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博碩士論文 etd-0721112-150411 詳細資訊
Title page for etd-0721112-150411
論文名稱
Title
暗視野架構下利用光色相差效應於微管道內快速偵測流動樣本深度之應用
Rapid Detection of Flowing Objects in Microchannel Utilizing the Chromatic Aberration Effect under a Dark-field Illumination Scheme
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
83
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
2012-07-09
繳交日期
Date of Submission
2012-07-21
關鍵字
Keywords
動態深度測量、色散效應、暗視野照明、微流道系統、微流式細胞儀
Dynamical depth detection, Dark-field illumination, Chromatic aberration, Microfluidic channel, Micro flow cytometer
統計
Statistics
本論文已被瀏覽 5681 次,被下載 1349
The thesis/dissertation has been browsed 5681 times, has been downloaded 1349 times.
中文摘要
本系統旨為發展一利用色相差機制進行Z軸方向深度偵測系統。近年微全分析系統(Micro-Total-Analysis-Systems, μ-TAS)快速發展,利用微流道整合多種微機械系統之晶片大為盛行,其中微流道內流體行為甚為重要,尤其是管道內流動之固體樣本行為,更高度引發研究者的興趣。傳統檢測方法多利用流場可視化技術,此種方式對於X-Y平面之檢測非常有效,但對於流動樣本之Z軸方向行為卻無法進行有效評估。部分研究利用數值模擬的方法進行預測,或使用外部機械(如自動掃描平台等)進行管道內之Z軸成像掃描,但此方式之偵測速度較慢,無法適用於高流速流動物體之Z軸深度偵測。
本研究利用一典型物理光學:色相差效應,結合一物鏡式暗視野顯微鏡系統,當流動物體通過色相差所成的偵測區域,透過分析其特定散射光波長的強度差異,即可解析微流動樣本之Z軸方向位置。色相差(chromatic aberration)主要發生於光穿越不同折射系數的介質時,因不同頻率的光束具有不同的折射係數,因此各個光波長將聚焦於特定深度位置,而造成不同Z軸深度物體會散射相對應的波長。利用此種特性,可對於流動物體進行軸向深度的檢測。為驗證此一想法,本研究初步利用具備連續波長光譜的氙氣燈作為光源,並使用低阿貝數(Abbe number)之壓克力(PMMA)透鏡作為色散插件。透過正規化光訊號後,建立其零位面,並選取450 nm及670 nm兩波長訊號作為散射光訊號中藍光以及紅光區域的消長指標,並進行運算得到深度以及其短、長波長強度比例關係,並以此建立波長強度比與深度位置之檢量線。於動態偵測中,將偵測物鏡定於零位面,利用光譜儀記錄流動樣本之散射光譜,並對照該檢量資料線即可快速獲得樣本於微管道內之深度。
實驗成果顯示,使用20X偵測物鏡,並以PMMA為色散插件透鏡時,適用樣本尺寸為20~5 μm,且其線性偵測範圍為±15 μm(R square=0.9779);使用高阿貝數透鏡BK7時,因色相差程度較低,所以可得到較高的尺寸解析度,並可適用於較小的粒子尺寸(2~5 μm),及較小的深度偵測範圍(約±10 μm)。由於光譜儀之光敏感度較低,致使可量測之粒子流動速率受限,實驗結果顯示使用光譜儀作為光偵測端系統,可量測流速小於0.5 mm/s粒子之深度。本研究為偵測更高流速之粒子,系統改以兩支雪崩式光電二極體作為偵測端,配合一低通濾鏡(CWL=450 nm, ±20 nm)及一高通濾鏡(CWL=650 nm, ±20 nm),並最佳化光檔插件後,建構一新光偵測系統,以提高可偵測之粒子速度。量測結果顯示,該系統可成功偵測流速為4.167 mm/s之流動樣本深度,且偵測範圍增大至±25 μm (R square=0.9929),且解析能力(throughput)可達126 particles/s。此系統並成功解析於流速約為2.778 mm/s環境下,人類紅血球深度位置之測定,其可偵測流速約為一般micro-PIV對紅血球進行偵測的5-8倍,證實此系統確實可運用於生醫應用範疇。
本篇研究利用普遍在光學系統中被視為是缺陷之色相差,將其轉化成適用於Z軸微距改變量偵測之優勢系統。其可運用於對微機構或流道構造之性能評估、微管道內粒子流動Z軸位置動態分析,並可應用於生物細胞、組織增生量測等定點靜態偵測,本研究所發展之創新光學系統將提供未來生物醫學及微流體系統研究更廣泛之應用。
Abstract
This research mainly develops a new z-position measurement based on the chromatic aberration effect. An objective-type dark-field illumination scheme is built to produce diascopic chromatic aberration light, and aimed to enhance the signal-to-noise ratio. The xenon lamp is adapted to create white light with continuous spectrum, besides, lens with low Abbe number is needed to extend the degree of chromatic aberration, so lens made of PMMA is as a chromatic aberration component. In the proposed system, the depths of samples in micro-channel is illuminated by the dispersed light and scatter the optical signals, which are captured by a low numerical aperture (N.A.) objective lens. After the simple normalization, the intensity ratio of two selected wavelengths 450 nm (blue light) and 670 nm (red light) from the scattered spectrum becomes a reliable index for the depth information of the detecting objects. By means of establishing the relationship between depth and intensity ratio, every object flowing through diagnosed spot is able to be determined the depth level by cross-referencing the database. By using spectrometer as detector, delicate moving components for light filtering or electrical stage for light scanning can be excluded for high-speed z-position detection. Furthermore, in order to identify the depth level of sample with high flowing rate, avalanche photodiodes are adapted to achieve rapid detection.
