神經系統在精密的人體中扮演極為關鍵的角色，一旦神經因外傷及病變而受損，會造成生活不便，甚至是家庭的負擔，近年來隨著微機電製程及動物實驗的進步，常用神經導管來誘導和支持神經纖維的生長，幫助受損神經再生。由於周邊神經組織由多種細胞和胞外基質所構成，研究這些細胞的生物力學特性及細胞間和胞外基質間的生物力學作用將有助於瞭解周邊神經生長及再生機轉。
本研究以細胞力學角度，以PC-12神經細胞及許旺細胞為樣本，探討神經細胞及許旺細胞個別及共培養力學特性。首先將PC-12細胞及許旺細胞個別培養，再將細胞共培養成PC-12細胞-許旺細胞單元。接著，改良本研究團隊先前僅對細胞分區單點壓痕實驗，採用自選之力-集壓痕實驗模式(flexible force volume mode)，提供壓痕點選擇法則，即壓痕點須考量細胞內微結構分佈及符合選擇之接觸力學模型之正向壓痕(normal indentation)假設。根據此法則，對個別及共培養PC-12神經細胞與許旺細胞獲得其高解析形貌及彈性響應，並藉由影像處理方法及所提供細胞標記法，整合原子力顯微鏡及細胞螢光染色骨架影像獲得同顆細胞樣本結果。以四面角錐接觸力學模型估測神經細胞局部之視楊氏模數(彈性模數)，試找出神經細胞局部之彈性模數與微結構骨架密度之關係。在糖尿病臨床應用方面，將PC-12細胞培養於高葡萄糖環境下，以相同實驗方法獲得形貌及彈性性質，搭配曠時攝影記錄軸突生長長度，並與正常PC-12細胞比較差異。另一方面，將原子力顯微鏡掃描共培養之PC-12細胞與許旺細胞之形貌及細胞材料特性，對模型結構分層，建構PC-12細胞-許旺細胞單元之二維有限元素模型，其中許旺細胞假設為超彈性材料，PC-12細胞軸突設為線性彈性材料，由最佳化程序搭配逆向有限元素法與壓痕實驗擬合，找出各層材料參數，並分析細胞單元內應力分佈及各層間應力變化與所使用之接觸力學結果進行比較。
由光學顯微鏡及原子力顯微鏡量測形貌結果，可發現PC-12細胞分化軸突長度可達60um，許旺細胞較PC-12細胞寬長，而PC-12細胞-許旺細胞單元形貌高度大於細胞軸突及生長錐。而高葡萄糖PC-12細胞，與正常細胞比較發現其軸突長度因培養時間增長而受到抑制，細胞本體高度高於正常細胞。根據細胞分區及進行力-集壓痕實驗，由細胞標記及影像處理方法，成功整合同樣本細胞之原子力顯微鏡形貌及細胞螢光染色影像，於選擇壓痕點法則中，亦成功驗證形貌傾斜影響估測視楊氏模數之正確性。另外，在PC-12細胞-許旺細胞單元螢光染色證實形成髓鞘化之細胞單元。由接觸力學模型估測結果可發現PC-12細胞軸突生長錐前端之視楊氏模數大於軸突與生長錐傳遞區及細胞核區，許旺細胞應力纖維之視楊氏模數大於細胞質及細胞核，而PC-12細胞-許旺細胞單元其視楊氏模數大於細胞軸突。本研究亦成功找出視楊氏模數與螢光影像之骨架密度的關係，由許旺細胞實驗結果中顯示細胞之視楊氏模數與肌動蛋白密度有線性正向關係。高葡萄糖神經細胞，因受到醣化影響與正常細胞比較，發現分區之生長錐視楊氏模數較高而細胞核較低。由有限元素模擬結果，證實許旺細胞之多層髓鞘膜為非線性超彈性材料，PC-12細胞軸突為線性彈性材料，外層許旺細胞為主要應力承受區，其次為軸突，顯示許旺細胞於單一神經纖維中，具有保護作用，此二維有限元素模型可應用當神經細胞單元承受較大形變(0.1~0.2 strain)之力學問題。
本研究發展之力學分析與模型建立的方法可應用至其他個別培養細胞或是共培養細胞的測試，將相關分析之機械性質以評估細胞內或是細胞與細胞間特性的變化與病變的影響，提供臨床上相關研究的理論基礎。

The nervous system plays an important role in the human body. The nerve injuries due to trauma and diseases can affect patient’s life and can increase burden of the patient’s family. Recently, advancements of MEMS technology and animal experiments enhance nerve conduit to help a wounded nerve to regenerate. Peripheral nerve tissue is composed of many kinds of neural cells and extracellular matrix. Study of biomechanical properties of the cells, cell-cell interaction, and extracellular matrix can help to understand the mechanism of peripheral nerve growth and regeneration.
