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博碩士論文 etd-0214107-125020 詳細資訊
Title page for etd-0214107-125020
論文名稱
Title
應用多重粒子追蹤功能及雷射光鉗技術之微流體自動細胞分類/計數系統
Microfluidic Cell Counter/Sorter Utilizing Laser Tweezers and Multiple Particle Tracing Technique
系所名稱
Department
畢業學年期
Year, semester
語文別
Language
學位類別
Degree
頁數
Number of pages
131
研究生
Author
指導教授
Advisor
召集委員
Convenor
口試委員
Advisory Committee
口試日期
Date of Exam
2007-01-20
繳交日期
Date of Submission
2007-02-14
關鍵字
Keywords
數位影像辨識、電滲透流、細胞分類、計數、光鉗、微流體
optical tweezers, electrokinetic focus, digital image processing, microparticle, microfluidics
統計
Statistics
本論文已被瀏覽 5622 次,被下載 2474
The thesis/dissertation has been browsed 5622 times, has been downloaded 2474 times.
中文摘要
本研究為一整合型細胞/粒子分類及計數之生醫光電系統,該系統整合即時多重粒子數位影像擷取及辨識系統,並利用紅外光光鉗控制及電驅動式微流體晶片進行粒子補捉及分類。此研究提出利用影像控制光鉗之自動控制系統,其將藉由自行撰寫之數位影像辨識及追蹤軟體對微流道之粒子進行即時影像特徵之辨識及粒子計數。與傳統影像辨識系統之單一粒子辨識不同處在於本研究將同時對流道內之多個粒子進行影像辨識,並同時追蹤其流動軌跡來計算其行進速度,並藉由電腦判斷光鉗是否作動,及其作動之精確時機以及作動之時間長短,因而可以對微流道中之粒子進行連續式偵測、計數及分類。
本實驗所開發之微流體細胞分類器,其製作之微管道晶片皆採用成本低廉之鈉玻璃製程,利用電滲透流在十字管道內聚焦並驅動粒子,以數位攝影機,捕捉微流體管道內粒子流動之即時影像,配合實驗室開發之影像辨識軟體來對粒子進行判讀,並利用雷射光鉗改變目標粒子流線,達到分離粒子之效果。結果顯示,光鉗可成功的在流速為300 μm/sec的5 μm與10 μm之混合粒子流中,經由影像辨識系統,成功辨識並啟動光鉗,使5 μm以及10 μm之微粒子產生約20 μm之側向位移,流向後端收集槽儲存,達成分離與計數。
本研究所提之創新方法,係一非接觸式之微粒子操控法,其可容易地被運用於樣品中特殊致病性細胞之捕捉、分類及計數,並且免除接觸式操作可能發生之交互感染,並因此可解決微全程分析系統領域內的細胞/粒子操控問題。
Abstract
This study proposes a novel microfluidic system based on a computer controlled digital image processing (DIP) technique and optical tweezers for automatic cell/microparticle recognition, counting and sorting in a continuous flow environment. In the proposed system, the cells/microparticles are focused electrokinetically into a narrow sample stream and are then driven through the region of interest (ROI), where they are recognized and traced in real time using a proprietary DIP system. Synchronized control signals generated by the DIP system are then used to actuate a focused IR laser beam to displace the target cells from the main sample stream into a neighboring sheath flow, which carries them to a downstream collection channel where they are automatically counted. The proposed approach makes possible the continuous sorting and counting of cell samples without the need for any moving parts or embedded transducers. The experimental results show that the proposed system is capable of sorting 5 μm or 10 μm PS bead from a mixture of 5 μm and 10 μm samples in the flow speed 300 μm/sec. The proposed system provides a simple, low-cost, high-performance solution for cell manipulation in microfluidic devices.
目次 Table of Contents
目 錄 I
圖目錄 V
表目錄 IX
中文摘要 X
Abstract XII

第一章 緒論 1
1.1 前言 1
1.2 研究動機及目的 2
1.3 文獻回顧 4
1.3.1 粒子計數器 5
1.3.1.1柯爾特粒子計數器(Coulter particle counters, CPC) 5
1.3.1.2 微型光學偵測裝置之粒子計數器 7
1.3.2 流式細胞偵測技術 8
1.3.3 整合型微流體細胞分類系統 11
1.4 本文架構 13
第二章 全自動分類系統之基礎理論 15
2.1 影像擷取理論 15
2.1.1影像擷取系統組成 15
2.1.2 影像運算與影像比對法則及原理 16
2.1.3 常規化相關匹配法(Normalized Cross Correlation,NCC) 17
2.2 光鉗之基本原理 19
2.3 微管道液態流體系統 24
2.3.1 電滲透流聚焦 25
第三章 全自動細胞分類控制系統整合與實驗架設 27
3.1視覺系統軟硬體架構 31
3.1.1 視覺系統硬體架構 31
3.1.2 視覺系統影像辨識演算法 32
3.1.3 多重質點細胞影像辨識 35
3.1.4 細胞質點移動位置及速度之計算及應用 38
3.1.5 細胞質點即時粒子計數之概念及其方法 41
3.2 PC-Based電腦控制及自動分類系統人機介面操作 43
3.2.1 PC-Based電腦控制系統 43
3.2.2 自動分類系統人機介面操作說明 44
3.3 雷射光鉗光學系統 46
3.4 微流體晶片系統 47
3.4.1 微流體晶片設計 48
3.4.2 微流體晶片製程 51
3.4.3晶片測試 53
第四章 結果與討論 55
4.1 微流體樣本流聚焦情形之數值分析與實際實驗結果圖 55
4.2 雷射光鉗有效捕捉力與粒子位移關係之實驗量測結果 57
4.3 樣本流聚焦寛度之控制 58
4.4 即時辨識分類操作畫面 59
4.5 影像即時辨識處理 60
4.6 不同目標粒子之混合樣品流分類結果 62
4.7 使用軟體偵測方式之粒子計數器 66
4.8 軟體計數器之性能分析 69
第五章 結論與未來展望 73
5.1 結論 73
5.2 未來展望 73
參考文獻 75
Appendix A. Image Capture Sub-Program 78
Appendix B. Image Display Sub-Program and Image Recognition Sub-Program 81
B.1 Program Initial 82
B.2 Image Display and Main Recognition Function 95
B.3 White Particle Software Counter Sub-Function 101
B.4 Black Particle Software Counter Sub-Function 103
B.5 Laser-Output Shutter Control Sub-Function 104
B.6 Laser-Closed Shutter Control Sub-Function 105
B.7 RS 232 CommPort Available Check Sub-Function 105
B.8 Miscellaneous 106
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