人體組織大多呈現不透明的狀態，其主要原因為組織的環境中富含水分子與不同高折射率的物質，如脂質、膠原纖維等，組織之間不均勻分布的折射率性質造成光線行經時發生高度散射。而在組織結構的三維顯微影像觀察中，散射使光線難以穿透至樣本深處，顯微鏡裝置將難以解析樣本深層區域的訊號。因此，為了減少光線的散射，我們發展出一種高折射率之水膠-組織包埋澄清系統，並將組織透明化，應用於消化道與腎臟組織的三維顯微影像建立。這些高折射率水膠是由丙烯醯胺的衍生化合物作為單體，以三乙二醇二甲基丙烯酸酯作為交聯劑，經由紫外光反應催化交聯，並與糖類共同聚合而成。在本篇論文中，藉由水膠包埋小鼠結腸與腎臟組織，定性與定量分析水膠系統的組織澄清效果，並利用其透光性，建立組織結構與血管網絡的三維顯微影像。此系統的優點為組織製備與光催化的反應性快，在一個小時之內即可達成組織透明化的效果，整個透明化的過程並於組織學的各類型染色技術有高度的相容性。我們發展的水膠-組織包埋澄清系統，經由糖類的添加修飾，能夠建立小鼠消化道與腎臟組織微環境的高解析度三維影像，且固體包埋的透明組織具有穩定的支撐結構，更利於組織樣本的保存和運輸。

Background and Aims: Unlike the transparent lens of the eyes, the internal organs of our bodies are intrinsically opaque. The opacity is caused by the mismatch of the refractive index between the chemical components of the tissues (mainly proteins and lipids) and the surrounding water molecules, leading to scattering as light encounters the tissue boundary. In microscopy, scattering creates difficulties in photon penetration into the specimen for 3-dimensional (3-D) visualization of the tissue structure. To reduce scattering in optical imaging, in this research we develop a hydrogel-based tissue clearing method with high-refractive-index components to prepare transparent specimens for 3-D gastrointestinal and renal histology.

Methods and Results: Solutions consisting of the acrylamide-based monomers, triethylene glycol dimethacrylate (cross-linker), and sugars were used to synthesize a series of the high-refractive-index hydrogels via ultraviolet polymerization. The optical-clearing capacity of the hydrogels is demonstrated by their permeability and polymerization within the specimens (mouse colon and kidney) to facilitate photon penetration for 3-D visualization of the tissue microstructure and vasculature in an integrated fashion. The advantage of this clearing method is its fast clearing kinetics -- within one hour to complete the tissue preparation and clearing process -- and its compatibility with fluorescence immunohistochemistry.

Conclusion: We developed a hydrogel-based tissue clearing method with sugar supplements for 3-D mouse tissue imaging. The solid, transparent tissue slab provides a suitable condition for tissue preservation, transportation, and optical imaging to reveal the details of the gastrointestinal and renal microenvironment with high definition.

摘要 i
Abstract ii
目錄 iv
表目錄 vii
圖目錄 viii
縮寫名詞對照表 ix
1. 緒論 1
1.1 組織光學澄清技術 1
1.2 三維組織影像技術 2
1.3 高折射率水膠 5
1.4 實驗目的 6
2. 實驗材料與方法 7
2.1 實驗材料與儀器 7
2.1.1 實驗材料 7
2.1.2 實驗儀器 9
2.1.3 實驗動物模型 9
2.2 高折射率水膠之製備 10
2.2.1 AMEGA水膠 11
2.2.2 AMEGA-S水膠 12
2.2.3 AMEGA-M水膠 12
2.2.4 HAMEGA-M水膠 12
2.3 AMEGA水膠系統之光學性質及組織澄清效果分析 13
2.3.1 水膠折射率量測 13
2.3.2 組織-水膠之光學穿透度量測及動力學分析 14
2.3.3 小鼠消化道之光學澄清 15
2.4 灌流系統及血管染色標記 15
2.4.1 血管染劑之配製 15
2.4.2 小鼠解剖與灌流 16
2.4.3 器官組織之後固定 16
2.5 組織樣本之切片 17
2.6 組織樣本之染色標記 17
2.6.1 免疫染色 17
2.6.2 化學染劑染色 18
2.7 水膠包埋之樣本封片 18
2.8 顯微影像之建立 19
2.9 影像處理及三維投影影像 19
2.10 梅納反應檢驗 20
3. 實驗結果 21
3.1 高折射率AMEGA-S水膠之合成與光學性質分析 21
3.2 AMEGA-S水膠包埋澄清技術於三維顯微組織學之應用 23
3.3 AMEGA-S水膠之組織澄清能力及化學性質之優化 25
4. 討論 28
5. 結論 33
6. 未來工作 34
7. 參考文獻 57
8. 附錄 62

