| Summary: | 博士 === 國立陽明大學 === 生醫光電研究所 === 100 === The first part of this thesis is to develop a method which can fine induce the spontaneous movement of cells. This study is based on the research of guiding the neural growth by using the infrared laser light. We illuminate the lamellipodia of cells by using 405 nm and 1064 nm laser light then observe the movement of lamellipodia. In the result, the 405 nm and 1064 nm laser light induce cell lamellipodia retraction and protrusion respectively. The most effective parameters of 405 nm laser light to induce lamellipodia retraction are 0.1 mW of power and 100 msec of illumination duration. On the other hand, the most effective parameters of 1064 nm laser light to induce lamellipodia protrusion are 4 mW of power and 10 sec of illumination duration. We also calculate the increment of local temperature in DMEM culture medium around the 405 nm and 1064 nm laser light. The results are = 5×10-7 oC and = 2×10-2 oC so the increment of local temperature is negligible. We find that the 405 nm laser light reduces the actin distribution near the illuminated site, while the 1064 nm light increases the density of actin.
In the second part of this thesis, we improve the system which was projected the 473 nm optical micropattern with 0.4 W/cm2 of power on the bottom of culture dish. The optical pattern will generate the light fence to culture and confine the region of cell growth. The lung cancer cells can migration and proliferation as normal cultured cells inside a 50 um width of triangle light fence but can not migrate to the outside of the light fence. The cells inside the light fence form a complete triangle colony after a few days. In order to confirm the correlation between the pattern of cell colony and light fence, we create a 50 um width of rectangle light fence on the bottom of culture dish. The cells inside the light fence form a complete rectangle colony after a few days. The results are similar to the experiment of the triangle light fence. We change the cell line with A549 and demonstrate that the micropattern culture is cell line independent. After the data analysis and quantification, the discrepancy between the pattern of cell colony and light fence is about 15 %. It means that the pattern of cell colony has a great resemblance to the light fence. Furthermore, the effect of cell viability affected by light fence is very small. We perform a complete process of the wound healing by using the dynamic light fence. The wound healing is appropriate for demonstrate the controlled ability of the light fence in the spatial and temporal domains. A complete process of the wound healing includes the three parts. First is to generate two independent cell colonies by using light fence (0-24 hr). Second is to maintain the micropattern of two independent cell colonies (24-48 hr). Finally is to carry out the wound healing (48-96 hr). It also manifests that the system can maintain and control the micropattern of the adhere cells in a long-term experiment. We also estimate the effect of the local risen temperature of DMEM culture medium around the 473 nm light fence at 0.4 W/cm2. The result shows that the local risen temperature is 4×10-6 ºC. Therefore we exclude the effect of the local risen temperature from the mechanism which affects the cell behaviors. On the other hand, we observe that the intercellular mitochondria were damaged by the 473 nm light fence. We surmise that 473 nm light fence destroy the mitochondria may be the mechanism of the micropattern culture of adhere cells by using light fences.
In future works of this thesis, there are three parts of the applied studies. The first part is using the light fence to study the cell dynamics, cell behaviors and cell-cell interaction. The second part is combining the light fence and biochip which generates the electric field or delivers the drugs. We aspire to develop a technique which can perform multi-stimulation on the cells.
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