多層暨波長選擇性體積全像顯微鏡

Abstract

傳統的光學顯微鏡在一次觀測中只能獲得單層焦平面的影像，目前有數種光學系統被提出來以獲得不同深度的影像，然而這些系統中大多需要使用機械(Mechanical)或電光(Electro-Optic)的軸向掃描機構才能獲得多層深度的圖像。 本論文使用多路複用體積全像光柵(Multiplexed Volume Holographic Gratings, MVHGs)以及非對稱照明(Asymmetric Lllumination)為基礎，提出多層暨光譜選擇性體積全像顯微鏡(Multi-depth and Wavelength Selective Volume Holographic Microscopy)。 本系統使用寬帶照明不需要透過軸向掃描在單次拍攝中就能獲得多層深度的影像，而針對具有弱相位特徵(Weak Phase Features)的透明樣本，本論文使用薄膜電晶體面板(Thin Film Transistor panel, TFT-panel)以及聚光器(Condenser)在非相干光源的情況下產生非對稱的結構光，當相機取得兩張不同方向的非對稱照明影像後，系統透過Matlab數學軟體的計算就能直接獲得多層深度的DPC影像。為了要減少單層DPC影像的擷取時間，本研究利用波長編碼體積全像光柵(Wavelength Coded Volume Holographic Gratings, WC-VHGs)的獨特性質，開發出具有光譜選擇性的波長編碼微分相位差體積全像顯微鏡(WC-VHGs based DPC-VHM)，有助於系統在單次拍攝就可以直接獲得兩種顏色的非對稱照明圖像，以減少單層DPC成像時間。 波長編碼體積全像光柵能提供良好光譜選擇的特性，本論文也開發出波長編碼體積全像光柵螢光顯微鏡(WC-VHGs based Fluorescence Microscopy)，螢光影像可以提供非活體細胞較多的細節，而此系統能夠同時觀察來自組織樣本內不同特徵的多色螢光圖像，以提供一對不重疊的雙重波長螢光圖像。為了要改善系統的光學切片能力，本研究使用HiLo的影像處理技術將物體中非焦平面影像的訊號抑制以獲得光學切片的影像。 本研究首先為了驗證多路複用光柵的多焦段透鏡和角度選擇性的效果，將分辨率測試片、花粉粒以及玉米莖當作樣本，以在單次拍攝中直接獲得八層深度的影像，並測量多路複用光柵的衍射效率對角度的曲線，其中半峰全寬（FWHM）的範圍為0.03°至0.07°，而光柵之間的隔離係數範圍從7.1到16.7，顯示其具有足夠的角度選擇性。在驗證系統提升影像對比能力的部分，本論文觀察分辨率測試片和洋蔥皮的明場和微分相位差影像，實驗結果顯示微分相位差影像比起明場的影像，影像對比能夠提升2至3倍，此外透過測量系統的調製傳遞函數，能發現在各空間頻域下微分相位差比起明場影像都能提升影像的對比。在加快影像擷取速度的部分，本論文結合波長編碼體積全像片的技術，透過分辨率測試片的圖像以驗證單層的影像擷取速度能提升一倍。而本研究也使用螢光小球來測試波長編碼體積全像片的光譜選擇性，其能將黃色的螢光影像區分成綠色和紅色的螢光影像，最後藉由HiLo的影像處理技術能將非焦平面的螢光球訊號抑制達到光學切片效果。 結論，本研究所提出的多層暨光譜選擇性體積全像顯微鏡可以觀測非螢光和螢光的影像，在非螢光影像的部分，本系統不需軸向掃描能在單次拍攝中獲得多層深度影像，透過和非對稱照明技術的結合，系統也可以觀測多層的微分相位差影像，並減少單層微分相位差影像的擷取時間。在螢光的部分，本系統透過光譜選擇性可以提供一對不重疊的雙重波長螢光圖像，並結合HiLo技術提升光學切片能力。

Several optical microscopy systems have been proposed to obtain three-dimensional (3D) images for biomedical application. Nevertheless, most of these systems require mechanical or electro-optic axial scanning mechanism to construct multi-depth images. In this thesis we based on the multiplexed volume holographic gratings (MVHGs) and asymmetric illumination techniques to present the multi-depth and wavelength selective volume holographic microscopy. Under the broadband LED illumination, the system can obtain multi-depth images without axial scanning in one shot. For transparent weak phase objects, we thesis make a Thin Film Transistor (TFT) panel and condenser to generate the asymmetric illumination in different directions. After obtaining two different direction asymmetric illumination images, the system can use the MATLAB to directly acquire multi-depth Differential Phase Contrast (DPC) images. To further reduce the DPC image acquisition time, the thesis uses the properties of Wavelength Coded Volume Holographic Gratings (WC-VHGs) to consist the WC-VHGs based DPC-VHM that has the wavelength selectivity. The system is able to acquire two color images of an object corresponding to two different asymmetric illumination directions in a single shot to reduce one depth DPC imaging time. Furthermore in this study, we also develop the WC-VHGs based Fluorescence Microscopy, which can simultaneously observe multi-color fluorescence images from different features in tissue samples to provide a pair of dual wavelength images without overlap. To improve the optical sectioning ability of the system, the study utilize the HiLo imaging process to suppress the image out of focus signal. In the experimental results, first the study take the eight depths images of resolution test chart, pollen grains, and corn stems to verify the multi-focal lens ability of the MVHGs. To check the angular selectivity of each grating in MVHGs, the research measures the diffraction efficiency of the MVHGs that shows the full width at half maximum (FWHM) of diffraction efficiency and angle curve is 0.03° to 0.07°. In addition, the isolation coefficient between the gratings ranges from 7.1 to 16.7 that indicate the MVHGs has sufficient angular selectivity. To verify the system can enhance the image contrast, the thesis observe the bright-field and DPC images of the resolution test chart and onion skin samples. The image results show that the DPC images compare to the bright field images, the images contrast can be improved by 2 to 3 times. Through the modulation transfer function analysis that suggests the DPC-VHM can enhances the images contrast in most frequency. Final, the study utilizes the microfluorescent beads to test the wavelength selectivity and optical sectioning ability of the system. In conclusion, the proposed system enables to obtain multi-depth images without axial scanning, and it can combine with the asymmetric illumination technique to obtain the DPC images. Furthermore, the system can provide a dual-wavelength fluorescence images without overlap and combine HiLo technique to enhance optical sectioning capabilities.