1. D, Z., et al., Recent progress in tissue optical clearing. Laser Photon Rev. Laser Photon Rev, 2013. 7: p. 732-757.
2. AK, B., et al., Revisiting optical clearing with dimethyl sulfoxide (DMSO). Lasers Surg Med, 2009. 41(2): p. 142-148.
3. G, V., et al., Use of an agent to reduce scattering in skin. Lasers Surg Med, 1999. 24: p. 133-141.
4. Choi, B., et al., Determination of chemical agent optical clearing potential using in vitro human skin. Lasers Surg Med, 2005. 36(2): p. 72-5.
5. Richardson, D.S. and J.W. Lichtman1, Clarifying Tissue Clearing. Cell., 2015 July 16. 162(2): p. 246-257.
6. YY, F. and T. SC, At the movies: 3-dimensional technology and gastrointestinal histology. Gastroenterology. Imaging and Advanced Technology, 2010. 139: p. 1100-1105.
7. Genina, E.A., A.N. Bashkatov, and V.V. Tuchin, Tissue optical immersion clearing. Expert Rev Med Devices, 2010. 7(6): p. 825-42.
8. Yeh, A.T. and J. Hirshburg, Molecular interactions of exogenous chemical agents with collagen--implications for tissue optical clearing. J Biomed Opt, 2006. 11(1): p. 014003.
9. MH, K., et al., Optical clearing of in vivo human skin: implications for light-based diagnostic imaging and therapeutics. Lasers in Surgery and Medicine, 2004. 34(2): p. 83-85.
10. Edler, D., Immunohistochemically detected thymidylate synthase in colorectal cancer: an independent prognostic factor of survival. . Clin Cancer Res, 2000. 6(2): p. 488-492.
11. H, H., et al., Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain. Nat Neurosci, 2011. 14: p. 1481-1488.
12. MT, K., F. S, and I. T, SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction. Nat Neurosci 2013. 16: p. 1154-61.
13. Erturk, A., et al., Three-dimensional imaging of the unsectioned adult spinal cord to assess axon regeneration and glial responses after injury. Nat Med, 2012. 18(1): p. 166-71.
14. Conchello, J.A. and J.W. Lichtman, Optical sectioning microscopy. Nat Methods, 2005. 2(12): p. 920-31.
15. Juang, J.H., et al., Three-dimensional islet graft histology: panoramic imaging of neural plasticity in sympathetic reinnervation of transplanted islets under the kidney capsule. Am J Physiol Endocrinol Metab, 2014. 306(5): p. E559-70.
16. Juang, J.H., et al., 3-D Imaging Reveals Participation of Donor Islet Schwann Cells and Pericytes in Islet Transplantation and Graft Neurovascular Regeneration. EBioMedicine, 2015. 2(2): p. 109-19.
17. Tang, S.C., et al., Plasticity of Schwann cells and pericytes in response to islet injury in mice. Diabetologia, 2013. 56(11): p. 2424-34.
18. Liu, Y.A., et al., 3-D imaging, illustration, and quantitation of enteric glial network in transparent human colon mucosa. Neurogastroenterol Motil, 2013. 25(5): p. e324-38.
19. Liu, Y.A., et al., 3-D illustration of network orientations of interstitial cells of Cajal subgroups in human colon as revealed by deep-tissue imaging with optical clearing. Am J Physiol Gastrointest Liver Physiol, 2012. 302(10): p. G1099-110.
20. Oldham, M., et al., Optical clearing of unsectioned specimens for three-dimensional imaging via optical transmission and emission tomography. J Biomed Opt, 2008. 13(2): p. 021113.
21. Fu, Y.Y., et al., Microtome-free 3-dimensional confocal imaging method for visualization of mouse intestine with subcellular-level resolution. Gastroenterology, 2009. 137(2): p. 453-65.