The experimental results show that the relationship between depth and intensity ratio is a parabola curve, but in this research, the region which tends to behavior linearly is adapted. The proposed system provides a linear detection range of ±15 μm for particles with a diameter of 20 μm. The lens with high Abbe number only obtains ±10 μm with linear detection range though, the resolution for size is better than PMMA. The BK7 lens is capable to discriminate the depth change of 2 μm micro-beads, note that there is no limitation of depth discrimination in this system, because of the measurement is achieved by cross-referencing the linear line. The use of UV-Vis-NIR spectrometer enable this system to analyze the depths of the samples in flow rate 0.5 mm/s. To gain the higher performance, the two avalanche photodiodes are utilized, and the short(CWL=450 nm, ±20 nm) and long(CWL=650 nm, ±20 nm) band pass filter are also equipped to represent enhancements of blue and red ray. The effective detection range extends to ±25 μm and has high linearity(R square=0.99285) after the optimization of light stop. In high flowing rate detection, this system is able to identify the depth of sample when the flow velocity is 4.167 mm/s, the calculated throughput is 126 particles/s. It also successfully analyzes the depth of flowing human erythrocytes under the flow velocity is 2.778 mm/s, the velocity which the developed system is capable to analyze is about 5-8 folds to the conventional micro-PIV system.
With this novel and simple approach, there will be the quantified information from z-direction of flowing body for bio-analysis, and also benefits estimating the performance of micro structure or device in the microfluidic chip, also the analysis of flow field. Except for dynamical detection, this system also be capable to apply in a open and static situation, such as cell or tissue proliferation assay.
目次 Table of Contents
中文摘要…………………………………………………………….…… i
英文摘要………………………………………..……………………………... iii
目錄………………………………………………………….…………….…. vi
圖次………………………………………..……………………………..….... viii
表次………………………………………..…………………….……….... x
符號表……………………………………..………………….………..…….. xi
縮寫表……………………………………..…………………….…………… xii
第 一 章 緒論………………………………………………………………. 1
1-1研究動機…………………………………………………………….. 2
1-2研究目標……………………………………………………….……. 3
1-3論文架構……………………………………………………….……. 4
第 二 章 文獻回顧…………………………………………………………. 5
2-1背景知識…………………………………………………………….. 5
2-1-1流式細胞儀偵測方法…………………………...……………. 5
2-1-2暗視野照明…………………………...………………………. 8
2-1-3色相差原理…………………………...………………………. 9
2-2相關文獻…………………………...………………..………………... 13
2-2-1 PIV系統於偵測微管道內粒子行為應用…...……………… 13
2-2-2色相差於深度探測應用…………………………...………… 19
第 二 章 實驗架構以及偵測方法…………………….…..………………. 22
3-1 實驗架構…………………………………………………………… 22
3-1-1色相差深度偵測設計概念……………………….…………. 22
3-1-2基本實驗架構……………………….………………………. 23
3-1-3晶片製程………………………………………….…………. 26
3-1-4透鏡的材料選擇以及色散能力評估……………….………. 28
3-2 偵測方法……………………………………………….…………... 31
第 四 章 實驗結果與討論…………………………………….…………... 38
4-1光譜儀於動態偵測的使用……………………………..…………… 38
4-1-1系統對樣本尺寸解析能力…………………………………... 38
4-1-2動態樣本之深度位置偵測……………………….………….. 41
4-2動態樣本之深度位置偵測……………………….………….……... 42
4-2-1高速深度偵測系統架設及校正……………………………... 42
4-2-2快速深度位置偵測…………...………………….…………... 46
第 五 章 結論與未來展望……………………….………………….…….. 52
5-1結論及綜合討論…………………………………..…….…………. 52
5-2未來展望………………………………………………..………..…. 53
參考文獻…………………………………….…………………….……….…. 57
附錄…………………………………….…………………….……….…. 63
作者自傳…………………………………….…………………….……….…. 66

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