聚光鏡對利用前向散射光奈米級精確解析雷射鑷夾中微小粒子位置之影響

Abstract

近幾年中，雷射鑷夾（Optical Tweezers）以被捕捉的微小粒子為一探針，已被廣泛地應用於生物醫學的研究上。然而當受雷射鑷夾所捕捉搬運的微小粒子的直徑越來越小、且所需觀測微小粒子的位移量到達奈米級時，依據Rayleigh準則得知，利用顯微物鏡上方CCD攝影機所能解析到微小粒子的位移量極限為次微米，因此我們需要更靈敏的方式來得到微小粒子到達奈米級的位移量。利用偵測經過微小粒子的雷射光所產生的前向散射光影像，可以準確的描述微小粒子奈米級微小移動量。而此前向散射光影像是藉由聚光鏡收集於光偵測器上，因此聚光鏡對於利用前向散射光影像解析微小粒子奈米級位移量扮演很重要的角色。 本研究中，我們以實驗的方式，探討以下三種聚光鏡情形：一、 直接利用聚光鏡觀測前向散射光影像時，聚光鏡位置對微小粒子空間解析度之影響。二、 觀測聚光鏡後焦平面之前向散射光影像時，聚光鏡位置對微小粒子空間解析度之影響。三、 聚光鏡的數值孔徑對微小粒子空間解析度之影響。我們發現一、直接利用聚光鏡觀測前向散射光影像時，當聚光鏡與物鏡為共焦，我們可以得到最高微小粒子的空間解析度。且其空間解析度隨著聚光鏡位置的改變，也隨之大幅地改變，甚至得到反方向的空間解析度。二、觀測聚光鏡後焦平面之前向散射光影像時，當聚光鏡與物鏡為共焦時，我們一樣可以得到微小粒子最高空間解析度。然而隨著聚光鏡位置的改變，微小粒子空間位移量解析度並無大幅度的變化。三、微小粒子空間解析度與聚光鏡的數值孔徑為線性的關係。隨著聚光鏡的數值孔徑增大時，則微小粒子空間解析度越高。 在本研究中，所實驗的結果均有相當好的穩定性與重複性，其實驗結果與理論計算值亦有相當好的一致性。我們利用本實驗所得到的最佳聚光鏡條件參數，對於直徑為1.1μm微小粒子在空間的解析度，至少可到達9.05nm的空間解析度

In these years, using the particle trapped by an optical tweezers as a probe, has been widely used in Biological and medical research. However, with the size of the trapped particle is getting smaller, and the displacement of the particle goes into nano-meter range, it is difficult for us to observe the tiny moves of tiny particle. From the Rayleigh principle, the minimum distinguishable displacement of a particle by geometric optics imaging is about 0.1□m. Therefore, if we want to detect the displacement of a particle under 0.1□m, we need a more sensitivity technique. By detecting the forward scattering image formed by the scattering laser light of the particle, it is able to detect the displacement of a particle under 0.1□m. A condenser is used to collect the forward scattering light and form a forward scattering image on a photo detector. Therefore, the condenser plays an important role on nanometer positioning of a small particle. In this research, we will experimentally discuss the following issues: 1: The influence of the condenser position to the sensitivity of particle positioning by detecting forward scattering image. 2: The influence of the condenser position to the sensitivity of particle positioning by detecting forward scattering image when especially observing the back focal plane of the condenser. 3: The influence of the numerical aperture of the condenser to the sensitivity of particle positioning by detecting forward scattering image. We conclude that 1: The highest sensitivity to detect the particle’s displacement by forward scattering image shifts occurs when the condenser and objective lens are confocal. And the position of the condenser influences a lot on the sensitivity of particle positioning. Even more, when the condenser at some positions, the shifts of forward scattering image is opposite to the real particle shifts. 2: While the forward scattering image on the back focal plane of the condenser is being observed, the highest sensitivity to detect the particle’s displacement also occurs when the condenser and objective lens are confocal. However, the position of the condenser doesn’t barely affect the sensitivity of particle positioning. 3: The sensitivity to track the particle via forward scattering pattern is almost direct proportional to the condenser’s numerical aperture. In conclusion, we show that our experimental results are stable and repetitively. And the experimental result is in good agreement with the theoretically calculation. By applying the best condenser position and numerical aperture factors from our thesis, we can at least have a 9.05nm-spacial resolution of a 1.1um-in-diameter particle.