3D quantification of short vertical neurites in membrane pores and their differentiation from other cell parts

Scientific efforts towards nerve regeneration and nerve-electrode interfaces depend on the possibility of guided neurite growth. The quantification and tracing of neurite growth during experiments is therefore essential. While tracing is possible under 2D cell culture conditions, it gets more complex when analysing three dimensionally orientated neurite pathways resulting in the ongoing development of 3D neuron tracing software. However, the quantification of short vertical neurite sprouts remains complicated due to difficult distinction from small cell body parts. With this study, we present a new method for precise identification and quantification of short neurite sprouts growing vertically from the surface of a track-etched membrane into 8 μm diameter pores of the membrane. Based on collected radius data from identified horizontally orientated neurites, a 95% reference interval for average radii of neurites was established and the limits were applied to trace neurite sprouts in the membrane's pores. Following this procedure, neurites were successfully distinguished from small cell body parts. This study demonstrates how to identify short neurite sprouts by assessing number, length, and radius with an additional checkpoint for bias detection.