本論文中設計一款激發波長可調式的時域聚焦雙光子激發顯微鏡，利用掃描鏡、透鏡與光柵的搭配而實現激發波長可調的範圍為730至1000 nm，並使用數位微鏡裝置 (Digital Micromirror Device, DMD) 取代光柵以將單像素成像 (Single-Pixel Imaging, SPI) 引入系統，在實驗中透過模擬優化激發光源入射DMD的角度，使得全激發波長可以依照相同激發光路行進並達到最佳繞射效率。藉由此系統與光譜解析系統的結合，螢光染料分子都可使用其最佳激發波長進行激發，使得有最大雙光子激發效率，並從而獲取螢光光譜與光譜解析影像。在多光子激發螢光顯微術系統中，可以從多色螢光細胞中解析光譜影像，其中系統的光譜解析度可達到0.45 nm，且也利用螢光染料在不同環境中最大放射波長擁有偏移的特性，來量測螢光波長偏移量與溶液環境的關係。另也建構單像素成像之拉曼光譜系統結合，來量測循環腫瘤DNA (circulating tumor DNA, ctDNA) 之表面增強拉曼散射 (surface-enhanced Raman scattering, SERS) 影像，藉由此系統可以取得拉曼光譜並且解析各拉曼峰的光譜影像，其中系統的光譜解析度可達到1.41 cm-1，並藉由ctDNA於SERS基板上的拉曼訊號來分析SERS效應之空間分佈情形，以證實SERS基板的均勻增益特性。;In this thesis, we designed a temporal focusing two-photon excitation microscope with tunable excitation wavelength. The combination of scanning mirror, lenses, and grating realized the tunable excitation wavelength in the range of 730 to 1000 nm. A digital micro-mirror device (DMD) was adopted to replace the diffraction grating in the conventional temporal focusing two-photon excitation microscope for implementing single-pixel imaging (SPI) approach in the presented system. The angle of excitation light at the DMD was optimized according to the simulation result, so that the excitation light for the wavelength from 730 to 1000 nm can have the same excitation light path and achieve the optimal diffraction efficiency. All fluorescent molecules can be excited by their optimal excitation wavelength to have the maximum two-photon excitation efficiency. By combined with the spectrally-resolved detection system, fluorescence spectra and spectrally-resolved images of the specimens can be obtained. Spectrally-resolved images of multifluorophore-labeled cells through the multiphoton excitation fluorescence microscopy system demonstrated that the spectral resolution of the system could reach 0.45 nm. Moreover, the variation in the emission spectrum of fluorescent molecules in different environments was used to determine the relationship between the maximum emission wavelength and solvent environment. In addition, a single-pixel imaging Raman spectroscopy system was also constructed to measure the SERS images of circulating tumor DNA (ctDNA), through which the Raman spectrum and spectrally-resolved images of each Raman peak could be obtained with a spectral resolution of 1.41 cm-1. Finally, the spatial distribution of SERS effect was analyzed by the Raman signals of ctDNA on the SERS substrate to verify the uniform gain characteristics of SERS substrate.
