請用此 Handle URI 來引用此文件：
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/97070完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 孫啟光 | zh_TW |
| dc.contributor.advisor | Chi-Kuang Sun | en |
| dc.contributor.author | 陳冠穎 | zh_TW |
| dc.contributor.author | Guan-Ying Chen | en |
| dc.date.accessioned | 2025-02-26T16:18:47Z | - |
| dc.date.available | 2025-02-27 | - |
| dc.date.copyright | 2025-02-26 | - |
| dc.date.issued | 2025 | - |
| dc.date.submitted | 2025-02-05 | - |
| dc.identifier.citation | 1.Denk, W. & Svoboda, K. Photon Upmanship: Techreview Why Multiphoton Imaging Is More than a Gimmick. Neuron vol. 18 (1997).
2. Helmchen, F. & Denk, W. Deep tissue two-photon microscopy. Nature Methods vol. 2 932–940 Preprint at https://doi.org/10.1038/nmeth818 (2005). 3. Zipfel, W. R., Williams, R. M. & Webb, W. W. Nonlinear magic: Multiphoton microscopy in the biosciences. Nature Biotechnology vol. 21 1369–1377 Preprint at https://doi.org/10.1038/nbt899 (2003). 4. Oheim, M., Beaurepaire, E., Chaigneau, E., Mertz, J. & Charpak, S. Two-Photon Microscopy in Brain Tissue: Parameters Influencing the Imaging Depth. Journal of Neuroscience Methods vol. 111 www.elsevier.com/locate/jneumeth (2001). 5. Rubart, M. Two-photon microscopy of cells and tissue. Circulation Research vol. 95 1154–1166 Preprint at https://doi.org/10.1161/01.RES.0000150593.30324.42 (2004). 6. Beaulieu, D. R., Davison, I. G., Kılıç, K., Bifano, T. G. & Mertz, J. Simultaneous multiplane imaging with reverberation two-photon microscopy. Nat Methods 17, 283–286 (2020). 7. Ouzounov, D. G., Wang, T., Wu, C. & Xu, C. GCaMP6 ΔF/F dependence on the excitation wavelength in 3-photon and 2-photon microscopy of mouse brain activity. Biomed Opt Express 10, 3343 (2019). 8. Cichon, J. et al. Imaging neuronal activity in the central and peripheral nervous systems using new Thy1.2-GCaMP6 transgenic mouse lines. J Neurosci Methods 334, (2020). 9. Kaszas, A. et al. Two-photon GCaMP6f imaging of infrared neural stimulation evoked calcium signals in mouse cortical neurons in vivo. Sci Rep 11, (2021). 10. Pernici, C. D., Kemp, B. S. & Murray, T. A. Time course images of cellular injury and recovery in murine brain with high-resolution GRIN lens system. Sci Rep 9, (2019). 11. Chien, Y.-F. et al. Dual GRIN lens two-photon endoscopy for high-speed volumetric and deep brain imaging. Biomed Opt Express 12, 162 (2021). 12. Yang, Y. et al. A Two-Step GRIN Lens Coating for In Vivo Brain Imaging. Neurosci Bull 35, 419–424 (2019). 13. Cai, D. J. et al. A shared neural ensemble links distinct contextual memories encoded close in time. Nature 534, 115–118 (2016). 14. Yang, Y. et al. Improved calcium sensor GCaMP-X overcomes the calcium channel perturbations induced by the calmodulin in GCaMP. Nat Commun 9, (2018). 15. Tian, L. et al. Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nat Methods 6, 875–881 (2009). 16. Barbera, G. et al. Spatially Compact Neural Clusters in the Dorsal Striatum Encode Locomotion Relevant Information. Neuron 92, 202–213 (2016). 