In this dissertation, PC-12 cells and rat Schwann cells were employed for biomechanical study of individual-cultured and co-cultured peripheral neural cell and Schwann cell. First, PC-12 cells and rat Schwann cells were individually cultured and then co-cultured to yield PC-12-Schwann cell unit. Second, a single indentation test of our previous study which was only applied for cellular region was improved and replaced with flexible force volume mode. A criterion of selecting multi-indentation sites was postulated by considering the ultrastructure distribution and normal indentation assumption of contact mechanics model. From the selecting criterion, high-resolution topography and elastic response of individual-cultured and co-cultured PC-12 and Schwann cells were obtained. The results of topography scanned by atomic force microscope (AFM) and immunofluorescence image of the same individual cell were integrated by using image processing and a cellular marking technique proposed in this study. A pyramidal contact mechanics model was employed to estimate apparent Young’s modulus (elastic modulus) of the cells and to find relationship between local elastic modulus and related ultrastructure density. Third, PC-12 cells were cultured under high D-glucose medium to examine effects of diabetic mellitus. The topography and elastic modulli were obtained by the same integrated method and the axonal growth was recorded by time-lapse photography and was compared with normal PC-12 cells. Finally, the topography of PC-12-Schwann cell unit scanned by the AFM was imported into a finite-element software. A bilayer structure was defined to construct a two-dimensional finite-element model of PC-12-Schwann cell unit. The Schwann cell was assumed as a hyper-elastic material and PC-12 axon was assumed as a linear elastic material. The material parameters were estimated by solving an inverse finite-element problem. The simulation results were also compared with the fitting results of the pyramidal contact mechanics model.
The results of topography and morphology by using optical and atomic force microscope shows PC-12 axon can grow length of 60um. Schwann cell was longer and wider than PC-12 cell, and the height of PC-12-Schwann cell unit was higher than the axon. The axon cultivated under high D-glucose medium was restricted to grow, and the height of cell body was higher than the normal PC-12 cell. The AFM topography was successfully integrated with the immunofluorescence image of the same cell by cell marking technique and image processing method. From the criterion of selecting indentation sites, the accuracy of elastic modulus estimation depended on the slope of cellular surface. Furthermore, PC-12-Schwann cell unit was proved to become a myelinated cell unit by immunofluorecence staining. The apparent Young’s modulus of front region of PC-12 growth cone was larger than those of the rear growth cone and the nucleus. The apparent Young’s modulus of Schwann cell’s stress fiber was larger than those of the cytosol and the nucleus. The apparent Young’s modulus of PC-12-Schwann cell unit was larger than the growth cone of PC-12 cell. The relationship between elastic modulus and structural density was found and it shows the elastic modulus of Schwann cell is linearly correlated to actin density. Because of the glycation of high D-glucose, the PC-12 cells cultivated under high D-glucose medium shows apparent Young’s modulus of the growth cone was higher and that of nucleus was lower than the control group. From the simulation results of FEM, Schwann cell was a nonlinear hyper-elastic material and PC-12 axon was a linear elastic material. The outer layer of Schwann cell was mainly compressed by the rigid tip and that shows Schwann cell can protect a peripheral nerve fiber against the exterior load. The simulation results also shows the FEM model can be applicable for the finite strain problem (0.1~0.2 strain).
