Comparing the average roughness of PDMS 184 and PDMS blends 10:1, 5:1 and 1:1

Figure 41 – Average Roughness Ra of the surface of PDMS 184 and PDMS blends 10:1, 5:1 and 1:1 (mean ± SD, n=3) * shows a significant decrease in surface roughness from PDMS 5:1 to PDMS 1:1 p = ≤ 0.04. (One- way ANOVA using Tukey analysis), Minitab.

The differences in surface roughness (average roughness Ra) were compared across the different blends of PDMS used in experiments, as shown in Figure 44. It was observed that there was a general trend between PDMS 184, 10:1 and 1:1 in that the average roughness Ra of the PDMS samples decreased overall as the amount of PDMS 527 in the blends increased. However it was found that the average roughness Ra for PDMS 5:1 (ratio of Sylgard 184: Sylgard 527) increased

0.00 50.00 100.00 150.00 200.00 250.00 300.00 350.00 400.00 PDMS 184 PDMS 10:1 PDMS 5:1 PDMS 1:1 A ve rag e R o u gh n e ss R a (n m ) *

144 when compared to the other three PDMS samples with an average roughness of 261 nm ± 99.6 nm (Figure 44).

The average roughness of PDMS 1:1 was found to be statistically lower than the roughness of PDMS 5:1 surface (p = 0.04). This could have been due to the ratio of the two components that made up these blends, Sylgard 184 and Sylgard 527. PDMS 1:1 contained equal parts of both types of Sylgard whereas PDMS 5:1 contained 5 parts Sylgard 184 to 1 part Sylgard 527. However this was greater than in PDMS 10:1, which contained 10 parts Sylgard 184 to 1 part Sylgard 527. The varying amounts of Sylgard 527 appeared to have an impact on the surface topography of the resulting PDMS blend, and a significant decrease in average roughness Ra of PDMS between PDMS 5:1 and PDMS 1:1 was observed.

To further investigate the differences observed in surface roughness of PDMS samples, Figure 45 shows a three – dimensional (3D) view of a representative image of each PDMS type giving the height (measured). This equates to the overall peak to valley roughness Rt across the sample, which is the total height of the roughness profile for each sample shown. The differences in surface roughness using the height (measured) can also be seen here and a similar trend is apparent when compared to the average roughness Ra shown in Figure 45 in that the height (measured) for PDMS 5:1 is higher in the images in Figure 45 and Figure 44.

Further image processing carried out on the 3D images in Figure 45 was Gaussian smoothing (width of 0.261 µm X and Y direction). The differences in the 3D images when comparing PDMS 1:1 to the other PDMS types could have been due to the lower stiffness of PDMS 1:1 which may have caused increased lateral deflection of the cantilever during scanning as the cantilever may have been sticking to the PDMS 1:1 surface causing lines or grooves to appear on the scan image. However, for each condition the same setpoint and z-length was used to maintain consistency in the tip-sample separation and to apply the same settings of force to each sample. When calibrating the spring constant of the tip, the tilt angle is considered by the SPM software as this is important in spring constant calibration. However additional correction factors can be used to take into consideration the effects of the measurements in liquid.

145

Figure 42 – AFM images showing a representative 3D scan image for each PDMS type. Scale bars show the scan size of 50 x 50 µm and the height (measured) of the sample, representing the peak to valley roughness Rt (µm).

5.2.1.2. Comparing the average roughness of the nucleus and cell body of RCE cells attached to different PDMS samples and tissue culture plastic

The average roughness Ra was also measured for RCE cells cultured on the different PDMS samples and tissue culture plastic. The specific areas of the cell that were studied were the nucleus and the cell body. The substrate on which the RCE cell was attached to was also measured for surface topography (see figure 48). Figure 46 compares the average surface roughness of the nuclear and cell body regions of the RCE cell when cultured on different PDMS samples and tissue culture plastic (TCP), as well as the PDMS and TCP surface roughness measurements as controls to compare the substrate topographies to RCE cell topographies.

PDMS 184

PDMS 5:1

PDMS 10:1

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Figure 43 – Average roughness Ra of RCE cells attached to different PDMS sample surfaces and tissue culture plastic comparing the average roughness Ra of the nucleus, cell body and surrounding substrate (mean ± SD, n=3). *** shows significant differences between RCE cell roughness of the nucleus on different PDMS blends and TCP when compared to the cell body of an RCE cell on the same type of PDMS and the substrate. P value for all comparisons ≤ 0.001 (one-way ANOVA using Tukey analysis, Minitab).

It was found that there was an overall trend in average roughness of the selected features of the RCE cell and the roughness of the surface decreased from the nucleus to the cell body. This may have been the result of the cell body and cell edge of the RCE cell becoming thinner and spreading across the surface of the substrate the further away from the nucleus. The setpoint height values have been compared later on in the Chapter.

Statistical analysis showed that there were no significant differences between the different PDMS roughness measurements and the general trend of decreasing roughness between the nucleus and the cell body was observed across all PDMS samples (Figure 46). Differences were found between the roughness of the nucleus and cell body when comparing RCE cells on different types of PDMS and TCP. This supported the observed trend of a significant decrease in roughness from nucleus to cell body, showing that it was present across the different substrates and was a characteristic of the RCE cell.

When nuclei were compared across the different PDMS substrates and TCP, differences were found in average roughness of the nuclei on PDMS 10:1 compared to PDMS 184 and TCP nuclei, showing that on PDMS 10:1, RCE cell nuclei were significantly rougher. The roughness of the nuclei region on

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Nucleus Cell body Substrate

A ve rag e R o u gh n e ss R a ( n m ) PDMS 184 PDMS 10:1 (184:527) PDMS 5:1 (184:527) PDMS 1:1 (184:527) Tissue culture plastic

147 TCP was also significantly lower than the nuclei roughness of RCE cells on PDMS 5:1 (Figure 46). The introduction of the softer Sylgard 527 to Sylgard 184 to produce PDMS blends appeared to have an effect on PDMS surface roughness, which was more obvious for PDMS 5:1 (see Figure 45), and therefore may have impacted on the attachment of the RCE cell to the substrate, altering the cell shape and cell mechanics, lending to the differences observed in nuclei surface roughness (Zamani et

al., 2013). However, when comparing the cell body across the different PDMS types, no significant

differences were found between the roughness data. This showed that the surface roughness properties of the RCE cell body remained within a similar range across different PDMS substrates including TCP, suggesting that the surface properties of the substrate did not have significant effects on the RCE cell body surface roughness.

For PDMS 184, significant differences were found between the roughness of the PDMS surrounding the cell and the nucleus and also between the cell body and the nucleus of the RCE cell attached to this type of PDMS. This was observed across the four types of PDMS and a similar trend was found. The way in which the RCE cell attached to the PDMS, determined this pattern in surface roughness and it was clear that this was inherent to the RCE cell and not due to the influence of the PDMS. Comparing RCE cells cultured on TCP, the trend observed previously of a decrease in average roughness between the nucleus and cell body was also found in RCE cells cultured on TCP (Figure 46). The average roughness of the RCE cell nucleus on TCP was found to be significantly higher in comparison to the average roughness of the TCP surface. Significant differences in roughness were also found between the cell body and nucleus on TCP and between the cell body and the TCP surface. No significant differences between the roughness of the RCE cell body and the PDMS or TCP surface surrounding the RCE cell were found, across all samples that were measured. Significant differences were also found between the roughness of the RCE cell body and the PDMS or TCP surface surrounding the RCE cells and this was expected due to the different materials used and the RCE cell cytoskeletal structures that would be measured.