17. Zhu, M. H., Jang, J., Milosevic, M. M. & Antic, S. D. Population imaging discrepancies between a genetically-encoded calcium indicator (GECI) versus a genetically-encoded voltage indicator (GEVI). Sci Rep 11, (2021). 18. Roome, C. J. & Kuhn, B. Simultaneous dendritic voltage and calcium imaging and somatic recording from Purkinje neurons in awake mice. Nat Commun 9, (2018). 19. Allen, W. E. et al. Global Representations of Goal-Directed Behavior in Distinct Cell Types of Mouse Neocortex. Neuron 94, 891-907.e6 (2017). 20. Zhang, Y. et al. Fast and sensitive GCaMP calcium indicators for imaging neural populations. Nature 615, 884–891 (2023). 21. Akerboom, J. et al. Optimization of a GCaMP calcium indicator for neural activity imaging. Journal of Neuroscience 32, 13819–13840 (2012). 22. Dombeck, D. A., Harvey, C. D., Tian, L., Looger, L. L. & Tank, D. W. Functional imaging of hippocampal place cells at cellular resolution during virtual navigation. Nat Neurosci 13, 1433–1440 (2010). 23. Resendez, S. L. et al. Visualization of cortical, subcortical and deep brain neural circuit dynamics during naturalistic mammalian behavior with head-mounted microscopes and chronically implanted lenses. Nat Protoc 11, 566–597 (2016). 24. Liu, X., Yang, P. S., Yang, W. & Yue, D. T. Enzyme-inhibitor-like tuning of Ca 2+ channel connectivity with calmodulin. Nature 463, 968–972 (2010). 25. Bandeen, R. & Maran, S. P. References and Notes 1. W. https://www.science.org (1979). 26. Surmeier, D. J., Guzman, J. N., Sanchez-Padilla, J. & Schumacker, P. T. The role of calcium and mitochondrial oxidant stress in the loss of substantia nigra pars compacta dopaminergic neurons in Parkinson’s disease. Neuroscience vol. 198 221–231 Preprint at https://doi.org/10.1016/j.neuroscience.2011.08.045 (2011). 27. Jahanshahi, M., Jones, C. R. G., Dirnberger, G. & Frith, C. D. The substantia nigra pars compacta and temporal processing. Journal of Neuroscience 26, 12266–12273 (2006). 28. Nielsen, M. S., Sørensen, J. C. & Bjarkam, C. R. The substantia nigra pars compacta of the Göttingen minipig: An anatomical and stereological study. Brain Struct Funct 213, 481–488 (2009). 29. Da Cunha, C. et al. Evidence for the substantia nigra pars compacta as an essential component of a memory system independent of the hippocampal memory system. Neurobiol Learn Mem 79, 236–242 (2003). 30. Dorsey, E. R. et al. Projected Number of People with Parkinson Disease in the Most Populous Nations, 2005 through 2030. www.neurology.org (2007). 31. Braak, H., Ghebremedhin, E., Rüb, U., Bratzke, H. & Del Tredici, K. Stages in the development of Parkinson’s disease-related pathology. Cell and Tissue Research vol. 318 121–134 Preprint at https://doi.org/10.1007/s00441-004-0956-9 (2004). 32. Campos, P., Walker, J. J. & Mollard, P. Diving into the brain: Deep-brain imaging techniques in conscious animals. Journal of Endocrinology vol. 246 R33–R50 Preprint at https://doi.org/10.1530/JOE-20-0028 (2020). 33. Chien, Y.-F. et al. Dual GRIN lens two-photon endoscopy for high-speed volumetric and deep brain imaging. Biomed Opt Express 12, 162 (2021). 