The methods of biomechanical analysis and model construction developed in this study may be applied to other individual-cultured and co-cultured cells. The mechanical properties of related analysis may be used for assessing the changes of mechanical characteristics of intracellular ultrastructures and cell-cell interaction and the effects of pathology. In addition, this technique and methodology will serve as a theoretical basis of related biomechanical studies for clinical applications.

[1] 馬大元, “最新神經系統解剖學, ”合記圖書出版社, 1995.
[2] S. Martinoia, M. Bove, M. Tedesco, B. Margesin, M. Grattarola, “A simple microfluidic system for patterning populations of neurons on silicon micromachined substrates,” Journal of Neuroscience Methods, Vol. 87, No.1 pp. 35-44, 1999.
[3] J. Kimura, “Peripheral Nerve Diseases, ”Elsevier, 2006.
[4] C. A. Bollini, J. A. Wikinski, “Anatomical review of the brachial plexus, ” Techniques in Regional Anesthesia and Pain Management, Vol.10, 2006.
[5] G. J. Siegel, B. W. Agranoff, R. W. Albers, S. K. Fisher, M. D. Uhler, “Basic Neurochemistry: Molecular, Cellular and Medical Aspects, ”6th ed., Lippincott-Raven, 1999.
[6] K. J. V. Vliet, G. Bao, S. Suresh, “The biomechanics toolbox - experimental approaches for living cells and biomolecules, ” Acta Materialia Vol. 51, No. 19, pp. 5881–5905, 2003.
[7] V. J. Morris, A. R. Kirby, A. P. Gunning, “Atomic Force Microscopy for Biologists,” 2nd ed., Imperial College Press, London, 2010.
[8] M. Radmacher, M. Fritz, C. M. Kacher, J. P. Cleveland, P. K. Hansma, “Measuring the viscoelastic properties of human platelets with the atomic force microscope,” Biophysical Journal, Vol. 70, No. 1, pp. 556–567, 1996.
[9] C. Rotsch, K. Jacobson, M. Radmacher, “Dimensional and mechanical dynamics of active and stable edges in motile fibroblasts investigated by using atomic force microscopy, ” Proceedings of the National Academy of Sciences, Vol. 96, pp. 921-926, 1999.
[10] R. E. Mahaffy, C. K. Shih, F. C. MacKintosh, J. Käs,“Scanning probe-based frequency-dependent microrheology of polymer gels and biological cells,” Physical Review Letters, Vol. 85, No. 4, pp. 880-883, 2000.
[11] A. Mathur, “Endothelial, cardiac muscle and skeletal muscle exhibit different viscous and elastic properties as determined by atomic force microscopy, ” Journal of Biomechanics, Vol. 34, No. 12, pp. 1545-1553, 2001.
[12] G. T. Charras, P. P. Lehenkari, M. A. Horton, “Atomic force microscopy can be used to mechanically stimulate osteoblasts and evaluate cellular strain distributions, ” Ultramicroscopy, Vol. 86, No. 1-2, pp. 85-95, 2001.
[13] J. Alcaraz, L. Buscemi, M. Grabulosa, X. Trepat, B. Fabry, R. Farré, D. Navajas, “Microrheology of human lung epithelial cells measured by atomic force microscopy,” Biophysical Journal, Vol. 84, No. 3, pp. 2071–2079, 2003.
[14] R. E. Mahaffy, S. Park, E. Gerde, J. Käs, C. K. Shih, “Quantitative analysis of the viscoelastic properties of thin regions of fibroblasts using atomic force microscopy, ” Biophysical Journal, Vol. 86, No. 3, pp. 1777–1793, 2004.
[15] F. Rico, P. Roca-Cusachs, N. Gavara, R. Farré, M. Rotger, D. Navajas, “Probing mechanical properties of living cells by atomic force microscopy with blunted pyramidal cantilever tips,” Physical Review, E Vol. 72, 2 Pt. 1, pp. 021914-1~10 , 2005.
[16] S. Park, D. Koch, R. Cardenas, J. Käs, C. K. Shih,“Cell motility and local viscoelasticity of fibroblasts,” Biophysical Journal, Vol. 89, No. 6, pp. 4330–4342, 2005.
