Automated cell segmentation in FIJI® using the DRAQ5 nuclear dye

Background: Image segmentation and quantification are essential steps in quantitative cellular analysis. In this work, we present a fast, customizable, and unsupervised cell segmentation method that is based solely on Fiji (is just ImageJ)®, one of the most commonly used open-source software packages for microscopy analysis. In our method, the "leaky" fluorescence from the DNA stain DRAQ5 is used for automated nucleus detection and 2D cell segmentation. Results: Based on an evaluation with HeLa cells compared to human counting, our algorithm reached accuracy levels above 92% and sensitivity levels of 94%. 86% of the evaluated cells were segmented correctly, and the average intersection over union score of detected segmentation frames to manually segmented cells was above 0.83. Using this approach, we quantified changes in the projected cell area, circularity, and aspect ratio of THP-1 cells differentiating from monocytes to macrophages, observing significant cell growth and a transition from circular to elongated form. In a second application, we quantified changes in the projected cell area of CHO cells upon lowering the incubation temperature, a common stimulus to increase protein production in biotechnology applications, and found a stark decrease in cell area. Conclusions: Our method is straightforward and easily applicable using our staining protocol. We believe this method will help other non-image processing specialists use microscopy for quantitative image analysis.