雙光子轉盤式共軛焦顯微鏡及全像刺激系統: 果蠅體內神經迴路分析工具

簡易檢索 / 詳目顯示

回結果列表

研究生：	鄭旭峰 Cheng, Shiu-Feng
論文名稱：	雙光子轉盤式共軛焦顯微鏡及全像刺激系統: 果蠅體內神經迴路分析工具 Advanced Two-Photon Spinning Disk Confocal Microscopy and Holographic Stimulation for In Vivo Neural Circuit Analysis in Drosophila melanogaster
指導教授：	江安世 Chiang, Ann-Shyn 朱士維 Chu, Shi-Wei
口試委員:	羅中泉 Lo, Chung-Chuan 高輔仁 Kao, Fu-Jen
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 系統神經科學研究所 Institute of Systems Neuroscience
論文出版年：	2024
畢業學年度：	113
語文別：	英文
論文頁數：	32
中文關鍵詞：	雙光子顯微鏡、腦功能影像、電腦生成全像術、光遺傳學、轉盤式共軛焦顯微鏡
外文關鍵詞：	two-photon microscopy, brain functional imaging, computer generated holography, optogenetics, spinning disk confocal microscopy
相關次數：	點閱：62 下載：0
分享至:	分享至facebook 分享至twitter

查詢本校圖書館目錄查詢臺灣博碩士論文知識加值系統勘誤回報

探索活體神經迴路的複雜性在神經科學研究中面臨巨大的挑戰，特別是在理解腦神經的功能。果蠅（Drosophila melanogaster）作為一種被廣泛研究的模式生物，因其相對簡單的神經結構、豐富的遺傳工具和詳細的連接組圖譜，成為研究腦功能和行為基本原理的理想系統。
我們的研究開發了一個整合了雙光子旋轉盤共聚焦顯微鏡(spinning disk confocal microscope)和雙光子電腦生成全像術（Computer generated holography）光刺激系統的成像和刺激平台，顯著提升了對果蠅神經活動的觀測和控制能力。我們展示了這個系統的快速成像能力以及光遺傳學神經調控的功能。另外在研究中我們觀測到高強度雙光子激發對果蠅多巴胺神經元振盪活動的頻率有影響，強調了在使用生物樣本時限制雷射光強的重要性。最後我們還發現激光引起的干擾可被利用來實現無需光遺傳學的神經元激活，為實驗方法開闢了新的途徑。

Understanding neural circuits in vivo is a formidable challenge in neuroscience, particularly at cellular and subcellular levels. Drosophila melanogaster, with its simple neural architecture and extensive genetic tools, is an ideal model for studying brain function and behavior. Optical microscopy, while essential, faces challenges in imaging deep brain regions due to light scattering. Two-photon microscopy overcomes this with longer wavelength lasers, advancing our ability to visualize and manipulate neural activity. However, indicators like GCaMP are needed for functional insights, requiring high-speed imaging. Spinning disk confocal microscopy meets this need, providing high temporal resolution and three-dimensional imaging. For active neuron control, optogenetic tools and computer-generated holography (CGH) offer precise manipulation. Our study combines these technologies, integrating a two-photon spinning disk confocal microscope with a CGH laser stimulation system, enabling targeted neuronal observation and manipulation. Despite its advantages, we found that high-intensity two-photon excitation affects neural activity, highlighting the need for careful calibration. Notably, laser-induced perturbation can be exploited for optogenetic-less activation, opening new avenues in neuroscience research.