[17] V. Lulevich, T. Zink, H. Y. Chen, F. T. Liu, G. Y. Liu, “Cell mechanics using atomic force microscopy-based single-cell compression,” Langmuir, Vol. 22, No. 19, pp. 8151-8155, 2006.
[18] D. C. Kevin, J. S. Alan, F. C. P. Yin, “Non-Hertzian approach to analyzing mechanical properties of endothelial cells probed by atomic force microscopy,” Journal of Biomechanical Engineering, Vol. 128, No. 2, pp. 176-184, 2006.
[19] E. M. Darling, S. Zauscher, F. Guilak, “Viscoelastic properties of zonal articular chondrocytes measured by atomic force microscopy, ” OsteoArthritis and Cartilage, Vol. 14, No. 6, pp. 571-579, 2006.
[20] E. M. Darling, S. Zauscher, J.A. Block, F. Guilak, “A thin-layer model for viscoelastic, stress-relaxation testing of cells using atomic force microscopy: do cell properties reflect metastatic potential? ” Biophysical Journal, Vol. 92, No. 5, pp. 1784–1791, 2007.
[21] B. A. Smith, H. Roy, P. D. Koninck, P. Grütter, Y. D. Koninck, “Dendritic spine viscoelasticity and soft-glassy nature: balancing dynamic remodeling with structural stability,” Biophysical Journal, Vol. 92, No. 4, pp. 1419–1430, 2007.
[22] L. Lu, Sara J. Oswald, H. Ngu, F. C. P. Yin, “Mechanical properties of actin stress fibers in living cells,” Biophysical Journal, Vol.95, No. 12, pp. 6060-6071, 2008.
[23] 李宗翰, “單軸應變對3T3纖維母細胞機械特性影響之研究, ”國立成功大學微機電系統工程研究所碩士論文, 2005.
[24] 張嘉峰, “應用原子力顯微術於PC-12類神經細胞之生物力學研究, ” 國立成功大學微機電系統工程研究所碩士論文, 2006.
[25] 馮俊雄, “應用原子力顯微鏡與免疫螢光染色法探討PC-12類神經細胞之黏彈力學性質, ”國立成功大學機械工程研究所碩士論文, 2007.
[26] 藍宏銘, “原子力顯微鏡術於PC-12類神經細胞軸突再生研究, ” 國立成功大學機械工程研究所碩士論文, 2008.
[27] 劉孟璋, “以原子力顯微鏡量測生命材料機械性質之研究-活體細胞地驗證實例, ” 國立成功大學機械工程研究所碩士論文, 2009.
[28] 郭玉宏, “施加電場對PC-12類神經細胞生長定位與軸突機械性質之影響, ” 國立成功大學奈米科技暨微系統工程研究所碩士論文, 2010.
[29] N. Ratner, L. Glaser, R. P. Bunge, “PC-12 cells as a source of neurite-derived cell surface mitogen, which stimulates Schwann cell division, ”Journal of Cell Biology, Vol. 98, No. 3, pp. 115–1155, 1984.
[30] C. Fernandezvalle, N. Fregien, P. M. Wood, M. B. Bunge, “Expression of the protein zero myelin gene in axon-related Schwann-cells is linked to basal lamina formation, ” Development, Vol. 119, No. 3, pp. 867-880, 1993.
[31] Y. F. Xu, L. Liu, Y. Li, C. Zhou, F. Xiong, Z. S. Liu, R. Gu, X. M. Hou, C. Zhang, “Myelin-forming ability of Schwann cell-like cells induced from rat adipose-derived stem cells in vitro, ”Brain Research, Vol. 1239, pp. 49-55, 2008.
[32] G. Keilhoff, A. Goihl, K. Langnase, H. Fansa, G. Wolf, “Transdifferentiation of mesenchymal stem cells into Schwann cell-like myelinating cells, ”European Journal of Cell Biology, Vol. 85, No. 1, pp. 11-24, 2006.