22. Fu, Y.Y. and S.C. Tang, Optical clearing facilitates integrated 3D visualization of mouse ileal microstructure and vascular network with high definition. Microvasc Res, 2010. 80(3): p. 512-21.
23. Tang, S.C., et al., Vascular labeling of luminescent gold nanorods enables 3-D microscopy of mouse intestinal capillaries. ACS Nano, 2010. 4(10): p. 6278-84.
24. Liu, Y.A., et al., Optical clearing improves the imaging depth and signal-to-noise ratio for digital analysis and three-dimensional projection of the human enteric nervous system. Neurogastroenterol Motil, 2011. 23(10): p. e446-57.
25. Chiu, Y.C., et al., 3-D imaging and illustration of the perfusive mouse islet sympathetic innervation and its remodelling in injury. Diabetologia, 2012. 55(12): p. 3252-61.
26. Fu, Y.Y., et al., Three-dimensional optical method for integrated visualization of mouse islet microstructure and vascular network with subcellular-level resolution. J Biomed Opt, 2010. 15(4): p. 046018.
27. Chien, H.J., et al., 3-D imaging of islets in obesity: formation of the islet-duct complex and neurovascular remodeling in young hyperphagic mice. Int J Obes (Lond), 2016. 40(4): p. 685-97.
28. Lin, P.Y., et al., PanIN-associated pericyte, glial, and islet remodeling in mice revealed by 3D pancreatic duct lesion histology. Am J Physiol Gastrointest Liver Physiol, 2016. 311(3): p. G412-22.
29. Tang, S.C., S.J. Peng, and H.J. Chien, Imaging of the islet neural network. Diabetes Obes Metab, 2014. 16 Suppl 1: p. 77-86.
30. Hua, T.E., et al., 3-D neurohistology of transparent tongue in health and injury with optical clearing. Front Neuroanat, 2013. 7: p. 36.
31. Wang, R.K., et al., Investigation of optical clearing of gastric tissue immersed with hyperosmotic agents. IEEE Journal of Selected Topics in Quantum Electronics, 2003. 9(2): p. 234-242.
32. Tuchin, V.V., R.K. Wang, and A.T. Yeh, Optical clearing of tissues and cells. J Biomed Opt, 2008. 13(2): p. 021101.
33. Xu, X. and R.K. Wang, Synergistic effect of hyperosmotic agents of dimethyl sulfoxide and glycerol on optical clearing of gastric tissue studied with near infrared spectroscopy. Physics in Medicine and Biology, 2004. 49(3): p. 457-468.
34. Azaripour, A., et al., A survey of clearing techniques for 3D imaging of tissues with special reference to connective tissue. Prog Histochem Cytochem, 2016. 51(2): p. 9-23.
35. Hama, H., et al., ScaleS: an optical clearing palette for biological imaging. Nat Neurosci, 2015. 18(10): p. 1518-29.
36. Hoffman, A.S., Hydrogels for biomedical applications. Advanced Drug Delivery Reviews, 2002. 54(1): p. 3-12.
37. Jafari, B., F. Rafie, and S. Davaran, Preparation and characterization of a novel smart polymeric hydrogel for drug delivery of insulin. Bioimpacts, 2011. 1(2): p. 135-43.
38. Langer, R. and J. Vacanti, Tissue engineering. Science, 1993. 260(5110): p. 920-926.
39. Tanigo, T., R. Takaoka, and Y. Tabata, Sustained release of water-insoluble simvastatin from biodegradable hydrogel augments bone regeneration. J Control Release, 2010. 143(2): p. 201-6.
40. Silva, A.K., et al., Growth factor delivery approaches in hydrogels. Biomacromolecules, 2009. 10(1): p. 9-18.
41. Matsumoto, A., et al., A synthetic approach toward a self-regulated insulin delivery system. Angew Chem Int Ed Engl, 2012. 51(9): p. 2124-8.
42. Yahia, L., History and Applications of Hydrogels. Journal of Biomedical Sciencies, 2015. 04(02).
43. Chai, Q., Y. Jiao, and X. Yu, Hydrogels for Biomedical Applications: Their Characteristics and the Mechanisms behind Them. Gels, 2017. 3(1): p. 6.
44. Azad, A.K., et al., Chitosan membrane as a wound-healing dressing: characterization and clinical application. J Biomed Mater Res B Appl Biomater, 2004. 69(2): p. 216-22.
