Figure 3. Flow chart for screen and examples of neuron/neurite detection by software.

(a) Sixteen cochleas are sufficient to plate 192 wells of a 384 well plate. Each condition is tested in quadruplicate and each plate has its own positive and negative controls. Automatic imaging on the ArrayScan VTi provides 16 non-overlapping images per well (about 80% of the surface area). Neurites are detected and measured by the software HCA Vision. Only neurites longer than 20 pixel length, about the diameter of the neuron, are measured. (b–g) Example photographic fields (b,d,f) and corresponding depiction of software detected neurons and all segments of the neurites (c,e,g). The neurites are color coded to their neurons. Very occasionally, neurites are incorrectly segmented as in the upper right pair in E, although it does not seem to interfere with reproducible detection of neurite elongating activity. The longest, continuous path of a neurite from the cell body to the end of the neurite is calculated for each neuron. Scale: 100 μm.