[33] W. C. Huang , J. D. Liao, C. C. K. Lin, M. S. Ju, “Depth-sensing nano-indentation on a myelinated axon at various stages, ” Nanotechnology, Vol. 22, No. 27, pp. 275101-1~8, 2011.
[34] A. J. M. Boulton, A. I. Vinik, J. C. Arezzo, V. Bril, E. L. Feldman, R. Freeman, “Diabetic neuropathies - a statement by the American Diabetes Association,” Diabetes Care, Vol. 28, No. 4, pp. 956-962, 2005.
[35] P. J. Dyck, C. Giannini, “Pathologic alterations in the diabetic neuropathies of humans: a review,” Journal of Neuropathology and Experimental Neurology, Vol. 55, No. 12, pp. 1181-1193, 1996.
[36] K. Koshimura, J. Tanaka, Y. Murakami, Y. Kato, “Involvement of nitric oxide in glucose toxicity on differentiated PC12 Cells: prevention of glucose toxicity by tetrahydrobiopterin, a cofactor for nitric oxide synthase, ” Neuroscience Research, Vol. 43, No. 1, pp. 31-38, 2002.
[37] A. M. Sharifi, S. H. Mousavi, M. Farhadi, and B. Larijani, “Study of high glucose-induced apoptosis in PC12 Cells: role of bax protein, ” Journal of Pharmacological Sciences, Vol. 104, No. 3, pp. 258-262, 2007.
[38] M. S. Ju, C. C. K. Lin, J. L. Fan, R. J. Chen, “Transverse elasticity and blood perfusion of sciatic nerves under in situ circular compression,” Journal of Biomechanics, Vol. 39, No. 1, pp. 97-102, 2006.
[39] R. J. Chen, C. C. K. Lin, M. S. Ju, “In situ biomechanical properties of normal and diabetic nerves: An efficient quasi-linear viscoelastic approach,” Journal of Biomechanics, Vol. 43, No. 6, pp. 1118-1124, 2010.
[40] L. A Greene, A. S. Tischler, “Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor, ” Proceedings of the National Academy of Sciences of the United States of America, Vol.73, No. 7, pp. 2424-24288, 1976.
[41] “Stage Top Incubator-Instructions Manual, ” Tokai Hit Co.
[42] MSCT Probe, Veeco Instruments, https://www.veecoprobes.com.
[43] J. E. Sader, I. Larson , P. Mulvaney, L. R. White, “Method for the calibration of atomic force microscope cantilevers, ” Review of Scientific Instruments, Vol. 66, No. 7, pp. 3789-3798, 1995.
[44] G. Wolberg, “Digital Image Warping, ” IEEE Computer Society Press, 1990.
[45] H. Hertz, J. Reine Angew. Mathematik, Vol. 92, pp. 156-171, 1882.
[46] G. G. Bilodeau, “Regular pyramid punch problem, ” Journal of Applied Mechanics-Transactions of the ASME, Vol. 59, pp. 519-523, 1992.
[47] E. L. Grzywa, A. C. Lee, G. U. Lee, D. M. Suter, “High resolution analysis of neuronal growth cone morphology by comparative atomic force and optical microscopy, ” Journal of Neurobiology Vol. 66, No. 14, pp.1529–1543, 2006.
[48] J. A. Weiner, N. Fukushima, J. J. A. Contos, S. S. Scherer, J. Chun, “Regulation of Schwann cell morphology and adhesion by receptor-mediated lysophosphatidic acid signaling, ” Journal of Neuroscience, Vol. 21, No. 18, pp.7069-7078, 2001.
[49] “ABAQUS User’s Manual, ” Version 6.11, ABAQUS Inc., 2011.
[50] 愛發股份有限公司編著, “ABAQUS實務入門引導, ” 全華圖書股份有限公司, 2007.
[51] C. T. Chang, C. C. K. Lin, M. S. Ju, “Morphology and ultrastruture-related local mechanical properties of PC-12 cells studied by integrating atomic force microscopy and immunofluorescence imaging, ” Journal of Mechanics in Medicine and Biology Vol. 12, No. 5, pp. 1250032-1~21, 2012.
