具共光程移相干涉術之表面電漿共振顯微儀(A common-path phase-shift interferometry surface plasmon resonance microscope)蘇園登，陳顯禎*國立成功大學工程科學系 台南市701大學路一號Y.-T. Su and S.-J. Chen*Department of Engineering Science, National Cheng Kung University, Tainan 701, Taiwan.*Tel: +886-6-2757575 ext. 63351; Fax: +886-6-2766549; E-mail: sheanjenmail.ncku.edu.tw.摘要表面電漿共振(surface plasmon resonance，SPR)顯微儀可即時且靈敏地量測於界面間所造成之厚度與介電常數微量變化，並可獲得空間量測資訊。本系統為一共光程(common-path)移相干涉術(phase-shift interferometry，PSI)之相位量測設計，由於P偏振入射光才可激發表面電漿波(surface plasmon wave，SPW)形成共振現象，而且SPR現象會隨界面條件微量改變而造成反射相位驟變，同時利用S偏振光相位保持不變為參考光，將兩者形成干涉圖案，利用電光調變器(electro-optical modulation)來造成相位移干涉，並經由相位重建方法取得空間相位之變化情形。此共光程相位技術可減低外界環境擾動、機械振動與雷射不穩定等因素，提供長時間穩定之相位量測(於兩個小時內可達10-3之穩定度)，目前此系統可精密偵測到1x10-6 RIU折射率之變化，其空間側向解析度(lateral resolution)約10m左右。AbstractA common-path surface plasmon resonance (SPR) phase-shift interferometry (PSI) imaging system is proposed based on the integration of SPR and common-path PSI techniques to measure the 2-D spatial phase variation caused by biomolecular interactions upon a sensing chip. The common-path SPR-PSI imaging system offers high resolution, high-throughout screening, and long-term stability capabilities to analyze microarray biomolecular interaction without the need for additional labeling, and provides valuable real-time quantitative information. Currently, the common-path PSI technique by means of a five-step phase reconstruction method with a liquid crystal device (LCD) can provide an extreme long-term stability (這段有點像廣告文字，若投稿的目標非常technical, 要不要換成中文版 說明為什麼 會有這樣好的特性, 的那一段) even with external disturbances such as mechanical vibration, buffer flow noise, and laser unstable issue to match the demand of real-time kinetic study in drug discovery. The SPR-PSI imaging system has achieved a detection limit of 1 10-6 refraction index change, a long-term phase stability of 10-3 p in over 120 minutes, and a spatial phase resolution of 2 x 10-3 p with a lateral resolution of 50m.一、前言 1. INTRODUCTION表面電漿共振(surface plasmon resonance，SPR)感測器可量測在固體與液體或固體與氣體界面之奈米膜層之介電常數或厚度的微量變化1,2。由於SPR技術具有快速地、靈敏度高、動態分析、無須對待測分子做任何的標記及定性和定量生物分子交互作用之優點，自1992年來便被廣泛地應用在生物分子診斷或奈米膜層分析的領域上3。然傳統SPR技術主要為單點或區域性檢測，要在未來取代其他檢測技術，不只應具有方便性、經濟性、高速化之感測器與系統之外，更應往具超高靈敏度、高解析與高可靠性之影像系統來發展，使之用於生物分子診斷上提供大量平行篩檢資訊，並於奈米膜層空間分析上提供顯微影像資訊3-10，提供一於奈米尺度高靈敏與高解析的量測平台。Surface plasmon resonance (SPR) sensors can be applied to measure slight variations in dielectric constant or thickness of nanometer membrane at the interface of solid and liquid or solid and gas 1,2. Applying SPR for detection, there is no need for labeled molecules and it is suitable for both qualitative and quantitative analysis on bio-molecular interactions. The process is also prompt, of high sensitivity and good for real-time analysis. It has been widely applied to bio-molecular diagnosis and nanometer membrane examinations 3. SPR technology has been applied to localized examination. Therefore, in addition to convenience, economy, fast speed, developing SPR technology for high sensitivity, high resolution, and reliable microscopic imaging would make vast parallel bio-molecular screening possible 3-10.傳統SPR影像系統主要以在入射角度於SPR角附近時，以一平行準直的單色光源照射有金膜與樣品組合的耦合稜鏡或光柵上，經由一光學影像系統處理後，由CCD偵測器取得SPR影像資料。這種系統雖然具備大量平行篩檢能力，然其偵測解析度卻大大被降低，難以偵測於低濃度下，較低分子量的分析物。根據理論及文獻上的研究分析，SPR感測器在各種耦合及量測方式對於檢測物之折射率解析度以菱鏡藕合方式量測SPR之相位變化具有最高之靈敏度3，其量測方法是利用信號光相位與參考光相位干涉比較反應前後，相位改變情形分析而得。因此以量測SPR相位之影像系統可以獲得比傳統影像系統更高之偵測解析度。本文的主要目的是針對表面電漿共振所造成之高靈敏相位變化，開發用於分析奈米膜層於空間上之介電常數或厚度的微量變化情形並可用於高通量即時動態量測生物分子之間的交互作用平行檢測之表面電漿共振干涉影像系統，由於共光程(common-path) 干涉技術的特性，可於長時間的量測下減少外界環境擾動、機械振動與光源不穩定造成相位飄移之因素，獲得一穩定之相位資訊。本嶄新系統結合表面電漿共振、共光程移相干涉術(phase-shift interferometry，PSI)來完成於空間平面上之相位量測。Conventionally, SPR imaging system applies a parallel monochromatic light beam incident on the sample and gold membrane compound at the coupling prism or grating interface. The angle of incidence is close to the SPR resonance angle and the SPR interference pattern is captured by a CCD detector. Such system has massive parallel screening power, however, its resolution is low that it is difficult to detect samples of low molecular weight and low concentration. Comparing SPR detector of various configurations, applying prism couple to produce interference between SPR phase shift with respect to the reference light beam produces the best sensitivity 3. Therefore, we developed a SPR interference imaging system with common optical paths for high through put real-time dynamic measurement of bio-molecular interaction causing slight variation in the dielectric constant or thickness of a nanometer membrane. Since signal and reference beams pass through a common optical path, the phase shift drifts due to environmental change, mechanical vibration, and light source fluctuation are common in the two beams and would cancel each other. Therefore, we can achieve a spatial phase shift measurement system combining SPR and common path phase-shift interferometry (PSI) for long term stabilit