(d) AFM scanning. During the AFM observation (b) and (c), the cantilever was distorted because of image lag.

only when the AFM scanned (Figures 1(b) and (c)). This was because the reflected light prevented us from observing the weak contrast of steps by LCM-DIM. Figure 1(a) shows the two-dimensional (2D) nucleation growth on the {110} surface of the HEW lysozyme crystal. A comparison of Figures 1(a) and (d) shows that there is no difference in the step advancing before and after AFM scanning. If the cantilever had affected the step movements, the 2D step would have lost its shape. It is, therefore, confirmed that the dual images could be successfully observed without disturbing each other.

The corresponding AFM image of the LCM-DIM image (shown in Figure 1) and its height profile are shown in Figure 2. In order to reduce the image acquisition time, the scan region was set to be rectangular (25 μm × 10 μm). We observed the same, but not identical, shape of steps. This difference was caused by the growing steps during AFM scanning. From the AFM observation, the height of the steps was estimated to be about 5 nm.

Here we discuss the comparison between LCM-DIM imaging and AFM imaging and how to combine the two. LCM-DIM had the advantage over AFM in imaging time. In addition, LCM-DIM could observe almost the same height level with AFM. Therefore, presently, simultaneous LCM-DIM and AFM imaging seems to be worthless. However, in contrast to 2D imaging, one-dimensional (1D) imaging, i.e., the line scan of AFM, can enhance the value of this interactive observation. The acquisition time of the line scan is about several seconds. Furthermore, the line scan can quantify the step height, which is the weak point of LCM-DIM. On the other hand, LCM-DIM

(a)(b)

Figure 2. AFM image of the {110} surface (a), which is the same as that shown in Figure 1 and the height profile (b) indicated by the white line in Figure 2(a).

observations can also help the AFM line scan by locating the position. Therefore, LCM-DIM imaging with AFM line scan is one of the most productive application of this hybrid microscope.

Next, the in-situ observation of nano scratching was performed using an AFM cantilever tip that had a large spring constant. Figures 3(a)-(f) shows LCM-DIM images of the HEW lysozyme during approaching (Figure 3(a)), scanning (Figures 3(b) and (c)), and retracting (Figures 3(d)-(f)) tip. A hole and 2D nucleation site of different contrast were observed around the tip-approaching point, as shown Figure 3(a). The hole depth was about 1.5 μm, as shown in Figures 3(g). Figures 3(b)-(f) show

Figure 3. Snapshots of nano scratching using a hard cantilever tip (a)-(f) and the AFM profiles of the first scan (g). The time period of the images was 60 s (b), 120 s (c), 180 s (d), 270 s (e) and 600 s (f) after the LCM-DIM observation started (a). The AFM scan was repeated 40 times. After scanning, the cantilever stayed on the surface for 2 min (b) and (c) and moved away from the crystal. After scratching, the ditch was buried by the growing steps (c)-(f).

the time course of scratching and recovering of the surface. The scratching was performed by scanning the cantilever at a normal force in the intermittent mode. The scanning length was 10 μm and the tip scanned 40 times. Many steps were generated at the scratched line, and the line was covered with growth steps. Some debris remained on the surface even after the surface seemed to be almost flat (Figure 3(f)). Kuznetsov et al. reported the AFM observation of nucleation after scratching of the thaumatin crystal surface with an AFM cantilever at different supersaturations [20]. They described that molecules detached from the crystal recrystallized again on the substrate in the supersaturated solution. Our results correspond to the most supersaturated conditions of their experiments.

In order to estimate the minimum force for scratching the crystal surface, we estimated the extent of the force of the tip that affects surface morphology by using a hybrid microscope. Figure 4 shows typical images of the surface where the cantilever tip approached and then retracted. We first searched the flat area shown in Figure 4(a) to detect small changes. After retracting, a small spot was observed, as shown in Figure 4(c) when the force of the approach exceeded a certain value. After several tens of seconds, the spot disappeared, as shown in Figure 4(d). The boundary force was different between increasing and decreasing the force. The observation of the spot started from 1.3 μN when the force was increased, and ended at 0.5 μN when it was decreased. This

Figure 4. Time series images of the indentation by the AFM tip and its recovering. The images are taken every 5 s and the tip was approached for 20 - 30 s. They are typical images of (a) a flat surface before tip approaching; (b) the tip approaching on the surface; (c) a small hole on the surface after the tip retracted; and (d) the recovered flat surface.

is believed to happen because the measured forces had two meanings. One is the force when the tip dig the lysozyme crystal, and the other is the force when the lysozyme crystal attached on the AFM cantilever tip crashed.

4. Conclusion

We demonstrated the joint operation of AFM and LCMDIM for studying HEW lysozyme crystal surface processes in a supersaturated solution. The simultaneous AFM and LCM-DIM observation was performed using a soft cantilever. On the other hand, the in-situ observation of surface scratching and recovering was performed using a hard AFM cantilever tip. The in-situ observation of nano indentation, the minimum force required to knock a hole on the crystal surface was about 1.3 μN.

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NOTES

*Corresponding author.

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