[52] M. R. K. Mofrad, R. D. Kamm, “Cytoskeletal Mechanics: Models and Measurements in Cell Mechanics, ” Cambridge University Press, New York, 2006.
[53] Y. C. Fung, “Biomechanics: Mechanical properties of living tissues, ”2nd ed., Springer-Verlag, 1993.
[54] K. D. Costa, F. C. P. Yin, “Analysis of indentation implications for measuring mechanical properties with atomic force microscopy, ”Journal of Biomechanical Engineering Transaction of the ASME, Vol. 121, No. 5, pp. 462-471, 1999.
[55] Y. C. Fung, “First course in continuum mechanics, ”3rd ed., Prentice-Hall, 1993.
[56] Y. Xiong, A. C. Lee, D. M. Suter, G. U. Lee, “Topography and Nanomechanics of Live Neuronal Growth Cones Analyzed by Atomic Force Microscopy, ” Biophysical Journal Vol. 96, No. 12, pp. 5060–5072, 2009.
[57] R. Lal R, B. Drake, D. Blumberg, D. R. Saner, P. K. Hansma, S. C. Feinstein, “Imaging real-time neurite outgrowth and cytoskeletal reorganization with an atomic force microscope, ” American Journal of Physiology, Vol. 269, 1 Pt. 1, pp. C275–C285, 1995.
[58] H. A. McNally, B. Rajwa, J. Sturgis, J. P. Robinson, “Comparative Three-dimensional Imaging of Living Neurons with Confocal and Atomic Force Microscopy, ” Journal of Neuroscience Methods, Vol. 142, No.2, pp.177–184, 2005.
[59] R. J. Chen, C. C. K. Lin, M. S. Ju, “Quasi-linear-viscoelastic properties of PC-12 neuron-like cells measured using atomic force microscopy,” Journal of Institute of Chinese Engineering, Vol. 34, No. 3, pp. 325-335, 2011.
[60] V. Parpura, P. G. Haydon, E. Henderson, “Three-dimensional imaging of living neurons and glia with the atomic force microscope, ” Journal of Cell Science, Vol. 104, No. 2, pp. 427–432, 1993.
[61] S. S. Schaus, E. R. Henderson, “Cell viability and probe-cell membrane interactions of XR1 glial cells imaged by atomic force microscopy, ” Biophysical Journal Vol. 73, No. 3, pp. 1205-1214, 1997.
[62] Y. X. Sun, D. Y. Lin, Y. F. Rui, D. Han, W. Y. Ma, “Three-dimensional structural changes in living hippocampal neurons imaged using magnetic AC mode atomic force microscopy, ” Journal of Electron Microscopy Vol. 55, No. 3, pp. 165-172, 2006.
[63] J. Laishram, S. Kondra, D. Avossa, E. Migliorini, M. Lazzarino, V. Torre, “A morphological analysis of growth cones of DRG neurons combining atomic force and confocal microscopy, ” Journal of Structural Biology, Vol. 168, No. 3, pp. 366–377, 2009.
[64] A. M. Rajnicek, L. E. Foubister, C. D. McCaig, “Temporally and spatially coordinated roles for Rho, Rac, Cdc42 and their effectors in growth cone guidance by a physiological electric field, ” Journal of Cell Science, Vol. 119, Pt. 9, pp. 1723-1735, 2006.
[65] R. J. Chen, C. C. K. Lin, M. S. Ju, “In situ transverse elasticity and blood perfusion change of sciatic nerves in normal and diabetic rats, ” Clinical Biomechanics, Vol. 25, No. 5, pp. 409-414, 2010.
[66] C. FernandezValle, D. Gorman, A. M. Gomez, M. B. Bunge, “Actin plays a role in both changes in cell shape and gene-expression associated with Schwann cell myelination, ” Journal of Neuroscience Vol. 17, No. 1, pp. 241-250, 1997.
[67] B. Alberts B, A. Johnson, J. Lewis, M. Raff, K. Roberts, P. Walter, “Molecular Biology of the Cell, ” Garland Science, New York, 2007.
