跳到主要內容

臺灣博碩士論文加值系統

(44.213.60.33) 您好!臺灣時間:2024/07/22 16:09
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

: 
twitterline
研究生:陳鴻羽
研究生(外文):Hung-Yu Chen
論文名稱:藉由液體填充法減少氣管像差/散射以增加果蠅腦光學穿透深度
論文名稱(外文):Enhance optical penetration depth in Drosophila brain – minimizing aberration/scattering from trachea by liquid filling method
指導教授:朱士維
指導教授(外文):Shi-Wei Chu
口試委員:江安世朱麗安賈世璿
口試委員(外文):Ann-Shyn ChiangLi-An ChuShih-Hsuan Chia
口試日期:2022-01-18
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:物理學研究所
學門:自然科學學門
學類:物理學類
論文種類:學術論文
論文出版年:2022
畢業學年度:110
語文別:英文
論文頁數:117
中文關鍵詞:功能成像光學顯微術果蠅氣管像差/散射滲透壓
外文關鍵詞:Functional imagingoptical microscopeDrosophilatracheaaberration/scatteringosmolarity
DOI:10.6342/NTU202200302
相關次數:
  • 被引用被引用:0
  • 點閱點閱:88
  • 評分評分:
  • 下載下載:5
  • 收藏至我的研究室書目清單書目收藏:0
為了了解大腦是如何運作的,全腦功能性成像技術是不可或缺的。在各種技術中,光學顯微術由於能捕抓個別神經元訊號的微米級空間解析度與毫秒級時間解析度而十分受歡迎,但是它典型的穿透深度僅有一毫米。因此,對於全腦研究,果蠅成為最具潛力的模式生物,它的腦小到理論上可以被光學顯微鏡所穿透。此外,它將近一半的腦神經結構性圖譜已經在Flycircuit被繪製,這個高比例完成度的結構圖譜成為果蠅功能成像珍貴的參考。然而,實際上,我們發現光學顯微鏡在果蠅腦中遇到意外的光損害,這造成我們無法對其做全腦成像,而其原因就源自於果蠅腦中的氣管。氣管裡的空氣與周圍組織的折射率的巨大差異,使得光遭遇強烈的像差/散射,因此無法抵達大腦的深層。
在本篇研究中,我們從醫學研究中的液體通氣(liquid ventilation)取得靈感,提出三種液體填充的方法試圖能降低氣管所造成的像差/散射。在所有方法中,滲透方法(osmosis method)成效最好,藉由間接改變果蠅腦內的滲透壓,使組織液因滲透壓差而流進微氣管裡取代原本的空氣。我們經由共厄焦顯微鏡及電刺激來分別檢視滲透方法對於影像穿透深度的提升及應用在功能性成像上的可行性。不過,我們的結果顯示,並非所有果蠅腦成像的穿透深度都有增強,我們歸因於那些會影響微氣管中液體的複雜生理現象。這這些現象需要活體氣管成像的技術,可能可以進一步檢測滲透方法。總而言之,這個研究為我們提供了能增加果蠅腦內光學穿透深度的方法,並且為全腦功能連接體研究鋪路。
To understand the brain, whole brain functional imaging techniques are indispensable. Among various techniques, optical microscopy is a popular imaging modality since it has sub-micrometer spatial and millisecond temporal resolution to capture individual functional responses of neurons, but the typical penetration depth is about 1 millimeter. Hence, for whole brain study, Drosophila is the most potential study target among the animal models because the tiny brain can be covered by optical microscopy “in principle”. Besides, nearly half of the structural connectome has been mapped in the Flycircuit database. The high covering ratio serves as an invaluable reference for functional study. However, in reality, when we image the Drosophila brain via optical microscopy, unexpected light distortion hinders us from whole brain imaging. The distortion comes from the trachea inside the brain. The difference of refractive index between the air in tracheas and the surrounding tissue cause strong aberration/scattering which leads to limited penetration depth.
