應用於生物黏附及延展特性之雷射鑷夾系統設計與研究

Abstract

雷射鑷夾由於其非侵入性及非機械破壞性的特點，且及所產生的捕捉力接大小接近許多生物力的範圍，因此常被應用於細胞與分子生物的研究中。在本論文中，我們利用雷射鑷夾研究克雷白氏肺炎桿菌單一根第三型線毛與第四型膠原蛋白的黏附力。線毛利用頂端的黏著素來黏附，而第三型線毛帶有四種不同的黏著素。藉由雷射鑷夾，我們首次量出了這四種黏著素對單一根線毛與膠原蛋白的黏附力:其中帶有MrkDv1黏著素的線毛幾乎與膠原蛋白沒有黏附力，而帶有MrkDv2、MrkDv3、及MrkDv4黏著素的線毛與膠原蛋白的黏附力分別為2.03±0.03 pN，3.79±0.12 pN，及2.87 ± 0.15 pN。我們也應用了雷射鑷夾於整合素alphaⅡbeta3與蛇毒蛋白的黏附力量測。結果發現單一個野生型的蛇毒蛋白與整合素相黏時的黏附力為4.5 pN，而單一個突變型的蛇毒蛋白與整合素相黏時的黏附力為1.81 pN。 由於在應用雷射鑷夾於生物實驗時，通常都會利用一微小球在其上塗覆一些生物分子，再使微小球去黏附另一生物材料。利用微小球被雷射鑷夾拉離生物材料的微小位移，即可反推生物黏附力。因此，對於微小球的微小位移，必須要精確的量測。在過去我們只考慮了一維位移的量測。然而，對於微小球的微小位移應該三維都有精確的解析。因此，我們建立了一套具有高三維空間解析度及時間解析度的單一粒子追蹤系統。並量測得其解析度在橫向可達5.5 nm，縱向可達11.5 nm。若只考慮微小球二維的追蹤，則可以三維可以解析的範圍為300 nm 300 nm。若考慮三維的追蹤，則可解析的範圍為200nm 200nm 200nm的空間。 最後，我們提出一雙光束雷射鑷夾的系統，其中至少有一道雷射光可在顯微鏡視野中是隨意移動。如此也許可以使雷射鑷夾應用於生物黏附或延展實驗只在二維的平面甚至一維的直線上進行。因而增加對微小球的解析範圍。我們希望這套系統，將來可被應用於更多生物的黏附及延展特性之研究。

Optical tweezers are appropriate for molecular or cell biology research because their properties of non-invasive and non-mechanical contact to biological sample and the trapping force is compatible to lots of biological materials’ forces. In this thesis, we used optical tweezers to study the adhesion between a single Klebsiella pneumoniae type 3 fimbria and collagen IV. Different MrkD adhesin variants on the fimbriae are known to display distinct adherence capability for the bacteria to bind extracellular matrix proteins. For this reason, we measured the adhesive force between different MrkD adhesin variants and collagen IV for the first time. The MrkDv1 adhesin and collagen IV are nearly not adhered. The adhesive force between each of the single fimbria carrying MrkDv2, MrkDv3, and MrkDv4 adhesin variants and collagen IV are 2.03 ± 0.03 pN, 3.79 ± 0.12 pN, and 2.87 ± 0.15 pN, respectively. We also used optical tweezers to measure the adhesive force between integrinαIIbβ3-expressing CHO cells and rhodostomin. The interesting result shows that the adhesive force of wild type and mutant rhodostomin are 4.5 pN and 1,81 pN, respectively. Since a molecular coated bead is usually used in the optical tweezers experiments. The tiny displacement of the bead in the optical trap needs to be precisely detected. In the previous biological experiments, we only measured the 1-D displacement. However, we realized that the 3-D displacements of the bead should be considered. Therefore, we built a single particle tracking (SPT) system which is capable of high spatial and temporal resolutions. Our SPT system has a spatial resolution of 5.5 nm in transverse direction and 11.5 nm in axial direction. If only 2D tracking of the bead is considered, the tracking range can reach 300 nm 300 nm. If the 3D tracking of the bead is needed, the tracking range would reduce to 200 nm 200 nm 200 nm. At last, we setup a dual beam optical tweezers system. At least one optical trap can be arbitrarily moved in the field of view. By delicately designing the methodology, the adhesion or extension experiment of biological materials may only need 2D or 1D tracking of the bead. Thus, the tracking range may increase. We sincerely hope that this dual beam optical tweezers with a SPT system can be applied to more adhesion and extension properties studies in the near future.