Abstract    p.2
中文摘要    p.3
Acknowledgment    p.4
Contents    p.5
I.    Introduction    p.6
II.    Materials and Methods    p.10
2.1    Optical Setup    p.10
2.2    Fly strains    p.11
2.3    Sample preparation for functional imaging    p.12
2.4    MV1 oscillation power dependency    p.12
III.    Results    p.14
3.1    Two-photon spinning disk microscopy for volume functional imaging.    p.14
3.2    Computer-generated holography (CGH) stimulation system.    p.15
3.3    Laser perturbation on MV1 oscillation.    p.16
3.4    Optimize imaging laser power.    p.17
3.5    Laser activation on optogenetic-less Drosophila neurons. p.17
IV.    Discussion    p.19
V.    References    p.23
VI.    Figure    p.26
VII.    Appendix    p.32

                                

1. Y. B. Chan, O. V. Alekseyenko, E. A. Kravitz (2015) Optogenetic Control of Gene Expression in Drosophila. in PLOS ONE, e0138181.
2. T. W. Chen et al. (2013) Ultrasensitive fluorescent proteins for imaging neuronal activity. in NATURE, pp 295-300.
3. P. Cheng et al. (2021) Direct control of store-operated calcium channels by ultrafast laser. in CELL RESEARCH, pp 758-772.
4. O. Hernandez et al. (2016) Three-dimensional spatiotemporal focusing of holographic patterns. in NATURE COMMUNICATIONS, pp 11928.
5. K. J. Hsu, Y. Y. Lin, A. S. Chiang, S. W. Chu (2019) Optical properties of adult Drosophila brains in one-, two-, and three-photon microscopy. in BIOMEDICAL OPTICS EXPRESS, pp 1627-1637.
6. A. Jeibmann, W. Paulus (2009) Drosophila melanogaster as a Model Organism of Brain Diseases. in INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, pp 407-440.
7. F. Li et al. (2020) The connectome of the adult Drosophila mushroom body provides insights into function. in ELIFE , e62576.
8. Lin, P. Y., Hwang, S. P. L., Lee, C. H., & Chen, B. C. (2021). Two-photon scanned light sheet fluorescence microscopy with axicon imaging for fast volumetric imaging. Journal of Biomedical Optics, pp 116503-116503.
9. I. Meloni, D. Sachidanandan, A. S. Thum, R. J. Kittel, C. Murawski (2020) Controlling the behaviour of Drosophila melanogaster via smartphone optogenetics. in SCIENTIFIC REPORTS, pp 17614.
10. P. Y. Plaçais et al. (2012) Slow oscillations in two pairs of dopaminergic neurons gate long-term memory formation in Drosophila. in NATURE NEUROSCIENCE, pp 592-599.
11. B. R. Rost, F. Schneider-Warme, D. Schmitz, P. Hegemann (2017) Optogenetic Tools for Subcellular Applications in Neuroscience. in NEURON, pp 572-603.
12. M. G. Shapiro, K. Homma, S. Villarreal, C. P. Richter, F. Bezanilla (2017) Infrared light excites cells by changing their electrical capacitance. in NATURE COMMUNICATIONS, pp 736.
13. M. Shin, J. M. Copeland, B. J. Venton (2018) Drosophila as a Model System for Neurotransmitter Measurements. in ACS CHEMICAL NEUROSCIENCE, pp 1872-1883.
14. J. H. Simpson, L. L. Looger (2018) Functional Imaging and Optogenetics in Drosophila. in GENETICS, pp 1291-1309.
15. Tsai Y-H, Liu C-W, Lin W-K, Wang C-S, Chiang C-H, Singh VR, So PTC, Chou C-F, Chu S-W (2020) Two-photon microscopy at >500 volumes/second. bioRxiv.
16. Wu, Jianglai, et al. (2020). Kilohertz two-photon fluorescence microscopy imaging of neural activity in vivo. Nature methods, pp 287-290.
17. Xiao, S., Giblin, J. T., Boas, D. A., & Mertz, J. (2023). High-throughput deep tissue two-photon microscopy at kilohertz frame rates. Optica, pp 763-769.
18. Y. Zhang et al. (2023) Fast and sensitive GCaMP calcium indicators for imaging neural populations. in NATURE, pp 884-+.

簡易檢索 / 詳目顯示

相關論文