the pattern design. This allows the precise evaluation of mitochondrial dynamics in individual neuronal processes in live neurons so that mitochondrial fission (Figure 3A, upper panel) or fusion (Figure 3A, lower panel) can be easily followed over time. To assess mitochondrial morphology in individual neuronal processes, we plated cells onto substrates patterned with lines 10μm wide and examined mitochondrial parameters after immunostaining with the mitochondrial marker TOM20 and the neuronal marker β-III-tubulin (Figure 3B). Conversion of the TOM20 signal into a binary image facilitated the visualization of mitochondrial structures for subsequent analysis with ImageJ software. The analysis of mitochondrial parameters in individual neuronal processes of patterned neurons at different time points revealed a change in mitochondrial morphology over time (Figure 3C). While mitochondria appeared punctated at day 40 of differentiation, they were more elongated and interconnected at day 100. We quantitated the length of individual mitochondria (also known as aspect ratio) as well as their interconnectivity (also known as form factor) using ImageJ analysis on the basis of the binarized TOM20 signal as described previously [28]. The results showed a time-dependent increase of mitochondrial length as well as interconnectivity when comparing 40 and 100 days old neurons (Figure 3C).
