Figure 2 Select threshold and identify cells. A, Grayscale maximum projection of original stack. Scale bar, 25 µm. B, Select threshold level, and noise filter value to remove small spots. C, “Output Threshold Image” is a 2D projection after threshold and noise filters are applied. To visualize 3D shapes of cells, hotter colors indicate thicker portions of cells. D, 3DMorph automatically identifies 3D connected components, and the user selects a maximum cell size. Objects larger than this value are considered to be erroneously connected cells (d′, d‴), and will be segmented into separate objects (d‴). E, Exclude remaining small cells, out-of-focus processes, or cells touching the x-y borders to isolate only full cells (F). G, At this point, the program records total occupied volume (calculated before excluding small cells, processes, etc.), the unoccupied volume, and the distance between cell centroids.