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Abstract
BackgroundIn mammalian cells the endoplasmic reticulum (ER) comprises a highly complex reticular morphology that is spread throughout the cytoplasm. This organelle is of particular interest to biologists, as its dysfunction is associated with numerous diseases, which often manifest themselves as changes to the structure and organisation of the reticular network. Due to its complex morphology, image analysis methods to quantitatively describe this organelle, and importantly any changes to it, are lacking.ResultsIn this work we detail a methodological approach that utilises automated high-content screening microscopy to capture images of cells fluorescently-labelled for various ER markers, followed by their quantitative analysis. We propose that two key metrics, namely the area of dense ER and the area of polygonal regions in between the reticular elements, together provide a basis for measuring the quantities of rough and smooth ER, respectively. We demonstrate that a number of different pharmacological perturbations to the ER can be quantitatively measured and compared in our automated image analysis pipeline. Furthermore, we show that this method can be implemented in both commercial and open-access image analysis software with comparable results.ConclusionsWe propose that this method has the potential to be applied in the context of large-scale genetic and chemical perturbations to assess the organisation of the ER in adherent cell cultures.
Highlights
In mammalian cells the endoplasmic reticulum (ER) comprises a highly complex reticular morphology that is spread throughout the cytoplasm
A high‐content and high‐resolution imaging strategy to visualise the endoplasmic reticulum Successful implementation of downstream image analysis relies upon the quality and consistency of the input images, and so the first step was to optimise our imaging protocol in such a way that it would allow for subsequent scale-up
Experiments were designed in 96-well optically clear plates, compatible with a fully automated confocal high-content screening microscope fitted with a 63 ×/1.15 NA water-immersion objective (Fig. 1A)
Summary
In mammalian cells the endoplasmic reticulum (ER) comprises a highly complex reticular morphology that is spread throughout the cytoplasm This organelle is of particular interest to biologists, as its dysfunction is associated with numerous diseases, which often manifest themselves as changes to the structure and organisa‐ tion of the reticular network. Even profiling of the Golgi and mitochondria in living cells allowing analysis of organelle fission and fusion events has been shown to be possible [7, 8] Despite this progress, the highly dynamic and morphologically complex endoplasmic reticulum (ER) network possesses a significant challenge to analyse using automated image analysis methods. There is a lack of available tools for the quantitative analysis of ER morphology, despite an increasing body of literature linking ER morphology with human disease [9, 10]
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