基於單雷射之同時四波長飛秒激發源之建構與其於雙光子顯微術之應用

Construction of a Single-laser-based Simultaneous Four-wavelength Excitation Femtosecond Source for Two-photon Fluorescence Microscopy

蕭仰廷(Yang-Ting Hsiao)

指導教授 ： 孫啟光

國立臺灣大學/電機資訊學院/光電工程學研究所/碩士(2021年)

https://doi.org/10.6342/NTU202100899

摘要

在複雜的大腦神經系統中可視化上億個神經元間錯綜的連結需仰賴同時多色標記以及高解析度的非線性顯微鏡。為了獲得從腦研究中獲得更全面的資訊，生物標記的顏色數量也從典型的三色在近年增加到十色。然而，現有的雙光子激發源最多只能提供三個激發波長，這也導致需要至少四色的研究發展仍很有限。
因此我在本論文中提出一基於單個雷射、具有同時四波長輸出的雙光子激發源。利用中心波長位於1,070奈米摻鐿光纖雷射以及具有弱負色散的短光子晶體光纖，通過以28.5%之波長轉換效率產生一個具有雙峰值於812與960奈米的藍移光譜。另外也經由脈衝壓縮使該兩波長在時間上重合，透過合頻產生位於880奈米的虛擬激發波長，以此產生一個子50飛秒的三波長脈衝。將1,070奈米做為第四波長並與三波長脈衝結合後，我利用該光纖型態四波長激發光源有效率地激發神經科學中染色常用之病毒中的四種螢光蛋白，使該神經標記工具可應用於光雙子顯微術。該四波長激發源具有的更多激發波長與高於一般商用雷射的激發效率可在神經科學、生物分子探測、或臨床應用等領域，作為一通用且易取得的雙光子四色激發工具。

關鍵字

雙光子螢光顯微術 ； 同時多色成像 ； 四激發波長 ； 自相位調製 ； 合頻

並列摘要

Visualization of intricate connections between billons of neurons in brain requires the simultaneous labeling of different neuronal phenotypes through multicolor labeling and subsequent observation under high-resolution nonlinear optical microscopes. To gain more comprehensive insights in brain studies, the number of fluorescent tags for labeling has been increasing from typical three colors to ten colors till recent. However, the current two-photon excitation sources provide at most simultaneous three wavelengths, which has hampered research requiring at least four-color labeling.
In this thesis, I propose a single-laser-based, four-wavelength two-photon excitation source. By using a 1,070 nm Yb:fiber laser to pump a short photonic crystal fiber in a weak negative dispersion region, I generated a blue-shifted spectrum with dual peaks at 812 and 960 nm through efficient self-phase modulation (28.5%). By compressing the blue-shifted spectrum to temporally overlap the dual peaks, I generated virtual wavelength effectively at 880 nm through sum-frequency generation, and thus produced a sub-50 fs three-wavelength pulse. Combined with the 1,070 nm laser source as the fourth excitation wavelength, the fiber-format four-wavelength excitation source enabled simultaneous two-photon fluorescence imaging of four-XFP-based Brainbow AAV, which is one of the four-XFP labeling tool kits for facilitating neuroscience studies, by efficiently exciting the four encoded fluorescent proteins (TagBFP, mTFP, EYFP, and mCherry). With increased excitation wavelengths and improved excitation efficiency than a typical commercial laser, our four-wavelength excitation source can serve as a versatile and easily-accessible tool for four-color two-photon fluorescence imaging in the field of neuroscience, biomolecular probing, and clinical applications.

並列關鍵字

two-photon fluorescence microscopy ； simultaneous multicolor imaging ； four-wavelength excitation ； self-phase modulation ； sum-frequency generation

參考文獻

1. J. Livet, T. A. Weissman, H. Kang, R. W. Draft, J. Lu, R. A. Bennis, J. R. Sanes, and J. W. Lichtman, "Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system," Nature 450, 56-62 (2007).

2. D. Cai, K. B. Cohen, T. Luo, J. W. Lichtman, and J. R. Sanes, "Improved tools for the Brainbow toolbox," Nature Methods 10, 540-547 (2013).

3. L. Abdeladim, K. S. Matho, S. Clavreul, P. Mahou, J.-M. Sintes, X. Solinas, I. Arganda-Carreras, S. G. Turney, J. W. Lichtman, and A. Chessel, "Multicolor multiscale brain imaging with chromatic multiphoton serial microscopy," Nature Communications 10, 1-14 (2019).

4. M. Inoue, A. Takeuchi, S. Manita, S.-i. Horigane, M. Sakamoto, R. Kawakami, K. Yamaguchi, K. Otomo, H. Yokoyama, and R. Kim, "Rational engineering of XCaMPs, a multicolor GECI suite for in vivo imaging of complex brain circuit dynamics," Cell 177, 1346-1360. e1324 (2019).

5. C. Ricard, and F. C. Debarbieux, "Six-color intravital two-photon imaging of brain tumors and their dynamic microenvironment," Frontiers in Cellular Neuroscience 8, 57 (2014).