In this study, we are inspired by liquid ventilation in medical research and propose three liquid filling methods to minimize the trachea-induced aberration/scattering. Among these methods, osmosis method is the most successful one. It changes the osmolarity of the brain and induces the tissue fluid into tracheoles to reduce aberration/scattering. The penetration enhancement and the feasibility for functional imaging were verified by confocal microscope and electric stimulation, respectively. Nevertheless, not all the penetration depths of Drosophila brain images enhance because of physiological complexity which affects the fluid in the tracheoles. The complexity calls for in vivo tracheal imaging that might further validate the osmosis method. Overall, the method provides an approach for optical penetration enhancement in the Drosophila brain and paves the way toward whole brain functional connectome study.
口試委員會審定書 i
謝辭 ii
摘要 iii
Abstract iv
Contents vi
Figure list ix
Table list xiii
Chapter 1. Introduction 1
1.1. From brain to connectome 1
1.2. Contemporary methods for whole brain functional connectome study 4
1.2.1. Technique for functional connectome: Why optical microscopy? 4
1.2.2. Animal models for whole brain connectome study: Why choosing Drosophila? 7
1.3. Bottleneck and approaches for Drosophila whole brain functional connectomics 9
1.3.1. Bottleneck: Shallow penetration depth due to aberration/scattering from trachea 11
1.3.2. Published approaches for better penetration in Drosophila brain 15
1.3.3. New approach for reducing reduce aberration/scattering from trachea: liquid filling method 17
1.4. Aim: Enhance optical penetration depth in Drosophila brain - minimizing aberration/scattering from trachea by liquid filling method 19
Chapter 2. Principle 20
2.1. Limited penetration depth in Drosophila brain: Effect from aberration and scattering 20
2.1.1. Effect from aberration and scattering in Drosophila brain 20
2.1.2. Quantify the penetration depth 23
2.2. Enhance penetration via aberration/scattering elimination: Osmosis method 26
2.3. Optical imaging technique for checking the penetration depth: Confocal laser scanning microscope 33
2.4. Optical imaging technique for imaging tracheas: Third harmonic generation microscopy 36
Chapter 3. Method 39
3.1. Sample preparation 39
3.1.1. Drosophila 39
3.1.2. Mounting the fly 40
3.2. Optics setup 43
3.2.1. Confocal laser scanning microscope 43
3.2.2. Third-harmonic generation microscope 43
3.3. Improve the penetration depth: liquid filling method 45
3.3.1. Liquid selection: PFCs, PFC emulsion, PBS 45
3.3.2. Filling method: Capillary method, Injection method, Osmosis method 48
3.4. Extend to functional imaging: survival test 53
3.4.1. Stimulation setup for survival test 53
3.4.2. Stimulation protocol 57
3.5. Experiment protocol for osmosis method and survival test 58
3.6. Analysis method for penetration depth 59
Chapter 4. Experiments and Results 65
4.1. THG imaging for trachea 65
4.2. Liquid filling method: PFCs and capillary method 67
4.3. Liquid filling method: PFC emulsion and injection method 69
4.4. Liquid filling method: PBS and osmosis method 71
4.4.1. Drosophila brain imaging with 100% PBS 71
4.4.2. Brain imaging in different concentration PBS from 90% to 70% 73
Chapter 5. Discussion 78
5.1. The feasibility and the advantage of THG microscopy for imaging trachea 78
5.2. The reliability and the shortcomings of our liquid-filling model 81
5.2.1. Compare our model with the published result 81
5.2.2. Compare our model with our results 84
5.3. Penetration enhancement via osmosis method 86
Chapter 6. Conclusion and Perspective 90
Chapter 7. Supplementary 91
7.1. Detailed results of brain images with hypotonic mounting medium 91
7.2. Optimal wavelength for Drosophila brain imaging 107
Reference 111
1.Freemon, F.R., Galen's ideas on neurological function. Journal of the History of the Neurosciences, 1994. 3(4): p. 263-271.
2.Horn, A., et al., The structural–functional connectome and the default mode network of the human brain. Neuroimage, 2014. 102: p. 142-151.