34. Sattin, A. et al. Aberration correction in long GRIN lens-based microendoscopes for extended field-of-view two-photon imaging in deep brain regions. Preprint at https://doi.org/10.1101/2024.07.24.604890 (2024). 35. Li, X. et al. Link Brain-Wide Projectome to Neuronal Dynamics in the Mouse Brain. Neurosci Bull (2024) doi:10.1007/s12264-024-01232-z. 36. Longitudinal Migration Tracking of Fluorescent Stem Cells in Vivo in the Mouse Brain. 37. Accanto, N. et al. A flexible two-photon fiberscope for fast activity imaging and precise optogenetic photostimulation of neurons in freely moving mice. Neuron 111, 176-189.e6 (2023). 38. Wall, K. F. & Sanchez, A. TItanium Sapphire Lasers. 39. Feng, G. et al. Neurotechnique Imaging Neuronal Subsets in Transgenic Mice Expressing Multiple Spectral Variants of GFP Variants with Altered Spectral Properties and Improved Translational Efficiency, Thermostability, and Quantum Yield. As a Result of These Favorable Properties, GFP and Its Variants Have Been Used to Follow Molecules and Cells in at Least a Dozen Species, Ranging from Slime. Neuron vol. 28 (2000). 40. Hadjantonakis, A. K. & Nagy, A. The color of mice: In the light of GFP-variant reporters. Histochem Cell Biol 115, 49–58 (2001). 41. Zhou, A. et al. Evaluation of resonant scanning as a high-speed imaging technique for two-photon imaging of cortical vasculature. Biomed Opt Express 13, 1374 (2022). 42. Liu, X., Yang, P. S., Yang, W. & Yue, D. T. Enzyme-inhibitor-like tuning of Ca 2+ channel connectivity with calmodulin. Nature 463, 968–972 (2010). 43. PIIS0092867402006827. | - |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/97070 | - |
| dc.description.abstract | 本研究聚焦於克服現有技術在觀測小鼠深層腦部影像時的挑戰，特別是在光學系統穩定性、重現性以及實驗流程簡化方面的困難。傳統的深層成像技術，雖然在橫向解析度與光路延伸上有所突破，但仍面臨樣本移動誤差導致無法觀測同一區域以及實驗耗時較長等問題。因此本研究設計並製作了一種自製GRIN rod holder與連接器，結合雙光子顯微鏡系統，目的是在快速、高效且穩定地對小鼠中腦黑質致密部（SNc）進行神經活動的觀測。
在系統性能測試中，使用標準螢光小球樣品進行解析度驗證，結果顯示本系統橫向解析度可穩定維持在1.40 ± 0.0025 微米。為了進一步驗證系統穩定性，我們設計了多種重現率測試，包括短時間內重接合、移動小鼠後的再連接影像分析，結果證明系統在這些情境下均能維持高準確性的影像重現，並且可以觀測到同一顆神經細胞。除此之外，利用活體小鼠的神經鈣離子指示劑（GCaMP6f），我們成功記錄了黑質致密部神經元的鈣離子濃度動態變化。 與其他文章內技術相比，本研究的新設計顯著縮短了實驗步驟以及所需時間，從原本的 30–60 分鐘減少至僅需 5–10 分鐘，極大提升了實驗效率，本系統不僅適用於觀測鈣離子濃度變化，亦具備延伸應用於記錄神經活動中的電位影像的潛力。 總而言之，本研究提出的雙光子顯微鏡結合梯度折射率透鏡系統，兼具高解析度、穩定性與操作便捷性，為未來神經科學研究中的深層腦部影像分析和動態活動觀測開啟了可能性。 | zh_TW |
| dc.description.abstract | This study focuses on overcoming the challenges in observing deep brain images of mice, particularly in terms of optical system stability, reproducibility, and the simplification of experimental procedures. While traditional deep imaging techniques have made breakthroughs in lateral resolution and optical path extension, they still face issues such as errors caused by sample movement, which prevent consistent observation of the same region, and prolonged experimental durations. Therefore, we designed and fabricated a custom-made GRIN rod holder and connector, combined with a two-photon microscope system, to achieve rapid, efficient, and stable observation of neural activity in the substantia nigra pars compacta (SNc) of the midbrain.
