利用全內反射式螢光顯微術(total internal reflection fluorescence microscopy，TIRFM)來觀測生化表面上的活體細胞不只能提供對細胞功能多一層的了解，更改善了影像的訊噪比(signal-to-noise ratio，SNR)。對於更動態的螢光分子影像而言，螢光訊號強度是必須被增強的。此論文提出一藉由奈米銀膜之表面電漿子(surface plasmons，SPs)來增強螢光訊號的表面電漿子增強螢光(surface plasmon enhanced fluorescence，SPEF)顯微術。實驗數據與模擬結果顯示相較於傳統的全內反射式螢光顯微術，表面電漿子增強螢光顯微術可獲得十倍螢光強化的活體細胞膜影像。但是單光子表面電漿子增強螢光顯微術沿傳播方向會漸漸失去侷限激發的特性，造成螢光成像為漸逝場(evanescent field)與散射光共同激發而後疊加所致。相較於單光子表面電漿子增強螢光顯微鏡，具較小激發體積與較低散射的雙光子表面電漿子增強螢光顯微鏡不僅可達成較高訊噪比的活體細胞膜螢光成像，更因可使螢光光子有較短的生命週期(lifetime)進而減少了光致漂白效應(photobleaching)並增加了光穩定性(photostability)。最後，此研究展示了廣視野(wide-field)物鏡式表面電漿子相位顯微術與表面電漿子增強螢光顯微術的首次結合，並用於同時觀測生化基材表面上活體細胞接觸區域的影像。整合移相干涉術(phase-shift interferometry)與共光程(common-path)光路設計之表面電漿子相位顯微術可提供具長時間穩定性的高靈敏相位資訊。同時，表面電漿子增強螢光顯微鏡可提供較亮的螢光影像。整合後的顯微鏡再透過厚度30 奈米的銀薄膜來完成高數值孔徑(numerical aperture)物鏡式的表面電漿子激發。此顯微鏡已成功發展並用於即時觀察轉染過含雙色胺酸功能區氧化還原酶(WOX1)/綠螢光蛋白(eGFP)基因的活體猴子腎臟纖維母細胞(COS-7 fibroblasts)的胞膜/基質接觸區域與同時取得此區域之高靈敏度相位影像。

Using a total internal reflection fluorescence microscopy (TIRFM) technique to image live cells on a biosurface not only provides an enhanced understanding of cellular functions, but also improves the signal-to-noise (SNR) ratio of the images. The intensity of the fluorescence signal must be increased if a more dynamic biomolecular imaging capability is required. Accordingly, this dissertation presents a surface plasmon enhanced fluorescence (SPEF) technique in which the fluorescence signals are enhanced via surface plasmons (SPs) offered by a silver nanolayer. The results demonstrate that live cell membrane images are brighter by approximately one order of magnitude than those provided by conventional TIRFM. However, the confinement of one-photon SPEF excitation is gradually loosed in its propagation direction, and therefore the biological fluorescent image is generated by the excitation of the superposition of an evanescent field and a scattered light component. In comparison with the one-photon SPEF, a two-photon SPEF can not only achieve higher SNR cell membrane imaging due to its smaller excitation volume and lower scattering, but also reduce photobleaching and increase photostability due to having a shorter fluorophore lifetime. Furthermore, the first combination for wide-field objective-based SPEF microscopy and surface plasmon phase microscopy has been developed to image living cells’ contacts on the surface of a bio-substrate simultaneously. The phase microscopy with a phase-shift interferometry and common-path optical setup can provide high-sensitivity phase information in long-term stability. Simultaneously, the SPEF imaging can supply bright fluorescent images. The combined microscope imposes a high numerical aperture oil-immersion objective upon the excitation of the SPs through a silver film with a thickness of 30 nm. The developed microscope is successfully applied to the real-time bright observation of living COS7 fibroblasts transfected with eGFP-WOX1 construct on the cell-substrate contact region and the high-sensitivity phase image of the cell-substrate contacts at the same time.

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