Automated analysis of intracellular calcium fluorescence in rat organotypic hippocampal cultures: Comparison to a manual, observer based method

详细信息    查看全文

• 作者：Orla Watters ; Mark Pickering ; Robert Murphy ; Keith J. Murphy ; John J. O鈥機onnor
• 关键词：TNF-伪 ; Tumor necrosis factor-伪 ; ROIs ; regions of interest ; EBSS ; Earles Balanced Salt Solution ; MEM ; Minimum Essential Medium ; BSS ; Buffered Salt Solution ; GUI ; graphical user interface ; PET ; positron emission tomography ; CT ; computerised tomography ; SEM ; standard error of the mean ; BSS ; Balanced Salt Solution
• 刊名：Journal of Neuroscience Methods
• 出版年：15 February, 2014
• 年：2014
• 卷：223
• 期：Complete
• 页码：20-29
• 全文大小：2570 K

文摘

The technical advances made in microscopy have been matched by an increase in the application of fluorescent microscopy to answer scientific questions. While analysis of fluorescent microscopy images represents a powerful tool, one must be aware of the potential pitfalls. Frequently, the analysis methods applied involve at least some manual steps which are dependent on an observers input. Typically these steps are laborious and time consuming, but more importantly they are also influenced by an individual observer's bias, drift or imprecision. This raises concerns about the repeatability and definitiveness of the reported observations. Using calcium fluorescence in organotypic hippocampal slices as an experimental platform, we demonstrate the influence that manual interventions can exert on an analysis. We show that there is a high degree of variability between observers, and that this can be sufficient to affect the outcome of an experiment. To counter this, and to eliminate the disagreement between observers, we describe an alternative fully automated method which was created using EBImage package for R. This method has the added advantage of being fully open source and customisable, allowing for this approach to be applied to other analyses.