In system performance testing, resolution verification using standard fluorescent microspheres demonstrated that the lateral resolution of the system could be stably maintained at 1.40 ± 0.0025 μm. To further validate system stability, we designed various reproducibility tests, including reassembly in a short time and reattachment after moving the mouse, confirming that the system could maintain highly accurate image reproduction under these conditions and observe the same neuron. Furthermore, using the neural calcium indicator GCaMP6f in live mice, we successfully recorded the dynamic changes in calcium ion concentrations in the neurons of the SNc. Compared to techniques discussed in other studies, the new design proposed in this study significantly reduces experimental steps and required time, from the original 30–60 minutes to just 5–10 minutes, greatly improving experimental efficiency. The system is not only suitable for observing calcium ion concentration changes but also has potential for extended applications in recording voltage images of neural activity. In summary, the two-photon microscope combined with a GRIN lens system proposed in this study offers high resolution, stability, and operational convenience, opening new possibilities for deep brain image analysis and dynamic activity observation in future neuroscience research. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2025-02-26T16:18:47Z No. of bitstreams: 0 | en |
| dc.description.provenance | Made available in DSpace on 2025-02-26T16:18:47Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | 口試委員審定書 I
誌謝 I 中文摘要 II ABSTRACT III 目 次 IV 圖次 VI 表次 X 第1章 引言 1 1.1 研究動機與目標 1 1.2 論文章節介紹 1 第2章 背景知識 3 2.1 非線性光學與雙光子顯微鏡 3 2.2 梯度折射率透鏡（GRADIENT INDEX (GRIN) LENS） 4 2.3 綠色鈣指示劑與神經活動觀測 5 2.4 黑質致密部（SNC）腦區研究背景 6 2.5 與其他系統之比較 7 第3章 研究方法 13 3.1 系統設計與設備製作 13 3.1.1 折射率梯度透鏡與雙光子顯微鏡系統整合 13 3.1.2 GRIN rod holder的設計 14 3.1.3 將GRIN rod holder與雙光子顯微鏡系統結合 23 3.2 實驗樣品準備 30 3.2.1 植入手術所需的自製夾具 30 3.2.2 小鼠的製備 32 3.2.3 實驗流程 34 第4章 結果 37 4.1 雙光子顯微系統搭配梯度折射率透鏡的橫向解析度 37 4.2 利用綠色鈣指示劑（GCAMP6F）觀測目標腦區神經細胞活動 41 4.3 系統重現率驗證 43 4.3.1 透過螢光小球當作樣品進行測試 43 4.3.2 透過標記綠色螢光蛋白（GFP）的小鼠進行測試 51 第5章 結論與未來目標 60 參考文獻 64 附錄一 圖 2.2之版權許可證明 68 附錄二 圖 2.2 之版權許可證明 74 附錄三 圖 2.3之版權許可證明 75 附錄四 圖 2.4之版權許可證明 76 | - |
| dc.language.iso | zh_TW | - |
| dc.subject | 重現率 | zh_TW |
| dc.subject | 梯度折射率透鏡 | zh_TW |
| dc.subject | 雙光子顯微鏡 | zh_TW |
| dc.subject | reproducibility | en |
| dc.subject | two-photon microscopy | en |
| dc.subject | GRIN lens | en |
| dc.title | 自製梯度折射率透鏡固定器用以觀測活體小鼠中腦區鈣離子成像 | zh_TW |
| dc.title | GRIN rod holder design for in vivo calcium imaging in mice mid-brain | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 113-1 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 高甫仁;宋孔彬 | zh_TW |
| dc.contributor.oralexamcommittee | Fu-Jen Kao;Kung-Bin Sung | en |
| dc.subject.keyword | 雙光子顯微鏡,梯度折射率透鏡,重現率, | zh_TW |
| dc.subject.keyword | two-photon microscopy,GRIN lens,reproducibility, | en |
| dc.relation.page | 83 | - |
| dc.identifier.doi | 10.6342/NTU202500410 | - |
| dc.rights.note | 同意授權(全球公開) | - |
| dc.date.accepted | 2025-02-05 | - |
| dc.contributor.author-college | 電機資訊學院 | - |
| dc.contributor.author-dept | 光電工程學研究所 | - |
| dc.date.embargo-lift | 2025-02-27 | - |
| 顯示於系所單位： | 光電工程學研究所 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-113-1.pdf | 38.81 MB | Adobe PDF | 檢視/開啟 |
系統中的文件，除了特別指名其著作權條款之外，均受到著作權保護，並且保留所有的權利。