指導教授：劉子銘臺灣大學：醫學工程學研究所李徹重Li, Tse-ChungTse-ChungLi2014-11-302018-06-292014-11-302018-06-292013http://ntur.lib.ntu.edu.tw//handle/246246/264468血液常規檢驗是一種在臨床診斷或評估個人健康上的重要指標，但由於此方式需要進行侵入式的抽血且檢驗過程耗時，因此利用顯微術發展一套非侵入性且即時量測血球資訊的檢驗方式是非常重要的。在眾多顯微術之中，光學顯微術擁有優越的次細胞解析度(200-300nm)、高掃描頻率和高敏感度。藉由染色或標定的幫助，光學顯微術可以用來觀察許多不同分子的資訊。因此在觀察次細胞尺度下分子的型態和細胞的動態上，光學顯微術扮演著非常重要的腳色。 現今，因非線性光學顯微術具有次微米三維空間的解析度，所以被廣泛地用於生物體內的研究。例如雙光子顯微術和倍頻顯微術。相較於雙光子顯微術，倍頻顯微術因其能量守恆的特性，擁有較小的傷害性，因此更適用於臨床的應用。此外，倍頻顯微術另一項最大的優點是不需要額外對比劑的幫助就能觀察到生物組織的型態，因此非常適合用於觀察活體生物影像。在影像擷取部分，雙光子螢光雖然其訊號和激發光沒有相干的特性，但由於訊號強，沒同步也能擷取到不低的訊號強度。而倍頻顯微術因其訊號較弱，在應用於高速掃描的情況下需要同步擷取來提高影像的品質，例如即時掃描微血管中血球的影像。 在本篇論文中，基於本實驗室建立的即時三倍頻影像系統上，我們利用倍頻訊號相干的特性，建立同步影像擷取系統，提升影像的清晰度以增加判別的正確性。相較於商用的影像擷取卡，我們使用現場可程式化閘陣列(FPGA)自己設計影像擷取的功能，提高調整的彈性，並透過比較器和FPGA內PLL的功能達成和雷射脈衝同步的影像擷取系統。另外，我們使用微軟提供的Microsoft Foundation Class (MFC)編輯使用者介面，提供雙通道每秒30張的影像顯示和存取。並可藉由此介面改變FPGA的參數，調整取像的範圍、頻率等功能。同時也提供另一種擷取模式(MAX mode)，用以改善訊號太弱造成的問題。此外也提供影像擷取後一些基礎的處裡功能。 藉由此同步的影像擷取系統，對於皮膚微血管中的血球訊號的擷取強度和對比度有大幅度的提升，進而降低了自動化影像判讀的難度。利用此和雷射脈衝同步的倍頻影像擷取系統，期望在未來臨床的應用上，能利用三倍頻顯微術快速且有效率的辨別分析出血液中的血球種類、速度和數目，藉以評估個人的健康狀況。Complete blood count (CBC) is a routine examination conducted in hospitals. It has an essential function in diagnosing diseases. CBC requires drawing blood from patients, which is an invasive method. This medical procedure requires patients to wait for the test results. Thus, having an on-site measurement method for blood cell counts to avoid the invasive drawing of blood and real-time analysis to save waiting time is highly desired. Compared to other types of medical imaging, optical microscopy has superior performance on sub-cellular resolution (200 nm to 300 nm) in general. It also has high frame rate and high sensitivity. Combined with staining or labeling dye, molecular information can be visualized in a microscopic scale. These features make optical microscopy an indispensible diagnostic tool for observing sub-cellular morphology and cellular dynamics. Nowadays, with the capability of sub-micron three-dimensional (3D) spatial resolution, nonlinear optical microscopies, such as two-photon fluorescence microscopy (2PFM) and harmonic generation microscopy (HGM), have been widely used for in vivo biological studies. Compared with 2PFM, HGM is a less invasive optical microscopic technique, which makes it more suitable for clinical applications. HGM can also be used to observe sub-cellular morphology without extra labeling. Thus, HGM is suitable for observing cellular dynamics in vivo. In terms of imaging acquisition, although 2PFM is an incoherent process, the sampled intensity will still be sufficiently high without synchronous sampling because of its high signal intensity. For harmonic generation microscopy, which has low signal intensity, synchronous sampling is necessary in high-speed scanning applications to obtain high-quality images. In this thesis, based on the third harmonic generation (THG) microscope system in our laboratory, the synchronous imaging acquisition system was built using the coherent feature of the TGH to improve the SNR of the image. We replaced the commercial frame-grabber with a field-programmable gate array (FPGA). Taking the flexibility of the FPGA, we designed acquisition and transfer functions, such as a frame-grabber. By using the PLL in FPGA and with the help of the comparator, we synchronized the sampling clock with the laser pulse. On the other hand, basing on the MFC, we programmed a graphical user interface with two-channel 30Hz frame rate windows to display and restore the image. The interface could adjust FPGA parameters, such as frame rate and time delay. We also provided a different acquisition mode, which is called MAX mode, to prevent the low intensity induced by shot noise. Several basic image process functions are also provided. With the aid of this synchronous imaging acquisition system, we can clearly observe the blood cells in human blood capillaries. We hope that for future clinical applications, the non-invasive automatic evaluation of speed and number of blood cells using the THG microscope system could be faster and more precise using this synchronous acquisition system.誌謝 i 中文摘要 ii Abstract iv Contents vii CHAPTER 1. INTRODUCTION 1 1-1 Application of Microscopy on Medical Imaging 1 1-2 Overviews on Different Types of Optical Microscopes 3 1-3 Optical Detection and Recording Systems in Microscopy 7 1-4 Motivation 9 CHAPTER 2. Basic Concepts 12 2-1 Conventional Optical Microscopy 12 2-2 Confocal Optical Microscopy 15 2-3 Nonlinear Optical Microscopy 18 2-3.1 Third Harmonic Generation (THG) 21 2-3.2 Two Photon Fluorescence (2PF) 28 2-4 Noise 35 2-4.1 Shot Noise 35 2-4.2 Johnson-Nyquist Noise (Thermal noise) 36 2-4.3 Flicker Noise ( 1/f Noise) 37 2-4.4 Non-Essential Noise Sources 38 CHAPTER 3. Experimental Setup 40 3-1 Setup of Excitation Laser Source 40 3-2 Optical Scanner 43 3-3 Imaging Acquisition System 44 3-3.1 Design of frame-grabber 48 3-3.2 Design of Graphical-User-Interface 58 3-4 Set up of the Third Harmonic Generation Microscope 61 CHAPTER 4. Results and Discussion 64 Summary 82 References 8435093835 bytesapplication/pdf論文公開時間：2019/01/27論文使用權限：同意有償授權(權利金給回饋學校)同步影像擷取三倍頻雷射訊噪比血液常規顯微術[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/264468/1/ntu-102-R00548025-1.pdf