3.Sporns, O., G. Tononi, and R. Kötter, The human connectome: a structural description of the human brain. PLoS Comput Biol, 2005. 1(4): p. e42.
4.Sonoda, T., et al., A noncanonical inhibitory circuit dampens behavioral sensitivity to light. Science, 2020. 368(6490): p. 527-531.
5.Zuo, X.-N., et al., Network centrality in the human functional connectome. Cerebral cortex, 2012. 22(8): p. 1862-1875.
6.Churchill, N.W., K. Madsen, and M. Mørup, The functional segregation and integration model: Mixture model representations of consistent and variable group-level connectivity in fMRI. Neural computation, 2016. 28(10): p. 2250-2290.
7.Vingerhoets, G., Phenotypes in hemispheric functional segregation? Perspectives and challenges. Physics of life reviews, 2019. 30: p. 1-18.
8.Friston, K.J., Functional and effective connectivity: a review. Brain connectivity, 2011. 1(1): p. 13-36.
9.Faingold, C.L., Emergent properties of neuronal networks, in Neuronal Networks in Brain Function, CNS Disorders, and Therapeutics. 2014, Elsevier. p. 419-428.
10.Alivisatos, A.P., et al., The brain activity map. Science, 2013. 339(6125): p. 1284-1285.
11.Park, S.H., et al., History of bioelectrical study and the electrophysiology of the primo vascular system. Evidence-based Complementary and Alternative Medicine, 2013. 2013.
12.Matt, C. and J. Shieh, Guide to research techniques in neuroscience, in Elsevier Inc. 2010.
13.Cogan, S.F., Neural stimulation and recording electrodes. Annu. Rev. Biomed. Eng., 2008. 10: p. 275-309.
14.Crosson, B., et al., Functional imaging and related techniques: an introduction for rehabilitation researchers. Journal of rehabilitation research and development, 2010. 47(2): p. vii.
15.Wilt, B.A., et al., Advances in light microscopy for neuroscience. Annual review of neuroscience, 2009. 32: p. 435-506.
16.Hillman, E.M., Optical brain imaging in vivo: techniques and applications from animal to man. Journal of biomedical optics, 2007. 12(5): p. 051402.
17.Tsai, Y.-H., et al., Two-photon microscopy at > 500 volumes/second. bioRxiv, 2020: p. 2020.10.21.349712.
18.White, B.H., What genetic model organisms offer the study of behavior and neural circuits. Journal of neurogenetics, 2016. 30(2): p. 54-61.
19.Stewart, A., et al., The developing utility of zebrafish in modeling neurobehavioral disorders. International Journal of Comparative Psychology, 2010. 23(1).
20.Weisenburger, S. and A. Vaziri, A guide to emerging technologies for large-scale and whole-brain optical imaging of neuronal activity. Annual review of neuroscience, 2018. 41: p. 431-452.
21.Chiang, A.-S., et al., Three-dimensional reconstruction of brain-wide wiring networks in Drosophila at single-cell resolution. Current biology, 2011. 21(1): p. 1-11.
22.Huang, S.-H., et al., Optical Volumetric Brain Imaging: Speed, Depth, and Resolution Enhancement. Journal of Physics D: Applied Physics, 2021.
23.Huang, C., et al., All-optical volumetric physiology for connectomics in dense neuronal structures. Iscience, 2019. 22: p. 133-146.
24.Patella, P. and R.I. Wilson, Functional maps of mechanosensory features in the Drosophila brain. Current Biology, 2018. 28(8): p. 1189-1203. e5.
25.Hancock, C.E., F. Bilz, and A. Fiala, In vivo optical calcium imaging of learning-induced synaptic plasticity in Drosophila melanogaster. JoVE (Journal of Visualized Experiments), 2019(152): p. e60288.
26.Theer, P., M.T. Hasan, and W. Denk, Two-photon imaging to a depth of 1000 µm in living brains by use of a Ti: Al 2 O 3 regenerative amplifier. Optics letters, 2003. 28(12): p. 1022-1024.
