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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 駱遠(Yuan Luo) | |
dc.contributor.author | Ju-Hsuan Chien | en |
dc.contributor.author | 簡如萱 | zh_TW |
dc.date.accessioned | 2021-06-17T04:41:08Z | - |
dc.date.available | 2021-08-30 | |
dc.date.copyright | 2018-08-30 | |
dc.date.issued | 2018 | |
dc.date.submitted | 2018-08-06 | |
dc.identifier.citation | 1. Engelbrecht, C.J. and E.H. Stelzer, Resolution enhancement in a light-sheet-based microscope (SPIM). Optics letters, 2006. 31(10): p. 1477-1479.
2. Boxem, M., Cyclin-dependent kinases in C. elegans. Cell division, 2006. 1(1): p. 6. 3. Benninger, R.K. and D.W. Piston, Two‐photon excitation microscopy for the study of living cells and tissues. Current protocols in cell biology, 2013: p. 4.11. 1-4.11. 24. 4. Olarte, O.E., et al., Light-sheet microscopy: a tutorial. Advances in Optics and Photonics, 2018. 10(1): p. 111-179. 5. Siedentopf, H. and R. Zsigmondy, Uber sichtbarmachung und größenbestimmung ultramikoskopischer teilchen, mit besonderer anwendung auf goldrubingläser. Annalen der Physik, 1902. 315(1): p. 1-39. 6. Huisken, J., et al., Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science, 2004. 305(5686): p. 1007-1009. 7. Preibisch, S., et al., Software for bead-based registration of selective plane illumination microscopy data. Nature methods, 2010. 7(6): p. 418. 8. Keller, P.J., et al., Fast, high-contrast imaging of animal development with scanned light sheet–based structured-illumination microscopy. Nature methods, 2010. 7(8): p. 637. 9. Tomer, R., et al., Quantitative high-speed imaging of entire developing embryos with simultaneous multiview light-sheet microscopy. Nature methods, 2012. 9(7): p. 755. 10. Chardès, C., et al., Setting up a simple light sheet microscope for in toto imaging of C. elegans development. Journal of visualized experiments: JoVE, 2014(87). 11. Lin, H.-C.A., et al. Light-sheet microscopy for quantitative ovarian folliculometry. in Diagnosis and Treatment of Diseases in the Breast and Reproductive System. 2017. International Society for Optics and Photonics. 12. Ahrens, M.B., et al., Whole-brain functional imaging at cellular resolution using light-sheet microscopy. Nature methods, 2013. 10(5): p. 413. 13. Wu, Y., et al., Inverted selective plane illumination microscopy (iSPIM) enables coupled cell identity lineaging and neurodevelopmental imaging in Caenorhabditis elegans. Proceedings of the National Academy of Sciences, 2011: p. 201108494. 14. Wu, Y., et al., Spatially isotropic four-dimensional imaging with dual-view plane illumination microscopy. Nature biotechnology, 2013. 31(11): p. 1032. 15. Weber, M., M. Mickoleit, and J. Huisken, Light sheet microscopy, in Methods in cell biology. 2014, Elsevier. p. 193-215. 16. Maruno, T., E. Toda, and K. Bennett. Comparison of CMOS and EMCCD Cameras for Computational Imaging with Application to Super-resolution Localization Microscopy. in Imaging Systems and Applications. 