Lui P P, Lee M M, Ko S, Lee C Y, Kong S K
Department of Biochemistry, Chinese University of Hong Kong, Shatin, NT, Hong Kong.
Biol Signals. 1997 Mar-Apr;6(2):45-51.
Confocal laser scanning microscopy (CLSM) is extensively used in the study of cellular activities through monitoring the temporal and spatial changes of biologically active molecules such as cAMP and Ca2+ which have been rendered visible by fluorescent labels. During our work with fluo-3 and Ca2+, we noticed two potential sources of artifacts which can make interpretation of the experimental observations difficult. Firstly, the excitation laser light generates heat that enhances the conversion of residual non-fluorescent acetoxymethyl (AM)-esterified indicator to the fluorescent form, thus giving rise to erroneous signals. Secondly, addition of reagents onto the coverslips alters the position of the focal plane, again causing error. In this paper, we present the phenomena and suggest ways to control and eliminate false images.
共聚焦激光扫描显微镜（CLSM）通过监测诸如cAMP和Ca2+等生物活性分子的时空变化，在细胞活动研究中得到广泛应用，这些生物活性分子已通过荧光标记变得可见。在我们使用fluo-3和Ca2+的工作过程中，我们注意到两个可能产生伪影的来源，这会使实验观察结果的解释变得困难。首先，激发激光会产生热量，这会增强残留的非荧光乙酰氧基甲基（AM）酯化指示剂向荧光形式的转化，从而产生错误信号。其次，将试剂添加到盖玻片上会改变焦平面的位置，同样会导致误差。在本文中，我们介绍了这些现象，并提出了控制和消除虚假图像的方法。