45. Kickhöfen, B., et al., Chemical and physical properties of a hydrogel wound dressing. Biomaterials, 1986. 7(1): p. 67-72.
46. Yoo, H.J. and H.D. Kim, Synthesis and properties of waterborne polyurethane hydrogels for wound healing dressings. J Biomed Mater Res B Appl Biomater, 2008. 85(2): p. 326-33.
47. Chen, J., H. Park, and K. Park, Synthesis of superporous hydrogels: Hydrogels with fast swelling and superabsorbent properties. Journal of Biomedical Materials Research, 1999. 44(1): p. 53-62.
48. Li, P., et al., A polycationic antimicrobial and biocompatible hydrogel with microbe membrane suctioning ability. Nat Mater, 2011. 10(2): p. 149-56.
49. Deng, C., et al., Collagen and glycopolymer based hydrogel for potential corneal application. Acta Biomater, 2010. 6(1): p. 187-94.
50. NA, P., et al., Physicochemical foundations and structural design of hydrogels in medicine and biology. Annu Rev Biomed Eng, 2000. 2: p. 9-29.
51. Matsuo, E.S. and T. Tanaka, Patterns in shrinking gels. Nature, 1992. 358(6386): p. 482-485.
52. Edman, P., B. Ekman, and I. Sjöholm, Immobilization of proteins in microspheres of biodegradable polyacryldextran. Journal of Pharmaceutical Sciences, 1980. 69(7): p. 838-842.
53. Dai, W.S. and T.A. Barbari, Hydrogel membranes with mesh size asymmetry based on the gradient crosslinking of poly(vinyl alcohol). Journal of Membrane Science, 1999. 156(1): p. 67-79.
54. Peppas, N.A. and R.E. Berner, Proposed method of intracopdal injection and gelation of poly (vinyl alcohol) solution in vocal cords: polymer considerations. Biomaterials, 1980. 1(3): p. 158-162.
55. Kim, Y.J., M. Ebara, and T. Aoyagi, A smart nanofiber web that captures and releases cells. Angew Chem Int Ed Engl, 2012. 51(42): p. 10537-41.
56. Matsukuma, D., K. Yamamoto, and T. Aoyagi, Stimuli-responsive properties of N-isopropylacrylamide-based ultrathin hydrogel films prepared by photo-cross-linking. Langmuir, 2006. 22(13): p. 5911-5.
57. Bozukova, D., et al., Hydrogel nanocomposites: a potential UV/blue light filtering material for ophthalmic lenses. J Biomater Sci Polym Ed, 2011. 22(14): p. 1947-61.
58. Huo, Y., H. Ketelson, and S.S. Perry, Ethylene oxide-block-butylene oxide copolymer uptake by silicone hydrogel contact lens materials. Applied Surface Science, 2013. 273: p. 472-477.
59. Zhou, C., et al., High water content hydrogel with super high refractive index. Macromol Biosci, 2013. 13(11): p. 1485-91.
60. Hennink, W.E. and C.F. van Nostrum, Novel crosslinking methods to design hydrogels. Advanced Drug Delivery Reviews, 2002. 54(1): p. 13-36.
61. Kamath, K.R. and K. Park, Biodegradable hydrogels in drug delivery. Advanced Drug Delivery Reviews, 1993. 11(1-2): p. 59-84.
62. Schacht, E.H., Polymer chemistry and hydrogel systems. Journal of Physics: Conference Series, 2004. 3: p. 22-28.
63. Yu, T., et al., A simple and rapid optical clearing method for improving optical imaging depth. 2015: p. 1-3.
64. Zhang, Q., et al., A perspective on the Maillard reaction and the analysis of protein glycation by mass spectrometry: probing the pathogenesis of chronic disease. J Proteome Res, 2009. 8(2): p. 754-69.
65. Mathew, A.P. and A. Dufresne, Plasticized Waxy Maize Starch: Effect of Polyols and Relative Humidity on Material Properties. Biomacromolecules, 2002. 3(5): p. 1101-1108.
66. O'Brien-Nabors, L., Alternative Sweeteners, Third Edition, Revised and Expanded. 2001: CRC Press.
67. Scaiano, J.C., K.G. Stamplecoskie, and G.L. Hallett-Tapley, Photochemical Norrish type I reaction as a tool for metal nanoparticle synthesis: importance of proton coupled electron transfer. Chem Commun (Camb), 2012. 48(40): p. 4798-808.