傳統的遠場光學顯微技術受限於光學的繞射極限(diffraction limit)，於軸向與側向空間解析度均無法達到小於二分之一波長的解析度，為了突破光學的繞射極限的限制，因此發展近場光學量測技術。雖然孔徑式近場掃描式光學顯微鏡(near-field scanning optical microscope，NSOM)可突破光學繞射極限，但解析度仍侷限在50 nm左右。為了達到更佳的空間解析度，本論文試圖發展一無孔式近場掃描式光學顯微鏡(apertureless near-field scanning optical microscope，aNSOM)，並主要將其應用於螢光樣品的影像量測。一般無孔式近場螢光偵測技術主要有兩個問題必須克服，一個為金屬探針所造成的螢光淬滅現象，另一個為遠場的螢光所造成的背景雜訊。為了克服這些問題，利用矽探針來避免螢光淬滅現象的發生，並以雙光子螢光激發技術來減少因遠場螢光信號所造成的背景雜訊。論文以商用之原子力顯微鏡(atomic force microscope，AFM)為基礎，並搭配矽材探針、光學、機械系統設計及資料擷取卡中的FPGA模組為控制系統的核心，建立一探針強化式雙光子螢光顯微鏡(tip-enhanced two-photon excited fluorescence microscopy)，期望能將空間解析度進一步提升。此外，針對近場螢光影像作相關之討論與量測，並分析探針與螢光分子的交互作用及螢光訊號的擷取，期望未來能將本系統應用在於分子生物領域上，得到分子尺度的螢光影像。

Due to the restriction of the diffraction limit in conventional optical microscopes, the axial and lateral spatial resolutions are hard to be achieved to a nanometer scale. Although an aperture near-field scanning optical microscope (NSOM) has been developed for breaking the diffraction limit, the spatial resolution is still limited in the range of 50 nm. To improve the current lateral spatial resolution, an apertureless near-field scanning optical microscope (aNSOM) has been developed. Furthermore, the aNSOM is applied to measure near-field fluorescence signal. The two major drawbacks of the near-field fluorescence detection which are fluorescence quenching by the metallic tip and background signals from the far-field fluorescence signal. Silicon tips and two-photon excitation allow us for quenching free detection and background suppression, respectively. In this thesis, a tip-enhanced two-photon excited fluorescence microscopy based on a commercial atomic force microscope (AFM) combined with commercially silicon tip, optical, mechanical and the FPGA module in data acquisition card of the control system has been developed to attempt to achieve a better spatial resolution. To test the performance of the system, a near-field fluorescent image from fluorescence molecules is grabbed. The interaction between the fluorescence signal and the tip is studied to assist how to obtain the fluorescent signal more efficiency. Eventually, the system could play a key role in biomolecular field, and provide fluorescence images with a molecule-scale resolution.

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