[68] H. Haga, S. Sasaki, K. Kawabata, E. Ito, T. Ushiki, T. Sambongi, “Elasticity mapping of living fibroblasts by AFM and immunofluorescence observation of the cytoskeleton, ” Ultramicroscopy Vol. 82, No. 1, pp. 253-258, 2000.
[69] C. Rotsch, M. Radmacher, “Drug-induced changes of cytoskeletal structure and mechanics in fibroblasts: An atomic force microscopy study, ” Biophysical Journal Vol. 78, No. 1, pp. 520-535, 2000.
[70] J. L. Hutter, J. Chen, W. K. Wan, S. Uniyal, M. Leabu, B. M. C. Chan, “Atomic force microscopy investigation of the dependence of cellular elastic moduli on glutaraldehyde fixation, ” Journal of Microscopy, Vol. 219, Pt. 2, pp. 61-68, 2005.
[71] Q. Q. Guo, Y. Xia, M. Sandig, J. Yang, “Characterization of cell elasticity correlated with cell morphology by atomic force microscope, ” Journal of Biomechanics Vol. 45, No. 2, pp. 304-309, 2012.
[72] B. C. Kieseier, “The Biology of Schwann Cells: Development, Differentiation, and Immunomodulation, ” Cambridge University Press, 2007.
[73] P. Morell, R. H. Quarles, “Myelin formation, structure and biochemistry. In G. J. Siegel Basic neurochemistry: molecular, cellular and medical aspects, ” Lippincott William & Wilkins, 1999.
[74] L. O. Heim, M. Kappl, H. J. Butt, “Tilt of atomic force microscope cantilevers: effect on spring constant and adhesion measurements, ” Langmuir, Vol 20, No. 7, pp. 2760-2764, 2004.
[75] A. R. Karduna, H. R. Halperin, F. C. P. Yin, “Experimental and numerical analyses of indentation in finite-sized isotropic and anisotropic rubber-like materials, ” Annals of Biomedical Engineering, Vol. 25, No. 6, pp. 1009-1016, 1997.
[76] D. R. Baselt, J. D. Baldeschwieler, “Imaging spectroscopy with the atomic force microscope, ” Journal of Applied Physics Vol. 76, No. 1, pp. 33-38, 1994.
[77] W. F. Heinz, J. H. Hoh, “Spatially resolved force spectroscopy of biological surfaces using the atomic force microscope, ” Trends Biotechnology, Vol. 17, No. 4, pp. 143–150, 1999.
[78] A. Heredia, C. C. Bui, U. Suter, P. Young P, T. E. Schäffer, “AFM combines functional and morphological analysis of peripheral myelinated and demyelinated nerve fibers, ” Neuroimage Vol. 37, No. 4, pp. 1218-1226, 2007.
[79] S. Kondra, J. Laishram, J. Ban, E. Migliorini, V. D. Foggia, M. Lazzarino, V. Torre, M. E. Ruaro, “Integration of confocal and atomic force microscopy images, ” Journal of Neuroscience Methods Vol. 177, No. 1, pp. 94-107, 2009.
[80] R. Kassies R, K. O. Van Der Werf, A. Lenferink, C. N. Hunter, J. D. Olsen, V. Subramaniam, C. Otto, “Combined AFM and confocal fluorescence microscope for applications in bio-nanotechnology, ” Journal of Microscope, Vol. 217, Pt. 1, pp. 109-116, 2005.
[81] D. W. Piston, “Choosing objective lenses: The importance of numerical aperture and magnification in digital optical microscopy, ” Biological Bulletin, No. 195, No. 1, pp. 1-4, 1998.
[82] I. T. Young, “Quantitative microscopy, ” IEEE Engineering in Medicine and Biology Magazine, Vol. 15, pp. 59-66, 1996.
[83] G. Karp, “Cell and Molecular Biology: Concepts and Experiments, ” 3rd ed, John Wiley & Sons, New York, 2002.
[84] M. B. Arturo, G. R. Ronald, “Atomic Force Microscopy in Liquid: Biological Applications, ” Wiley-VCH Verlag & Co. KGaA, Weinheim, 2012.