Young S R, Wong R K, Bianchi R
Department of Physiology and Pharmacology, State University of New York Health Science Center, Brooklyn 11203, USA.
Methods. 2000 Aug;21(4):373-83. doi: 10.1006/meth.2000.1026.
Fluorescent Ca2+ indicator dyes can be introduced into cells through the same microelectrode used for intracellular voltage recording. Simultaneous measurement of cell membrane potential and intracellular Ca2+ concentration can be very helpful in interpreting the mechanisms of Ca2+ increases. This chapter describes fluorescence image acquisition using a CCD camera and a computer program that also records a synchronized membrane potential trace. The same program allows for preliminary data analysis. More elaborate analyses can be accomplished with commercial programs. We also describe quantitative evaluations of sources of error in the use of the statistic deltaF/F as an indicator of Ca2+ concentration. Especially important errors to minimize are changes in background fluorescence and inappropriate autofluorescence corrections. Some improvement of fluorescence images of cells deep within slices may be accomplished by masking. One method is described for making a mask based on the raw fluorescence image. With another method, highly detailed cell morphologies may be conveyed by using masks based on neurobiotin injections and camera lucida drawings.
荧光钙指示剂染料可通过用于细胞内电压记录的同一微电极引入细胞。同时测量细胞膜电位和细胞内钙浓度对于解释钙增加的机制非常有帮助。本章介绍了使用电荷耦合器件（CCD）相机和计算机程序进行荧光图像采集，该程序还记录同步的膜电位轨迹。同一程序允许进行初步数据分析。更精细的分析可以通过商业程序完成。我们还描述了将统计量ΔF/F用作钙浓度指标时误差来源的定量评估。特别要尽量减少的重要误差是背景荧光的变化和不适当的自发荧光校正。通过掩膜可以对切片深处细胞的荧光图像进行一些改进。描述了一种基于原始荧光图像制作掩膜的方法。通过另一种方法，基于神经生物素注射和明箱绘图制作的掩膜可以传达高度详细的细胞形态。
