Optical clearing provides a useful means to enhance photon penetration into turbid tissues such as skins for optical examination. However, the integration of optical clearing with high-resolution (□m-level) microscopy for in-depth tissue imaging remains a field under development. In the present study, we apply an aqueous optical-clearing solution, FocusClearTM, on opaque protein gels of bovine serum albumin (BSA) and collagen to demonstrate its optical-clearing effect by transmittance quantification and fluorescence detection. Two commonly used optical-clearing reagents, glycerol and dimethyl sulfoxide (DMSO), were used for comparison. Significant increases in transmittance for both gels immersed in FocusClearTM were observed in visible wavelengths while there were fewer enhancements in glycerol and DMSO. Confocal images of fluorescent beads in BSA and fibroblasts in collagen gels showed that the application of FocusClearTM resulted in stronger fluorescence signal and better resolution in comparison to the use of glycerol or DMSO as the optical-clearing reagent. In conclusion, the application of FocusClearTM not only enhanced light transmittance but also increased the fluorescence intensity that formerly diminished due to the opacity of biological samples. The results demonstrated that the optical-clearing effect of FocusClearTM was superior to that of glycerol and DMSO in in-depth confocal imaging.

光學透明化對於混濁不易透光的生物組織，如皮膚，可有效提升光線於組織的穿透度，對組織於光學顯微鏡的觀察有所助益。目前，光學透明化結合高解析度(~μm)顯微鏡的使用以獲得組織深處影像的技術尚未發展完全。本研究中，我們將一新型光學透明化水溶液－FocusClearTM，分別應用於不透明的蛋白質膠體，牛血清蛋白及膠原蛋白，藉由透光度的定量及內部螢光的偵測來展現FocusClearTM光學透明化的效果。其他兩種常用的光學透明化溶液，glycerol與dimethyl sulfoxide(DMSO)也應用於實驗中作為比較的依據。於可見光範圍，FocusClearTM的應用使兩種蛋白質膠體透光度皆大幅提升，而glycerol與DMSO的效果則不如FocusClearTM顯著。利用共軛焦顯微鏡觀察牛血清蛋白膠體內部螢光小球及膠原蛋白內部螢光纖維母細胞的螢光影像，可發現隨著掃描的深度增加，FocusClearTM與Glycerol及DMSO相較起來，其應用可使影像維持較強的螢光訊號強度及較高的解析度。整體而言，FocusClearTM可使生物樣本透光度大幅提升，進而提升樣本內部反射回來的螢光訊號，使樣本深處之清晰螢光影像也可得。FocusClearTM光學透明化的效果優於常用的Glycerol與DMSO，於生物影像的應用十分具有潛力。

Abstract 2
中文摘要 4
謝誌 5
圖目錄 8
1. Introduction 9
1-1 Bioimaging 9
1-2 Introduction of confocal microscopy 10
1-2-2 Features of confocal microscope 11
1-2-3 Limitation of confocal microscope 12
1-3 Transmittance increase in biological tissues 13
1-3-1 Causes of biological tissue opacity 13
1-3-2 Optical-clearing mechanism of reagents 15
1-3-3 Common optical-clearing reagents 18
1-3-4 Introduction of FocusClearTM 24
1-4 Objective of research 25
2. Materials and Methods 27
2-1 Materials 27
2-1-1 Experimental materials 27
2-1-2 Experimental instruments 27
2-2 Quantification of the optical-clearing effect of FocusClearTM based on BSA system 27
2-2-1 Formation of BSA gel 27
2-2-2 Optical clearing of BSA gel using different optical clearing reagents 28
2-2-3 Imaging of Fluorescent microspheres dispersed in BSA gel 28
2-3 Quantification of the optical-clearing effect of FocusClearTM based on collagen system 30
2-3-1 Formation of collagen gel 30
2-3-2 Optical clearing of collagen gel using different optical-clearing reagents 31
2-3-3 Optical clearing of cells grow in collagen gel 31
2-3-4 Imaging of cells grow in collagen gel 32
3. Results and Discussion 33
3-1 Quantification of the optical-clearing effect of FocusClearTM 33
based on BSA system 33
3-1-1 Transmittance quantification using different concentrations of BSA solution 33
3-1-2 Transmittance quantification using different optical-clearing reagents 34
3-1-3 Fluorescence quantification post optical clearing 35
3-2 Quantification of the optical-clearing effect of FocusClearTM 37
based on collagen system 37
3-2-1 Transmittance quantification with no cells in collagen gels 37
3-2-2 Transmittance quantification with cells grow in collagen gels 38
3-2-3 Confocal imaging post optical clearing 39
4. Conclusion 40
5. Future Work 41

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