臺灣博碩士論文加值系統

雷射光鉗自Ashkin 於1970 年開始研發以來，廣泛應用在許多生物樣
本的非侵入式操縱，特別是在與細胞骨骼相關的蛋白質研究上有長足進展。在雷射光鉗的技術中，對於被操縱物的軸向位移偵測技術的發展並未如橫向位移偵測般成熟。在本論文中我們引入了1997 年Lee 等人發展的差動共焦顯微術。該技術具有軸向解析率高、測量的動態範圍大、工作距離長的特色。我們將之與雷射光鉗結合，用以偵測光鉗中乳膠小珠的軸向運動。
我們架設的差動共焦鏡可以量測到系統本身的背景擾動16 nm。在使
用NA 1.3 的物鏡與27 mW 的雷射功率所形成的光鉗中，本系統可偵測出直徑3 µm 的乳膠小球約50 nm 尺度的軸向熱擾動，並且計算出光鉗的軸向彈性係數為2.34 µN/m，此軸向彈性係數隨雷射功率呈線性變化。
接著我們進一步以肌凝蛋白與肌動蛋白為樣本，量測蛋白質對小珠在
緩衝溶液中自然擾動的影響。實驗結果顯示小珠在蛋白質的環境中有較大的擾動現象，但此擾動在肌凝蛋白與肌動蛋白的結合下隨著軸向抬升有顯著降低。我們也從光鉗動態抬升小珠的過程中，即時偵測到肌凝蛋白與肌動蛋白的結合，會延遲小珠的軸向運動的特性。
我們已成功量測到肌凝蛋白與肌動蛋白的結合作用對小珠擾動與運
動的影響。本系統的動態測量範圍達3 µm 以上，結合其奈米解析度，極適合應用在蛋白質彈性研究上。

Since the first invention in 1970 by Ashkin, optical tweezers have become a versatile non-invasive tool extensively used in controlling tiny biological samples, especially cytoskeleton related proteins. In optical tweezers, the precise measurement of axial motion of a trapped object in optical tweezers is not as well developed as that of the lateral motion. In this thesis we have
introduced differential confocal microscopy (DCM), developed by Lee in 1997, into optical tweezers for the axial detection. DCM has advantageous features such as high depth resolution, large dynamic range, and long working distance.
Combined with optical tweezers, we can detect the axial movement of an optically trapped latex bead.
The resolution of axial position detection is 16 nm, which is limited by the background fluctuations of the whole system. We have detected about 50 nm scale of thermal fluctuation of a latex bead of 3 µm in diameter, trapped in a beam with a power of 27 mW through 1.3-NA objective lens. From the thermal fluctuations of the bead, we calculate the axial tweezers stiffness to be 2.34 µN/m at this power. We also find that the axial stiffness is linearly proportional to the laser power.
With this setup, we measured the effect of myosin–actin binding to the fluctuations of a trapped bead. The result shows the fluctuations of a bead are lager with the presence of protein in solution. However, as myosin–actin bonds formed, the fluctuations of the myosin-coated bead gradually decreased when we elevated the bead from actin-coated dish bottom. Also, we have observed the motion retardation of bead, caused by the tension of myosin–actin binding between the bead and the bottom.
We have successfully detected the binding effect of proteins to the motion of a trapped latex bead. In our experiment, the dynamic range of detection is lager than 3 µm, which makes the system suitable for the application of protein elasticity research.

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