2012. Optical Society of America. 17. Becker, K., et al., Ultramicroscopy: light-sheet-based microscopy for imaging centimeter-sized objects with micrometer resolution. Cold Spring Harbor protocols, 2013. 2013(8): p. pdb. top076539. 18. Kaufmann, A., et al., Multilayer mounting enables long-term imaging of zebrafish development in a light sheet microscope. Development, 2012. 139(17): p. 3242-3247. 19. Schmid, B., et al., High-speed panoramic light-sheet microscopy reveals global endodermal cell dynamics. Nature communications, 2013. 4: p. 2207. 20. Arrenberg, A.B., et al., Optogenetic control of cardiac function. Science, 2010. 330(6006): p. 971-974. 21. Huisken, J. and D.Y. Stainier, Even fluorescence excitation by multidirectional selective plane illumination microscopy (mSPIM). Optics letters, 2007. 32(17): p. 2608-2610. 22. Vyas, S., Y.-H. Chia, and Y. Luo. Multiplexed volume holographic gratings for simultaneous generation of Airy and Dual Airy beams. in Optical Manipulation Conference. 2018. International Society for Optics and Photonics. 23. Vyas, S., Y.-H. Chia, and Y. Luo. Angular Multiplexed Volume Holograms for Simultaneous Generation of Airy Beam Shapes. in Digital Holography and Three-Dimensional Imaging. 2018. Optical Society of America. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/70855 | - |
dc.description.abstract | 即時顯微影像是研究生物解剖構造、機制和過程不可或缺的工具,其中傳統光學顯微鏡是最主要,也是最常用的工具,但礙於樣本的體積,傳統顯微鏡常無法取得合適的影像,因此有越來越多的影像技術被發展出來,例如共軛焦顯微鏡術,以配合研究上日益增高的需求。雷射共軛焦掃描顯微鏡需要以點對點的方式掃描標本因此掃描時間隨著樣本體積增大而延長,若掃描時間長,並不合適用來觀察活的標本,另外,由於雷射共軛焦掃描顯微鏡直接照明整個樣本,包括聚焦及失焦的部分,因此會增加光漂白和光毒性的可能。為了克服這個缺點,本研究設計及發展光切片顯微鏡,以得到快速的光切片影像,將有助於觀測有體積的活樣本。
光切片顯微鏡也被稱為層光螢光顯微術,是由兩條光路所構成,一條為照明光路,另一條為偵測光路,照明光路利用圓柱鏡將光單一方向的聚焦,使光束形成一層薄片,只有被薄片光照射或激發且同時位於偵測光路物鏡的焦點平面上的樣本才能成像,該層影像由二維偵測器(例如感光耦合原件或互補式金屬氧化物半導體)快速記錄下來,即可獲得即時非侵入性的光學切片影像。光切片顯微鏡具有低光漂白和低光毒性的優點,因此適合用來觀察活體樣本,並可進行影像的三度空間重組。 有很多論文及報告將光切片顯微鏡應用於斑馬魚、果蠅及秀麗隱桿線蟲的胚胎等,但卻一直無法使用光切片顯微鏡成功的觀察活體秀麗隱桿線蟲,主要是因為活體秀麗隱桿線蟲無法存於水中,而水是光切片顯微鏡的觀察環境。此外,在觀測過程中蟲體的抽動也是一大問題。要解決以上觀測的困境需要設計特別的容器來放置線蟲並配合適當的樣本處理方法。此篇論文,藉由自製的樣本容器及穩定的電動馬達平台,配合三種樣本處理方式,設計及發展出適合觀察活標本的光切片顯微鏡,成功的觀察活的秀麗隱桿線蟲的生殖系統,包含其活體內的卵及胚胎。 為了測試自製光切片顯微鏡的影像效果,我們比較同一個樣本在我們自製的影像系統與明視野顯微鏡及傳統螢光顯微鏡的影像,發現使用我們的系統能得到較高對比與較高解析度的影像,使我們得以觀察活體秀麗隱桿線蟲。此篇論文展示光切片顯微鏡的實驗設計與架構,並藉由觀察活體秀麗隱桿線蟲來測試系統的影像結果。 | zh_TW |
dc.description.abstract | Imaging a volumetric sample in real time is required to directly observe biological anatomy, mechanisms and process. There are a variety of microscopic imaging systems, such as confocal scanning microscopy, developed to provide fine optical sectioning capability. However, the main drawbacks of currently existing confocal systems are long aquisition time and severe photobleaching since confocal scans point by point and illuminates all the sample including in-focus and out-of-focus position. To overcome such problems and to obtain high-speed optical sectioning images, we have demonstrated light sheet microscopy (LSM) and shown real-time images which are benefit of thick live samples.
