複合原子力顯微鏡與共軛焦雷射掃描顯微鏡之新式掃描

Abstract

原子力顯微鏡是一種高精度的探針掃描儀器，能夠在奈米級的分辨率下繪製樣本的三維表面，而雷射共軛焦掃描顯微鏡是一種掃描具非破壞性的光學檢測系統，由於在垂直高度上有次微米級的分辨率，而被廣泛用於構築生物細胞及工程材料的三維輪廓。然而，兩種顯微鏡卻有各自的缺點，原子力顯微鏡雖具有高精度的掃描能力，但掃描時間過長且掃描範圍過小。雷射共軛焦掃描顯微鏡具有快速且大範圍掃描的能力，但解析度受限於光學繞射極限，解析度無法達到奈米級。因此，近年來有許多研究結合這兩種顯微鏡來達到高速大範圍且高精度的掃描。 然而，在結合這兩台顯微鏡時，卻有幾個整合上的問題，第一，兩台顯微鏡在掃描時間上具有數量級的差異，原子力顯微鏡需要數分鐘至數十分鐘來掃描長寬約為數十微米的範圍，而在相同範圍下，雷射共軛焦顯微鏡僅需數十秒，比較下來原子力顯微鏡的掃描時間過長會拖垮整體掃描速度。第二，若兩台顯微鏡的座標未經過校正的話，便無法正確的判斷彼此的相對位置，進而導致掃描上的對位問題。 本論文中，結合了上述兩種顯微鏡以及長行程 平台，並開發共同運作的演算法。首先在掃描前會用先利用共軛焦顯微鏡對原子力顯微鏡做位置校正，之後用原子力顯微鏡對長行程 平台作校正。校正完後，透過雷射共軛焦顯微鏡進行一次大範圍掃描，之後利用邊緣檢測法來找出感興趣的掃描範圍，並更進一步利用變速掃描節省原子力顯微鏡掃描的時間，藉此來提升整體的掃描速度，之後透過演算法來規劃兩顯微鏡與長行程 平台的路徑，並結合兩顯微鏡的掃描結果來構築一張快速大範圍且高精度的三維掃描影像。

Atomic force microscope (AFM) is a powerful technology that has ability to sketch the 3D topography of the sample in nanoscale resolution. On the other hand, confocal laser scanning microscope (CLSM) is a non-destructive optical inspection system in sub-micro resolution and widely used in three-dimensional profile of biological samples and engineering material. However, both of them have their own long-standing shortcomings. AFM suffers from a lower scanning speed and smaller scanning range. The resolution of the CLSM cannot reach nanometer level due to the optical diffraction. In order to overcome these limitations, many researches dedicated to the integration of AFM and CLSM so to achieve high-speed, large-range and high-precision scanning. However, there is an upcoming problems when AFM and CLSM are combined together. The scanning time in AFM is great more than that in CLSM. For micro-size scanning range, AFM takes several minutes to tens of minutes to scan while CLSM only takes tens of seconds. In addition, if the coordinates of the two microscopes are not correctly calibrated, the relative position of two microscopes cannot be accurately obtained, which may cause alignment problems. In this thesis, we design a hybrid microscope which combines AFM, CLSM and a long travel range positioning stage (LTRPS) and develop a novel cooperative fast algorithm to achieve high-speed, large-range and high-precision scan. First, the calibration of the microscopes will be implemented. Next, CLSM starts a large range scan first and then define the region of interesting (ROI) by edge detection. Next, the scan regions of the AFM are arranged based on the ROI and adaptive scanning region method is proposed to reduce the scanning time. Next, apply variable speed scanning to increase the AFM scanning speed. Finally, we compare sample features to build a fast, large-range, high-precision 3D scan image.