## 3 Results
### Transport of Cells
#### 3.2.3 Reduced Photobleaching  
Combining optical tweezers with fluorescence microscopy offers a powerful technique for studying the biochemical and biophysical properties of single cells, as well as single proteins and molecules [47; 48]. By incorporating a fluorescent label, we obtained direct information with regard to the spatial and temporal distribution and evolution of particles within cells. However, such integrated technology has remained elusive due to the dramatic photobleaching on common fluorescent dyes under exposure to optical trapping and fluorescence excitation beams [49; 50; 51]. Therefore, establishing combined optical manipulation and fluorescence microscopy with minimal photobleaching to cells is crucial.  
In this study, experiments were performed to further demonstrate that the source of photobleaching can be reduced by indirect manipulation with the proposed microtool. The mitoTracker Red probe (M7512, Invitrogen, Thermo Fisher, Waltham, MA, USA) stained yeast cells were indirectly manipulated with the microtool and directly manipulated with optical tweezers for 10 min. The output laser power from the laser source was set to 0.9 W. Fluorescence microscopy was employed to measure the fluorescence. The red dye was excited with a fluorescence illuminator (120 PC Q, X-cite) and imaged with a Nikon TRITC cube. For each cell, the fluorescence images at different time points were captured with the same exposure setting.  
For indirect manipulation, a yeast cell was trapped between the V-shaped heads of two microtools, as shown in Figure 13a. In this manner, the cells are stably captured between the two microtools and its  
Figure 12: Experiments of cell transport with an obstacle in the workspace. **(a)** t = 0 s: the cell in the initial position. **(b)** t = 8 s: the cell reached the waypoint in the path. **(c)** t = 17 s: the microtool was turned to the new direction. **(d)** t = 28 s: the cell was successfully transported to the desired position. **(e)** Absolute position errors of the cell in transport.  
fluorescent images could be expediently taken. Figure 13c shows that although the imaged cell shape slightly changed due to the Brownian motion, the size of the red area displayed no obvious reduction during the indirect manipulation. Additionally, the average fluorescence intensity, measured by ImageJ [52], at 10 min only dropped to 96% \(\pm\) 2% (mean \(\pm\) SEM) compared with that at 0 min. For direct manipulation, Figure 13d shows a series of the fluorescence images of a yeast cell that was directly trapped by optical tweezers. During the direct trapping, the posture of the cell remained unchanged. However, the size of the red area was considerably reduced. Moreover, the fluorescence intensity of the cell under direct optical manipulation dropped to 86% \(\pm\) 1% (mean \(\pm\) SEM) compared with that at 0 min. Figure 13b shows the time-dependent change of fluorescence intensity under direct and indirect optical manipulation. The results suggest that during the long-time trapping, indirect optical manipulation with the proposed microtool induces considerably less photobleaching to cells compared with direct optical manipulation. This finding also indicates that laser exposure to the cells is effectively reduced.