单细胞内Ca^（2＋）时空变化的激光共聚焦显微测定

A Powerful Method for the Determination of the Spatial and Temporal Changes of Intracellular Ca￣(2+) in Single Living Cells by Laser Scanning Confocal microscopy

应用激光扫描共聚焦显微系统（ＬＳＣＭ）和Ｆｌｕｏ－３／ＡＭ荧光探剂标记技术，测定了单个活细胞胞内游离Ｃａ２＋的动态变化与立体分布影像．结果显示，在３７℃，Ｆｌｕｏ－３／ＡＭ终浓度为６μｍｏｌ／Ｌ的条件下，Ｃ５７ＢＬ／６Ｊ小鼠巨噬细胞负载１ｈ左右即可获得良好的标记效果．相反，若探剂浓度太高或负载时间太长，胞内荧光强度太强，影响在共聚焦显微镜镜下分辨细胞内结构．因此用ＬＳＣＭ研究细胞内游离Ｃａ２＋变化时，荧光探剂的负载应以获得最适荧光信号而不是以最大荧光强度为标准．上述方法在其他如平滑肌细胞、卵母细胞中的测定亦获得满意的结果，这对进一步研究各种生理和病理条件下细胞内Ｃａ２＋信号的动态变化、与跨膜Ｃａ２＋梯差的关系及对活细胞功能活动的调节提供了一种可行的、直观的研究手段。

Procedures for the determination ofthe spatial distribution and the dynamic changesof intracellular Ca2+ signaling in single intact living cells are described using laser scanning con focal microscopy (LSCM) and the highly fluorescent Ca2+ -sensitive dye, fluo-3/AM. The duration, dye concentration, and requirement forPluronic are determined empirically for the loading conditions. It is especially important for theproper intracellular dye concentration to provideadequate signal strength for detection while notto disturb normal intracellular physiology. Forexample, in C57BL/6) macrophages, it wasfound that cells cultured on glass cover slips wereincubated for 1 h at 37℃ with 6 μmol/L fluo-3/AM, resulting in excellent LSCM imaging ofintracellular Ca2+. The similar results have beensuccessfully obtained by this method for othertypes of cells such as vascular smooth musclecells, Chinese hamstar oocytes. It may providean available, visual experimental approach forinvestigation into the dynamic changes of Ca2+ signal and their relationship to transmembraneCa2+ gradient in living cells under the physiological and pathological conditions.