# Hippocampal Neurons' Alignment on Quartz Grooves and Parylene Cues on Quartz Substrate
## 3 Results and Discussion  
The algorithm for neurons' alignment analyzed direction and length of individual neurons' segments, which were separated in points of neurons bifurcation and connection. For neurons cultivated on anisotropic structures, directional growth and suppression of random growth (e.g., growth in curve) were expected. This made the used algorithm effective for analyzing neurons' alignment in comparison with Principal Component Analysis (PCA), which can be used for general purposes. PCA for analyzing neurons' alignment would be based on the distribution of each pixel of a cell border if it is a horizontal, vertical, or diagonal pixel cell border. However, such PCA considers direction only in three directions (respectively, two directions) and thus, it would lead to more ambivalent results; even the trend would be similar. E18 neurons were used for experiments to obtain purer neurons' culture from other cell cultures. To qualitatively analyze neurons' growth, neurons were cultivated on various structures. The growth of neurons on fabricated test structures is shown in Figure 2. For the 4 \(\upmu\)m deep grooves, the neurons had a tendency to adhere on the bottom of the groove and stick to the wall. Two axons or dendrites could grow next to each other, stuck to the opposite walls on the bottom of the groove (Figure 2a). This effect decreases with decreasing groove depth. For the 0.25 \(\upmu\)m deep grooves, the neurons did not stick to the wall of the shallow groove, but the neuron growth direction was still affected by the direction of the grooves (Figure 2b). When a neuron growing on flat quartz reached the grooves, it would start to grow in alignment with these grooves (Figure 2c). Moreover, when a neuron was growing in alignment with the grooves and it reached the edge of the grooves, it had a tendency to turn and grow along the edges of the grooves (Figure 2d) rather than to continue to grow on the flat quartz. If the grooves were deep enough, the neuron even followed a bended groove (Figure 2e). The neurons' alignment on quartz grooves and on other structures was caused by the neurons' tendency to stick to the wall of the grooves. However, on the parylene structures on quartz substrate, neurons preferred the parylene surface to the quartz surface for adhering, due to their significantly higher affinity to parylene than quartz (Figure 2f,g). Unlike with quartz grooves, neurons on parylene structures grew freely and did not stick to the wall or the edge of the parylene structures. If parylene structures, as cues, were long and thin enough, then neurons would follow and align with the parylene pattern. Similarly to the quartz grooves, if a neuron grew on flat quartz and reached the parylene cues, it would align with these cues; when a neuron growing on parylene cues reached the edge of the cues, it would turn back or grow along this edge (Figure 2h). Parylene cues on quartz substrate had a chemical affinity effect on neuron alignment; neurons preferred to grow on parylene structures and they usually only overstepped quartz from one parylene structure to another parylene structure, while quartz grooves in bulk quartz substrate had a topographical effect on neuron alignment, so neurons had a tendency to stick to walls of quartz grooves. Except neurons, the parylene structure were also stained by green color; this was because parylene treated by oxygen plasma becomes active and binds the dye during staining. However, the fluorescence intensity of the stained parylene was lower than the fluorescence intensity of the stained neurons, meaning that neurons were distinguishable from parylene. of where the perpendicular alignment started. If it is considered that neurons should align in the longitude direction of ridges (that is angle 90\({}^{\circ}\)), then the lowest direction of neurons should be in angle 0\({}^{\circ}\) (respectively 2.5\({}^{\circ}\), as middle angle from the angle group 0-5\({}^{\circ}\)).