雙光子螢光超光譜顯微術於多螢光生物樣本之研究

The research of multi-fluorescence bio-specimens with two-photon fluorescence hyperspectral microscopy

陳亞婷(Ya-Ting Chen)

指導教授 ： 陳思妤

國立中央大學/理學院/光電科學與工程學系/碩士(2015年)

摘要

生物醫學工程之發展日益健全，而分子影像為其中相當重要的環節之一，為了解決傳統顯微鏡只能擷取樣本輪廓資訊的限制，我們將超光譜影像技術融合至雙光子螢光顯微系統中，研發出雙光子螢光超光譜顯微術，而此技術結合了雙光子螢光激發機制之優點，具有光學切片、高縱向解析度與較低光破壞等特性，以及超光譜影像能獲得光譜維度資訊的優點，希望可將此系統用於活體研究中，並藉由所得到之空間與光譜資訊以增加顯微技術之應用層面，使得研究人員對於實驗樣本有更多了解，同時減少單單依賴樣本輪廓進行判定所造成的失誤機率。
本論文欲使用先前開發出之平行接收雙光子螢光超光譜顯微術進行實驗，並測試系統效率與解析度分析。除此之外，本論文最主要目的是試著將此套顯微系統實際應用至生物研究中，欲解決傳統顯微技術對於具有多重螢光來源樣本之限制，以光譜線性分離法提升光譜重疊現象的判斷能力。在生物研究中將分為兩部分進行，第一部分以所培養之動物細胞進行多螢光標定作為實驗樣本，進行光譜線性分離法之驗證，第二部分則利用老鼠皮膚組織進行實驗，將系統與分析法實際應用於醫療研究，試著將可能影響醫生影像判斷之二倍頻訊號與自發螢光濾除以驗證系統效能。

關鍵字

雙光子螢光 ； 超光譜顯微術 ； 線性分離法 ； 多螢光標定 ； 生物研究

並列摘要

The development of biomedical engineering has gradually improved, and molecular imaging has been one of its most important techniques. To solve the limitation of a conventional microscope that merely detects the morphology of samples, we’ve combined hyperspectral imaging with two photon fluorescence microscopy and designed the setup of two photon fluorescence hyperspectral microscopy. This neoteric technique includes the advantages of both microscopies, providing deeper depth penetration, less photo damage to sample, higher axial resolution, and the addition of receiving spectral information of the targeted sample simultaneously. With these properties, it’s more suitable for biomedical research in vivo, it also provides more information of the samples to researchers, and reduces the misjudgment probabilities of researches that are dependent on the morphology.
Using the previously developed system, a non-de-scanned two-photon fluorescence hyperspectral microscope with parallel recording, experiments of cells and sectioning skin samples were carried out to test the efficiency of the system. Furthermore, the major aim is to try to actually apply this microscopy to biological researches and to overcome the limitation of multiple fluorescence labeling samples. Multispectral images might generate crosstalk and increase the difficulty of fluorescence discrimination. Therefore, we applied the applicably spectral analysis, linear unmixing, to improve the spectral discriminating ability.
In the biological research, there were two parts of experiments. In the first part, cultured cell lines were labeled with multiple fluorescence dyes and the hyperspectral images of these cells were analyzed to show the ability and correctness of linear unmixing. In the second part, mouse skin tissues with hair follicles being fluorescently labeled were used as samples. According to the experimental results and the spectral analyses, the fluorescence and second harmonic generation signals was easily separated. The unexpected signal sources, second harmonic generation, can be removed to avoid influencing doctor’s diagnoses. The two-photon hyperspectral imaging combined with linear unmixing has shown its ability to biomedical fields.

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