The LSM, which is also known as selective plane illuminaiton microscopy, consists of two light ways. One is the illumination part and the other is the detection part. The light source is modulated to a thin laminar light by a cylindrical lens which focuses light into a line. Only a sample at the detection objective lens, excited by the laser at the same time, can produce an image. The 2D image will be recorded rapidly by a detector, such as CCD or sCMOS, which obtains optical sectioning, non-invasive and real-time images. LSM is suitable for three-dimensional observation of a live sample because of its low photobleaching and phototoxicity. Many papers have applied LSM for observing zebra fish, dorsophila, Canenorhabditis elegans (C. elegans) embryo and so on. However, there has only rare reports using LSM to image live C. elegans because the worm is hardly to be maintained in water, which is an important medium for the water-immersed objective used in LSM to image. Besides, the movement of the worm may worsen the situation. To conquer such problems, it is necessary to design a special sample holder to keep the worms and to develop an appropriate sample mounting methods. In this thesis, we have designed a light sheet microscope using a highly precise motor stage and developed an unique sample holder and three sample mounting methods for observing live thick samples. By using our LSM, we have successfully observed the reproductive system of a C. elegans, including oocytes and embryos. To see if our LSM has higher image quality, we use our LSM, bright-field microscopy and conventional fluorescence microscopy to image the same sample of a C. elegans, and compared their image qualities. We found that through our LSM, we could obtain high contrast, high resolution and high-speed optical sectioning images. This thesis has experimentally presented and characterized LSM’s experimental design and setup. In addition, we demonstrated that this LSM was able to obtain three-dimensional images of a C. elegans in vivo. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T04:41:08Z (GMT). No. of bitstreams: 1 ntu-107-R05458001-1.pdf: 3030882 bytes, checksum: 49a1e235e52e91f964e32503da219863 (MD5) Previous issue date: 2018 | en |
dc.description.tableofcontents | 誌謝 i
中文摘要 ii ABSTRACT iv CONTENTS vi LIST OF FIGURES ix LIST OF TABLES xii LIST OF SYMBOLS xiii Chapter 1 Introduction 1 1.1 Motivation and Purpose 3 1.2 Optics Perspective of LSM 4 1.2.1 LSM is low photobleaching, phototoxicity and price 4 1.2.2 LSM is suitable for 3D imaging 7 1.2.3 LSM can image thick, low scattering sample 7 1.2.4 LSM has fast scanning rate 8 1.3 Literature reviews 8 1.4 Outline 9 Chapter 2 Principle of LSM 10 2.1 LSM Illumination 10 2.1.1 Light sheet property 10 2.1.2 How to generate a light sheet 11 2.1.3 Theory 12 2.2 LSM Detection 14 2.2.1 Theory 14 2.2.2 2D detective array 16 2.3 LSM Configurations 18 2.4 Sample Mounting 21 2.4.1 Solid gel cylinder 21 2.4.2 Tube embedding 22 2.5 Image Processing 23 Chapter 3 Optical Setup and Sample Preparation 25 3.1 Light Sheet Microscopy Setup 25 3.2 Home-made Components 26 3.2.1 Motorized stage 26 3.2.2 Chamber 28 3.2.3 Sample holder 28 3.2.4 Sample holder fixator 29 3.3 Sample Preparation 30 3.3.1 Sample preparation method I 31 3.3.2 Sample preparation method II 32 3.3.3 Sample preparation method III 34 Chapter 4 Results and Discussion 36 4.1 Light Sheet Thickness 36 4.2 Lateral Resolution 40 4.3 Photobleaching Measurement 44 4.4 C. elegans Image Results 45 4.4.1 Comparison between different sample preparation methods 45 4.4.2 Comparison in different microscopies 48 Chapter 5 Conclusions and Future Directions 51 5.1 Conclusions 51 5.2 Future directions 51 5.2.1 Change to two-sided illumination 51 5.2.2 Image C. elegans in 3D image by timelapse sequences 52 5.2.3 Use other beam shaping techniques 55 REFERENCES 57 | |
dc.language.iso | en | |
dc.title | 設計與發展光切片顯微鏡術並應用其觀察活體秀麗隱桿線蟲 | zh_TW |
dc.title | Design and Develop Light Sheet Microscopy for Live Canenorhabditis elegans | en |
dc.type | Thesis | |
dc.date.schoolyear | 106-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 蔡瑞章(Jui-Chang Tsai),吳瑞菁(Jui-Ching Wu) | |
dc.subject.keyword | 光學切片,光切片顯微鏡,層光螢光顯微術, | zh_TW |
dc.subject.keyword | Optical-sectioning microscopy,light sheet microscopy,selectve plane illumination microscopy, | en |
dc.relation.page | 58 | |
dc.identifier.doi | 10.6342/NTU201802345 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2018-08-06 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 醫療器材與醫學影像研究所 | zh_TW |
顯示於系所單位: | 醫療器材與醫學影像研究所 |
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