27.Hsu, K.-J., et al., Optical properties of adult Drosophila brains in one-, two-, and three-photon microscopy. Biomedical optics express, 2019. 10(4): p. 1627-1637.
28.Resh, V.H. and R.T. Cardé, Encyclopedia of insects. 2009: Academic press. p. 1011-1015.
29.Ji, N., T.R. Sato, and E. Betzig, Characterization and adaptive optical correction of aberrations during in vivo imaging in the mouse cortex. Proceedings of the National Academy of Sciences, 2012. 109(1): p. 22-27.
30.Wigglesworth, V., A new method for injecting the tracheae and tracheoles of insects. Journal of Cell Science, 1950. 3(14): p. 217-224.
31.Pedrazzani, M., et al., Sensorless adaptive optics implementation in widefield optical sectioning microscopy inside in vivo Drosophila brain. Journal of biomedical optics, 2016. 21(3): p. 036006.
32.Johnsen, S. and E.A. Widder, The physical basis of transparency in biological tissue: ultrastructure and the minimization of light scattering. Journal of theoretical biology, 1999. 199(2): p. 181-198.
33.Streich, L., et al., High-resolution structural and functional deep brain imaging using adaptive optics three-photon microscopy. Nature methods, 2021. 18(10): p. 1253-1258.
34.Schnabel, C., et al., Total liquid ventilation: a new approach to improve 3D OCT image quality of alveolar structures in lung tissue. Optics express, 2013. 21(26): p. 31782-31788.
35.Ahn, C., et al., Overcoming the penetration depth limit in optical microscopy: Adaptive optics and wavefront shaping. Journal of Innovative Optical Health Sciences, 2019. 12(04): p. 1930002.
36.Helmchen, F. and W. Denk, Deep tissue two-photon microscopy. Nature methods, 2005. 2(12): p. 932-940.
37.Smithpeter, C.L., et al., Penetration depth limits of in vivo confocal reflectance imaging. Applied optics, 1998. 37(13): p. 2749-2754.
38.Wigglesworth, V.B., The extent of air in the tracheoles of some terrestrial insects. Proceedings of the Royal Society of London. Series B, Containing Papers of a Biological Character, 1931. 109(763): p. 354-359.
39.Wigglesworth, V., Surface forces in the tracheal system of insects. Journal of Cell Science, 1953. 3(28): p. 507-522.
40.Davies, W.M., Memoirs: on the tracheal system of Collembola, with special reference to that of Sminthurus viridis, Lubb. Journal of Cell Science, 1927. 2(281): p. 15-30.
41.Orgeig, S., et al., The anatomy, physics, and physiology of gas exchange surfaces: is there a universal function for pulmonary surfactant in animal respiratory structures? Integrative and comparative biology, 2007. 47(4): p. 610-627.
42.Albers, M.A. and T.J. Bradley, Osmotic regulation in adult Drosophila melanogaster during dehydration and rehydration. Journal of experimental biology, 2004. 207(13): p. 2313-2321.
43.Boyd, R.W., Nonlinear optics. 2020: Academic press.
44.Oron, D., et al., Depth-resolved structural imaging by third-harmonic generation microscopy. Journal of structural biology, 2004. 147(1): p. 3-11.
45.Jenett, A., J.E. Schindelin, and M. Heisenberg, The Virtual Insect Brain protocol: creating and comparing standardized neuroanatomy. BMC bioinformatics, 2006. 7(1): p. 1-12.
46.Clark, L.C. and F. Gollan, Survival of mammals breathing organic liquids equilibrated with oxygen at atmospheric pressure. Science, 1966. 152(3730): p. 1755-1756.
47.Leach, C.L., et al., Partial liquid ventilation with perflubron in premature infants with severe respiratory distress syndrome. New England Journal of Medicine, 1996. 335(11): p. 761-767.
