- 學位論文
顯微造影技術在裸鼠微循環血管之血流速分析研究
Microscopic Imaging Technique for Blood Flow Velocity Analysis on Microcirculation Vessels of Nude Mice
摘要
微循環研究主要是瞭解微循環之機能表現，而透過觀察局部組織中微血管的血流速為其中一種重要途徑。為能研究局部的微血管之血流速，本研究提出一種以影像處理為基礎的血流量測方法，量測裸鼠之腫瘤成長過程中的表面血流速，顯微照影主要是利用型號M320之顯微鏡進行記錄，其空間解析度為1.42μm及影像取樣率為每秒30張影像，以獲得微循環血流之動態影像資訊。 在影像處理方面，為能解決因規律心跳所造成的晃動問題，以交互資訊之影像對位法可得到穩定之血流影像。然而，通常在分析血流影片過程中，需要處理大量的影像資訊，而影像對位為其中不可缺且耗時之處理步驟，因此為能獲得穩定的血流分析影像，以張對張的影像匹配之處理，以解決影像在獲得期間物體晃動的問題。在第一項研究中，針對現存一些影像匹配演算法進行評估，並結合以Powell最佳路徑搜尋法及低運算的自動影像對位演算法，同時透過模擬出不同程度的晃動影像，以驗證最佳的血管影像對位演算法，實際應用於血流影像對位時，以變異比率演算法有較佳的穩定性及準確性。 影像以張對張的處理也是一個常用的血流影像測速法，但其不同演算法的正確性比較卻很少被調查。因此第二項研究目的為評估現存的血流影像測速法，其中包含相互關聯法、霍夫轉換法及光流法，利用模擬出微循環血管各種不同流速變化特性之動態影像資訊，以評估量測速度演算法之準確性。根據實驗比較後，光流法比其他兩種演算法(相互關聯法及霍夫轉換法)較適合於量測血球的流動速度。 前兩項研究所獲得之結果應用於第三項研究，主要是以裸鼠腫瘤表面之血流為其觀察對象，觀察裸鼠植入腫瘤後，連續數週(1~ 5週)之成長及量測表面血流變化，由結果顯示，所量測的腫瘤樣本之微細血管及微靜脈之血流速介於40~350μm/s之間。而在腫瘤成長期間的微血管管徑與血流之差異比較，發現植入腫瘤前與腫瘤成長第二週時的血流速值具有統計意義(p<0.05)。本研究以影像分析微循環血流之技術，未來可應用於腫瘤發展之預測與機能評估，所獲得的多樣性活體小動物腫瘤之微循環血流資料，將作為腫瘤與血液生理相關領域之參考。
並列摘要
The study of microscopic imaging in microcirculation, by monitoring the blood flow information at each local micro-vessel, is important in understanding the amount of the blood present in local tissues. To investigate the speed of blood cell moving in each local micro-vessel, a new approach for measuring the microcirculation blood flow velocity at tumor surface of nude mice (SCID) as tumor growth based on image processing method has been developed. The blood flow images were acquired by M320 (JMC Corporation, Kyoto, Japan), with a special resolution of 1.42μm and an image sampling rate of 30 frames per second. Frame to frame image registration with mutual information feature matching was used to obtain stable images in order to solve the image movement caused by regular heartbeats. However, image registration of blood vessels is often a prerequisite and a time consuming step in blood flow analysis of large microscopic video sequence in vivo. In order to obtain stable images for blood flow analysis, frame-to-frame image matching as a preprocessing step is a solution to the problem of movement during image acquisition. In our first study, the performance properties of several matching metrics are evaluated through simulated image registrations. An automatic image registration program based on Powell’s optimization search method with low calculation redundancy was implemented. The matching method by variance of ratio is computationally efficient and improves the registration robustness and accuracy in practical application of microcirculation registration. Image processing for blood flow velocity measurement using a frame by frame analysis is a common approach. The accuracy of the calculations, which is algorithm dependant, has rarely been examined. In the second study, we evaluated the accuracy of the existing methods, which include cross correlation method, Hough transform method, and optical flow method, by applying these methods to simulated micro-vessel image sequences. Simulated experiments in various micro-vessels with random RBC motion were applied in the evaluation. The blood flow variation in the same micro-vessels with different RBC densities and velocities was considered in the simulations. The calculation accuracy of different flow patterns and vessel shapes were also examined respectively. Based on the comparison, the use of an optical flow method, which is superior to a cross-correlation method or a Hough transform method, is proposed for measuring RBC velocity. In the third study, the blood flow observation was focused on the tumor surface of nude mice. Multiple weekly data were acquired from each nude mouse for five weeks after tumor implantation. The averaged red blood cell velocities of capillaries were measured and calculated at different weeks for each tumor. The microvascular RBC velocity measurements in postcapillaries and venules in the surface of solid tumor were found that the distribution of blood flow velocity among all samples was ranged from 40 to 350 μm/s. The differences in vessel diameter and blood flow velocity during tumor growth were studied in comparison with the initial week. According to the obtained p value (p<0.05) of data, significant difference can be found between the velocity values at the initial week and the 2nd week. The results by using the presented imaging system and analysis method suggested an optical based process has the potential to develop new experimental protocols in numerical simulation of tumors growth. This measurement system may also be useful in many other biotechnological evaluations.