A Semi-Automatic Tool for the Standardized Analysis of Fluorescent Intensity Changes in Polarized Cells

TL;DR

This paper introduces a free, open-source tool for analyzing fluorescent intensity changes in polarized cells, focusing on subcellular regions that are often overlooked.

Contribution

The novel contribution is a semi-automatic, open-source program for defining ROIs in subcellular imaging experiments with complex cell morphology.

Findings

The program successfully validated ROI determination and movement correction in cochlear Deiters’ cells.

It is a free and open-source solution for subcellular imaging, which is currently underrepresented in research.

The tool was tested using ATP-induced test impulses to examine subcellular Ca2+ handling in Deiters’ cells.

Abstract

Imaging of intracellular messengers, like calcium, is one of the most reliable methods to follow real-time changes in several aspects of cellular activity, like receptor activation. However, the analysis could be influenced and biased by several factors like the location, shape, and size of the regions of interest (ROIs) and by the detection and correction of the movement of the preparation. Programs which are provided by the manufacturers are expensive and cannot be shared by collaborators. Many self-made programs have been implemented lately which have in-built cell recognizer ROI identification functions. These programs focus on the soma of the cells and neglect the processes, because in full tissue preparation finding cells is still challenging. Subcellular imaging experiments are still rare. To the best of our knowledge there is no program which can automatically define ROIs for subcellular imaging experiments even in single indicated cells with complex morphology. We developed and validated a program to address this gap using simple and understandable mathematical methods for ROI determination and simple statistics for movement correction. Validation experiments were conducted on cochlear Deiters’ cells. Deiters’ cells have processed morphology which connects two fluid compartments in the cochlea. Because of the function and the fine morphology of the cell, it could be interesting to examine the subcellular Ca2+ handling mechanisms of it. Test impulses were activated by ATP. With some limitations the program successfully fulfilled its purpose. As a free, easily understandable, and open-source program, we hope it will help to analyze and plan subcellular experiments.

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