Quantitative image analysis tool to study the plasma membrane localization of proteins and cortical actin in neuroendocrine cells

Background: Adrenal chromaffin cells are a widely used model system to study regulated exocytosis and other membrane-associated processes. Alterations in the amount and localization of the proteins involved in these processes can be visualized with fluorescent probes that report the effect of different stimuli or genetic modifications. However, the quantitative analysis of such images remains difficult, especially when focused on specific locations, such as the plasma membrane. New method: We developed an image analysis algorithm, named plasma membrane analysis in chromaffin cells (PlasMACC). PlasMACC enables automatic detection of the plasma membrane region and quantitative analysis of multi-fluorescent signals from spherical cells. PlasMACC runs in the image analysis software ImageJ environment, it is user-friendly and freely available. Results: PlasMACC delivers detailed information about intensity, thickness and density of fluorescent signals at the plasma membrane of both living and fixed cells. Individual signals can be compared between cells and different signals within one cell can be correlated. PlasMACC can process conventional laser-scanning confocal images as well as data obtained by super-resolution methods such as structured illumination microscopy. Comparison with existing method(s): By comparing PlasMACC to methods currently used in the field, we show more consistent quantitative data due to the fully automated algorithm. PlasMACC also provides an expanded set of novel analysis parameters. Conclusion: PlasMACC enables a detailed quantification of fluorescent signals at the plasma membrane of spherical cells in an unbiased and reliable fashion. (C) 2014 Elsevier B.V. All rights reserved.