制至关重要。各种遗传编码的Ca2+指标（GECIs）用于监测细胞内Ca2+变化在几种类型的显微镜系统。然而，目前还没有研究哪种显微系统是使用GECIs对Ca2+信号的时空模式进行长期成像的理想选择。我们在这里比较了使用三种不同的成像系统，由荧光共振能量转移（FRET）为基础的比率GECI报告的Ca2+信号，黄色cameleon 3.60 （YC3.60）在DT40 B淋巴细胞中稳定表达。这些系统包括一个宽视场荧光显微镜，一个多点扫描共聚焦系统，和一个单点扫描共聚焦系统。DT40细胞中YC3.60的光漂白程度和信噪比高度依赖于荧光激发方法，尽管在每个成像系统中总照明能量保持在恒定水平。更引人注目的是，b细胞抗原受体刺激在表达yc3.60的DT40细胞中引起的Ca2+反应在不同的成像系统中是不同的，并且受所用照明功率的显著影响。我们的研究结果表明，优化成像系统，包括照明和采集条件，对细胞内Ca2+信号的准确可视化至关重要。
Monitoring the pattern of intracellular Ca2+signals that control many diverse cellular processes is essential for understanding regulatory mechanisms of cellular functions. Various genetically encoded Ca2+indicators (GECIs) are used for monitoring intracellular Ca2+changes under several types of microscope systems. However, it has not yet been explored which microscopic system is ideal for long-term imaging of the spatiotemporal patterns of Ca2+signals using GECIs. We here compared the Ca2+signals reported by a fluorescence resonance energy transfer (FRET)-based ratiometric GECI, yellow cameleon 3.60 (YC3.60), stably expressed in DT40 B lymphocytes, using three different imaging systems. These systems included a wide-field fluorescent microscope, a multipoint scanning confocal system, and a single-point scanning confocal system. The degree of photobleaching and the signal-to-noise ratio of YC3.60 in DT40 cells were highly dependent on the fluorescence excitation method, although the total illumination energy was maintained at a constant level within each of the imaging systems. More strikingly, the Ca2+responses evoked by B-cell antigen receptor stimulation in YC3.60-expressing DT40 cells were different among the imaging systems, and markedly affected by the illumination power used. Our results suggest that optimization of the imaging system, including illumination and acquisition conditions, is crucial for accurate visualization of intracellular Ca2+signals.