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Automated analysis of intracellular calcium fluorescence in rat organotypic hippocampal cultures: comparison to a manual, observer based method.
Date Issued
2014-02
Date Available
2013-11-29T09:46:22Z
Abstract
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.
Sponsorship
Other funder
Other Sponsorship
The authors would like to thank Science Foundation Ireland (JOC- SFI: 09/RFP/NES2450), Enterprise Ireland (MP- CF20100068), Congressionally Directed Medical Research Programs (MP- MS100055), MS Ireland, and University College Dublin for financial support.
Type of Material
Journal Article
Publisher
Elsevier
Journal
Journal of Neuroscience Methods
Volume
223
Issue
15 February 2014
Start Page
20
End Page
29
Copyright (Published Version)
2014 Elsevier Ltd.
Language
English
Status of Item
Peer reviewed
This item is made available under a Creative Commons License
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