Dual-channel radially polarized surface plasmon microscopy for sensitive detection of fluorescent and non-fluorescent nano-objects

由於具有表面偵測高靈敏度的特性，至今已有許多關於表面電漿共振感測器的學術研究，並且已進駐到個人化醫療市場。其中影像式表面電漿共振顯微術具有直觀的優點，普遍使用稜鏡激發與陣列式的晶片來實現。然而在微觀上，此種架構的解析度並不高，所檢測到的陣列式區域也並非均勻分布，其中應包含更多的微觀信息有待仔細分析，而非只是區域性的選擇性統計結果。此外，雖然學術界正有許多超解析的顯微技術不斷被發表，但其整體系統還是偏向複雜化，整體製作費用也相當高。本研究將發揮表面電漿共振顯微術的優點，建置一套全新的顯微系統，好處是不僅可以偵測表面電漿強化後的螢光信號，更可以藉由表面電漿共振的吸收角度變化，以光學方式進階分析該待測物之奈米級物化特性。 針對此目的，我們提出並論證了一種雙通道徑向偏極化的表面電漿顯微術系統，首先應用在探測單一奈米粒子的特性。對於奈米螢光球，我們能夠同時收集螢光和表面電漿彈性散射兩種訊號。這兩種訊號構成的影像資訊可以互補。我們使用徑向偏極化入射光以及高數值孔徑的顯微物鏡，使所有入射角度的光聚焦至入射平面時皆為橫向磁場(transverse-magnetic)，從而在表面電漿彈性散射影像中得到一個黑色圓環，也改進了系統的分辨率和靈敏度。實驗發現，相較於線性偏極化光，此徑向偏極化光明顯提高了50%以上的螢光激發強度。 我們首先採用這種技術來探測直徑20奈米的單一螢光球體，它除了提供一種螢光訊號，同時亦有表面電漿彈性散射訊號。本系統也應用於量測非螢光物質，包括矽微米球、nano-KTP、LaPO4、CD-R的光柵結構等。針對生物性的樣本，我們選擇了DNA鏈和PC12細胞膜，結果證明本系統可在氣態或液態介面進行量測。此研究可繼續擴展到雙光子螢光顯微術，用以檢測螢光分子，以及二倍頻光學顯微術，用來檢測非對稱之奈米晶體。由於具有雙通道，可使用彈性散射的表面電漿共振反射影像，用來觀察螢光的閃爍效應。此外，金屬表面增強的螢光猝滅效應和量子點的發光特性也正在進一步研究中。Due to the advantage of surface sensitivity, various SPR biosensors for scientific research fields or personal medicine markets have been reported. However, especially for SPR imaging applications, the designs are usually based on prism-coupling method and ensuing chips with array patterns. In fact, these designs entail the disadvantages of a limited spatial resolution and non uniform detection regions. Although several super-resolution microscopes have been proposed and developed, systems are usually complicated and high-costs. In this thesis, we adopt the surface plasmon resonance technique to build a brand new imaging system. Alongside fluorescence, SPR absorption can also be exploited towards better imaging and understanding of the surface properties. Towards this aim, we demonstrate a dual-channel radially-polarized surface plasmon microscopy (SPM) system with capability down to single nanoparticle detection. For nanospheres stained with fluorescent molecules, we are able to simultaneously collect the fluorescence and elastic scattering images. These two complementary emitted signals lead to well co-localized images. The improved resolution and higher sensitivity of this system are enabled by use of a radial polarizer and a high numerical aperture objective. It provides TM-polarization status to the entire incident beam, which results in the formation of a dark circular ring in the reflected image. The fluorescence intensity is then clearly enhanced by more than 50% under radial polarization as compared to a linear one, while azimuthal polarization being fully TE is ineffective and serves as a reference. We first applied this technique to detect a single fluorescent sphere of 20 nm in diameter, which potentially reveals unique information as compared to other measurements on bulk films. Moreover, it also provides a way to compensate for the blinking characteristic of the fluorescence, which does not affect the elastic scattering channel. We are currently extending this technique to stained biological objects such as DNA strands and cell membranes in liquid environments. This technique has been extended to study two photon fluorescence (TPF) signals from organometallic nanospheres, as well as second harmonic generation (SHG) signals from non-centrosymmetric nanocrystals via a multiphoton confocal microscope. In relation with this research, metallic ion enhanced fluorescence and quenching effects from quantum dots are fundamental topics currently under investigation