工學院: 醫學工程學研究所指導教授: 劉子銘王靖尊Wang, Jing-ZunJing-ZunWang2017-03-062018-06-292017-03-062018-06-292016http://ntur.lib.ntu.edu.tw//handle/246246/277396人體血液常規檢驗是評估個人健康上的重要指標，透過流式細胞儀等分析儀器進行紅血球、白血球及血小板計數，其計數數量都各有鑑別診斷上的意義；然而血液的分析，需藉由侵入式抽血來進行檢驗，此舉不但會造成患者的負擔也可能因送檢過程使得檢體變質，造成健康評斷上的錯誤。 隨著醫療科技的發展，眾多的非侵入式生醫影像技術被開發出來，例如光學同調斷層掃描是利用干涉儀原理，來對生物組織進行成像，可提供微米等級解析度之二維活體組織斷層影像。共軛焦顯微術是利用針孔來阻擋不對焦的光線以及散射光的干擾，而形成次微米解析度的切片影像；運用高組織穿透深度的紅外光源，這些技術不需要標定，就能在活體內取得血球影像，但受限於光的繞射極限與組織散射，皆無法在組織深處取得次微米解析度的清晰影像，也因此缺乏鑑別各式血球的能力。而倍頻光學顯微術比起其它光學斷層掃描術，在深層組織仍擁有次微米三維空間解析能力，研究已經證實可以在人類活體內取得血球的流動影像，並判讀出白血球的數量，是目前最有潛力發展成為非侵入式活體影像流式細胞儀的技術。此外，當病患的血液進行流式細胞儀檢驗，血球計數比例有疑義，需進行更進一步的血液抹片檢驗時，得依靠染劑來觀察血球，而三倍頻光學顯微術不需進行染劑標定即可觀察血球並同時儲存影像，不僅省去血液抹片檢查的檢驗時程也呈現了血球的原始樣貌。 為了提供穩定的三倍頻顯微術光源，本實驗室架設一套波長1150 奈米的光纖飛秒雷射系統，比起鈦藍寶石及鉻貴橄欖石雷射，對溫度以及濕度變化較不敏感。在顯微鏡系統中可提供二倍頻、三倍頻、多光子螢光、共軛焦單光子反射光，四種類比訊號。然而現今的類比轉數位擷取板，無法直接擷取4種訊號，且在做類比轉數位時，會有直流訊號被截除的現象，導致訊號強度降低；在影像擷取的部分，雷射光源的脈衝重覆率僅11.25MHz，透過高速的掃描鏡快軸(8kHz)來移動雷射聚焦位置時，點與點之間的時間間格較大，所以必須擷取到每個訊號點的最大值，避免取得較弱的訊號，導致降低影像強度。因此，要獲取高解析度、即時測量血球資訊，在硬體方面，規格勢必要相容於光電訊號，保留直流訊號；在軟體方面，樣本經雷射激發出的類比訊號，需藉由雷射同步訊號來取得類比訊號的最大值，藉此提高影像的對比。 在本篇論文中，利用Altera提供的現場可編成邏輯閘陣列(FPGA)自己設計影像擷取的功能，FPGA不僅減少開發時間，也具有高效能及可靠性。透過FPGA內的鎖相迴路(PLL)功能達成和雷射脈衝同步的影像擷取系統，並設計符合此FPGA之類比數位轉換板(Analog to Digital Converter Board)。另外，我們使用微軟提供的Microsoft Foundation Class (MFC)設計使用者介面，提供多通道每秒15張影像顯示和存取，並可藉由此介面即時改變FPGA的參數，調整取樣的範圍、頻率以及雷射脈衝相位等功能。同時也提供另一種擷取模式(XYT mode)，用於長時間錄影，可彈性設定錄影間隔，達成影像經時間間隔後，存取一張影像或平均影像後再進行存取，減少存取影像的資料量。此外也提供基礎的影像處理功能。 同步雷射訊號以及使用自製的類比數位擷取卡後，小鼠耳朵微血管的血球影像訊號和對比度均有大幅度的提升，進而降低了自動化影像判讀的難度。利用和雷射脈衝同步之影像擷取系統，期望在未來臨床的應用上，能利用本實驗室架設之光纖飛秒雷射系統，以非侵入式方式，分析出血液中的血球種類和數目，藉以評估個人的健康狀況。The routine human blood test is an important indicator in the evaluation of personal health. Analytical instruments such as flow cytometry are applied to count the numbers of erythrocytes, leukocytes, and platelets, and the numbers all carry their own significance in the differential diagnosis. However, we usually examine blood status by using invasive procedures such as blood sampling, which not only burdens patients, but also lead to the deterioration of the specimens during the delivery process, and may cause errors in health evaluations. With medical technology advances, many experimental non-invasive biomedical imaging technology methods have been developed. By using an interferometer, near infrared light, and the interference principle to image the biological tissue. Optical Coherence Tomography provides two-dimensional tomographic images of in vivo biological tissue with micron grade resolution. By using the pinhole to block unfocused light and the interference of scattering light, confocal microscopy provides biopsy images with sub-micron level resolution, Use of high tissue penetration depth of infrared light, these techniques can get the blood cell imaging in vivo without calibration.. Owing to the restriction of light diffraction limitations and tissue scattering, confocal microscopy cannot provide sub-micron resolution of clear images with tissues in depth; therefore, Lack of ability to identify various types of blood cells. Comparing harmonic generation microscopy to other optical tomographic microscopies, it is characterized by sub-micron three-dimensional resolution with tissues in depth. The research has been verified that can get human flow of blood cell images and interpret of the number of leukocytes in vivo, it is currently the most potential to develop into a non-invasive in vivo imaging flow cytometer technology. In addition, there is also no need to use dye during the examination. When there is any doubt of the percent composition of blood cells with flow cytometry, the patient’s blood specimen should be sent for further blood smear, which is