48.Hood, C.I. and J.H. Modell, A morphologic study of long-term retention of fluorocarbon after liquid ventilation. Chest, 2000. 118(5): p. 1436-1440.
49.Al-Rahmani, A., et al., Effects of partial liquid ventilation with perfluorodecalin in the juvenile rabbit lung after saline injury. Critical care medicine, 2000. 28(5): p. 1459-1464.
50.Murgia, X., et al., Aerosolized perfluorocarbon improves gas exchange and pulmonary mechanics in preterm lambs with severe respiratory distress syndrome. Pediatric research, 2012. 72(4): p. 393-399.
51.de Abreu, M.G., et al., Comparative Effects of Vaporized Perfluorohexane and Partial Liquid Ventilation in Oleic Acid–induced Lung Injury. The Journal of the American Society of Anesthesiologists, 2006. 104(2): p. 278-289.
52.Rambaud, J., et al., Hypothermic total liquid ventilation after experimental aspiration-associated acute respiratory distress syndrome. Annals of intensive care, 2018. 8(1): p. 1-9.
53.Kohlhauer, M., et al., A new paradigm for lung-conservative total liquid ventilation. EBioMedicine, 2020. 52: p. 102365.
54.Riess, J.G., Understanding the fundamentals of perfluorocarbons and perfluorocarbon emulsions relevant to in vivo oxygen delivery. Artificial cells, blood substitutes, and biotechnology, 2005. 33(1): p. 47-63.
55.Moradi, S., A. Jahanian-Najafabadi, and M.H. Roudkenar, Artificial blood substitutes: first steps on the long route to clinical utility. Clinical Medicine Insights: Blood Disorders, 2016. 9: p. CMBD. S38461.
56.Littlejohn, G.R. and J. Love, A simple method for imaging Arabidopsis leaves using perfluorodecalin as an infiltrative imaging medium. Journal of visualized experiments: JoVE, 2012(59).
57.Heymann, N. and F.-O. Lehmann, The significance of spiracle conductance and spatial arrangement for flight muscle function and aerodynamic performance in flying Drosophila. Journal of Experimental Biology, 2006. 209(9): p. 1662-1677.
58.Seelig, J.D. and V. Jayaraman, Feature detection and orientation tuning in the Drosophila central complex. Nature, 2013. 503(7475): p. 262-266.
59.Jägers, J., A. Wrobeln, and K.B. Ferenz, Perfluorocarbon-based oxygen carriers: From physics to physiology. Pflügers Archiv-European Journal of Physiology, 2021. 473(2): p. 139-150.
60.Hayashi, S. and T. Kondo, Development and function of the Drosophila tracheal system. Genetics, 2018. 209(2): p. 367-380.
61.Chien, C.-H., et al., Label-free imaging of Drosophila in vivo by coherent anti-Stokes Raman scattering and two-photon excitation autofluorescence microscopy. Journal of biomedical optics, 2011. 16(1): p. 016012.
62.Socha, J.J., et al., Real-time phase-contrast x-ray imaging: a new technique for the study of animal form and function. Bmc Biology, 2007. 5(1): p. 1-15.
63.Heidenthal, G., The occurrence of x-ray induced dominant lethal mutations in Habrobracon. Genetics, 1945. 30(2): p. 197.
64.Grosch, D.S., Induced lethargy and the radiation control of insects. Journal of Economic Entomology, 1956. 49(5): p. 629-631.
65.Beyenbach, K.W. and P.M. Piermarini, Osmotic and ionic regulation in insects, in Osmotic and Ionic Regulation. 2008, CRC Press. p. 231-278.
66.Golovynskyi, S., et al., Optical windows for head tissues in near‐infrared and short‐wave infrared regions: Approaching transcranial light applications. Journal of biophotonics, 2018. 11(12): p. e201800141.
連結至畢業學校之論文網頁點我開啟連結
註: 此連結為研究生畢業學校所提供,不一定有電子全文可供下載,若連結有誤,請點選上方之〝勘誤回報〞功能,我們會盡快修正,謝謝!
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
無相關期刊