本論文整合了雷射導引直寫技術(Laser Guide Direct Writing,LGDW )與雷射光鉗(Optical Tweezers)系統來進行快速單一活體細胞 動態效應分析。本研究採用830 nm 雷射光源來建構雷射導引直寫系統，並將雷射引導入倒立式顯微鏡系統，進而與微流體樣本置換系統 以及雷射光鉗系統整合，得以透過非接觸式操控單一細胞於三維空間移動，並經排列後組成二維平面活細胞陣列。此細胞陣列可作為藥物 作用下的細胞型態即時監測，以及觀察細胞間的變化差異，因此單一細胞層級細胞陣列可作為最為直接且顯著的方式來觀察藥物與細胞 的交互作用。雷射導引直寫系統除了執行建構細胞陣列功能，本研究也將830 nm 雷射光源運用於探討低能量雷射光治療(Low-Level Laser Therapy ,LLLT)的光生物調節(Photobiomodulation)作用機制，藉以觀察細胞陣列在接受近紅外光雷射刺激的細胞反應與活性氧化物質 (Reactive Oxygen Species, ROS)的形成，並利用H2DCFDA螢光染劑來標定細胞經由LLLT 作用後所產生的活性氧化物質變化量，作為推測 低能量雷射光治療的細胞調控機制。此外也將H2DCFDA運用於觀察化療藥物艾黴素(Doxorubicin)作用於細胞後所產生之活性氧化物質 的形成，其中Doxorubicin 已被證實細胞經其作用所產生的過量活性 氧化物質會引起細胞乃至器官氧化壓力(Oxidative Stress)。本論文希冀透過雷射直寫系統與雷射光鉗系統的整合，可以延伸運用於單一活 體細胞層級的光電醫學與藥物快篩平台，作為探討低能量雷射光治療 與抗癌藥物以及其他治療方法的作用機制與功能驗證。;In this study, the integration of laser-guided direct writing(LGDW) and This thesis presents a new methodology that integrates a laser-guided direct writing (LGDW) system and optical tweezers system to analyze the dynamic response of single living cells. In this study, we apply 830 nm laser to construct a LGDW system that is integrated into an inverted microscope, together with microfluidic flow chamber, and incorporated an optical tweezers system to manipulate single living cell in three dimension and then pattern two dimension (2D) cell array in well-defined manner. This 2D living cell array provides a direct observation platform and allows us to monitor the variation of cell morphology during drug-cell interaction in real-time. On the other hand, the LGDW system will further applied to conduct the low-level laser therapy (LLLT) photobiomodulation at the single cell level and realize the effect of LLLT photobiomodulation on the generation of reactive oxygen species (ROS), where the ROS formed by LLLT will be identified with H2DCFDA fluorescence. Besides, H2DCFDA fluorescence is used to detect the ROS formed by doxorubicin when used as antitumor drug is attributed to the generation of ROS. We anticipate the proposed integrated system including LGDW system and optical tweezers system can be applied for photomedicine and drug screening platform at the single-cell level so as to investigate the mechanism and verify the effectiveness of LLLT, antitumor drugs, and other specific therapies.