where cells are stained to investigate the blood cell morphology. By using third harmonic generation microscopy (THG), there is no need to stain the blood cells before investigating while in the meantime the images can be saved. This method not only saves the examination time of the blood smear, but also presents the original morphology of the blood cells. To provide a stable light source for third harmonic generation microscopy, a 1150 nm femtosecond fiber laser system was built in our laboratory that is relatively insensitive, with temperature and humidity that are comparable to the Ti-Sapphire laser and Chromium-Doped Forsterite Laser. Four modes of analog signals are provided in the microscopy system, including second harmonic generation microscopy, third harmonic generation microscopy, multi-photon fluorescence microscopy, and confocal single-photon reflection microscopy. Today’s capture board that transform analog to digital cannot process the four types of the signal directly, and there is depletion phenomenon that takes place in the DC signals while transforming between the two signal types, causing a weakening of the signal amplitude. On the part of image capture, the pulse-repetition rate of laser light source is only 11.25 MHz. When shifting the focal point of the laser through fast-steering tilt-axis scan mirrors, (8 kHz) intervals time between points are larger. Thus, it is essential to capture the maximum of the every signal point, and to prevent weaker signal capture, which will cause reductions in image intensity. Therefore, to get high resolution and immediate investigations of the blood cells status, in respect to hardware, the norms of the microscopy system must be compatible with optical signals to preserve the DC signals. With respect to software, the analog signals of the specimens stimulated by the laser must be captured with the help of the synchronization of the laser signals to the get maximum level of analog signal to improve the contrast of the image. In this thesis, we use field-programmable gate array (FPGA) design imaging acquisition system. FPGA not only reduce development time, but also has high efficiency and reliability. By using the Phase-locked loops (PLL) in FPGA, we synchronized the sampling clock with the laser pulse, and designed to meet this FPGA board of the analog to digital converter board (ADCB). In addition, we programmed a graphical user interface with multi-channel 15Hz frame rate windows to display and restore the image. This interface can also be immediate changed FPGA parameters, such as imaging range, sampling clock frequency and laser pulse phase function. At the same time, we also provided another capture mode (XYT mode) for long video-recording that sets the video intervals flexibility. It can save single images or multiple images taken at certain time intervals over an average period, thus reducing the data size of the images saved. It can also provide basic image processing functions. Synchronized the sampling clock with the laser pulse and used our analog to digital converter board, we can clearly observe images of blood cells in mouse ear capillaries, Thereby reducing the difficulty of automated image interpretation. We hope that for future clinical applications, the non-invasive automatic evaluation of speed and number of blood cells using the fiber femtosecond laser microscope system could be faster and more precise using this synchronous acquisition system.9255819 bytesapplication/pdf論文公開時間: 2016/8/26論文使用權限: 同意有償授權(權利金給回饋學校)類比數位轉換板現場可編成邏輯閘陣列微軟基礎類庫ADC boardFPGAMFC[SDGs]SDG3thesis10.6342/NTU201602088http://ntur.lib.ntu.edu.tw/bitstream/246246/277396/1/ntu-105-R03548023-1.pdf