雷射鑷夾單一粒子追蹤系統之設計與分析

Abstract

自雷射鑷夾與以其為基礎之單一粒子追蹤的觀念被提出以來，業已成為最廣為使用於顯微鏡下對微小粒子進行操控與追蹤的方法，尤以細胞與分子生物的應用最為重要。 雖雷射鑷夾已在生物實驗中有多方應用，但目前仍無適當的雷射鑷夾捕捉理論適用生物系統中。因此，我們提出利用徑向對稱的多層微粒子來模擬類似生物細胞等非均質的微粒子，以之計算其所受雷射鑷夾的捕捉力，並對過去的理論進行修正與擴充。同時，藉由實驗量測雷射鑷夾對肺上皮細胞捕捉力，亦驗證了我們的理論模型。而在對細胞的捕捉力預測上，相較於過去的理論，我們的延伸模型可以減少超過20%的誤差。 另外，我們亦設計了一套具有離焦探測雷射光束的單一粒子追蹤系統，並針對在不同尺寸的微粒子下，探測雷射光束的離焦距離與偵測位移訊號之間的關係進行探討。我們不僅經由實驗的方式與散射理論模型，證明利用偵測離焦探測雷射光束之散射光斑，可以減少微粒子在不同方向位移之位移訊號耦合的現象，進而突破可追蹤之微粒子尺寸的限制。同時，我們也推測出最佳化的離焦距離與微粒子尺寸的關係。經由實驗證實，相較於沒有離焦偵測的情況，最佳化的離焦距離在追蹤上，微粒子追蹤之空間解析度、可量測的範圍與正確性，都會有明顯的提升。

Since the combination of optical tweezers with single particle tracking were proposed, it has become the most famous method applied in many research fields to manipulate and track the micro-particles in a microscope, especially in cellular and molecular biology. Although optical tweezers have been widely applied in the biological researches, there has not been a sophisticated model for their optical force for biological cells and organelles. Therefore, we extended the current model of optical tweezers to predict the optical force upon an non-uniform biological sample which is simulated as a spherically symmetric sphere. According to the comparison between the theoretical predictions of optical force on a lung epithelial cell and the experimental results, a 20% of reduction of prediction error will be obtained by using our extended model. We also designed a single particle tracking system with an off-focused probe laser beam, and studied the variations of position signals for different sizes of the tracked particles due to the focal offset between the probe laser beam and the trap laser beam. This study shows that the signal coupling between the trapped bead displacements in different axes may be significantly decreased via detecting the scattered pattern of the probe laser beam with a focal offset. Consequently, the limitation of the compatible size of the tracked particle can be improved. In this work, the relationship between the optimized focal offset and the tracked particle size is obtained theoretically and experimentally. It is confirmed that the spatial resolution, the tracking range, and the precision of the tracking can be improved with a probe laser beam at an optimized focal offset.