模組化之定量微分相位差顯鏡之設計開發

Design and Development of Modular Microscopy for Acquiring Isotropic Quantitative Differential Phase Contrast Images

林鈺梓(Yu-Zi Lin)

指導教授 ： 黃光裕

共同指導教授 ： 駱遠(Yuan Luo)

國立臺灣大學/工學院/機械工程學研究所/碩士(2018年)

https://doi.org/10.6342/NTU201801654

摘要

顯微鏡下觀察微薄且透明的細胞，常需要透過染色來提升對比和解析能力，但是染色卻會引發光毒(Phototoxicity)和光致褪色(Photo-bleaching)問題。染色光毒讓細胞會停止生長而逐漸死亡，為了不破壞細胞生存本質，免標記的顯微技術顯得格外重要。
免標記顯微技術可分為定性與定量兩種，定量免標記技術可以同時量測到細胞光學厚度，有助於分析探討細胞變化歷程，本論文研究目的在於開發定量免標記顯微技術。相較於其他免標記量化相位顯微技術，本論文以高效率影像擷取的寬場(Wide field)量測以及量化微分相位差(Quantitative Differential Phase Contrast, QDPC)顯微技術為基礎，進行模組化之定量微分相位差顯微鏡之設計開發。
本論文研究使用薄膜電晶體板放置於傅立葉平面來產生結構光，透過薄膜電晶體板的可變光圈控制達成光強度的調變。研究提出漸層結構光的概念，漸層式光強度調變圖樣由相機取得2組共4張的互補影像，再透過Matlab數學軟體來求得樣本的相位差資訊。相較於目前其他研究團隊提出的對比結構光(Contrast structured light)多次擷取影像方法，彩色漸層式的光強度調變圖樣可以在較低擷取次數下獲得等向性的相位轉換函式，大幅提升了相位重建的效率以及準確度。
實驗驗證使用微塑膠球來測試系統的定量量測能力和準確性，且展示完整的重建影像，並由圖片的相位差值和微塑膠球的折射係數，進而推得微塑膠球的幾何厚度。此外，透過開發的自動拍攝程式進行3T3小鼠纖維細胞和CYL2人體肺癌細胞長時間拍攝觀察，重建後的影像具有高對比且不受樣本角度影響，可以清楚地觀察到細胞萎縮過程和詳細構造變化。當3T3小鼠纖維細胞萎縮時，相位差值逐漸由~1.5 rad 增加至~2.5 rad。而當CYL2人體肺癌細胞萎縮時，相位差值逐漸由~4.5 rad降至~0.25 rad。由相位差值可以進一步推得細胞的光學厚度，相對的幾何尺寸變化數據有助於細胞歷程之探討分析。

關鍵字

光學顯微術 ； 結構光照明 ； 可變光圈控制 ； 相位重建 ； 微分相位差影像

並列摘要

Because cells are thin and transparent objects, they usually have to be dyed to be observable at a bright field microscope. But fluorescently labeled methodology will lead to a phototoxicity and photo-bleaching problem. After observing dyed cells, they will stop growing and gradually die. In order to observe a transparent cell without poisoning them, it is important to develop label-free microscopic techniques.
Label-free microscopic techniques include qualitative and quantitative types. Since a quantitative label-free microscope can provide quantitative optical thickness which is helpful to analyze the process of cells changes. The purpose of this thesis is to develop quantitative label-free microscopy. Due to the advantages of wide-field based measurement, quantitative differential phase contrast microscopy (QDPC), compared to other label-free quantification phase microscopic techniques, it is more efficient to capture images without point-by-point plane scanning or depth scanning.
In this study, a thin-film transistor (TFT) shield is placed on the Fourier plane to generate structured light by variable pupil control. After light passes through the TFT shield, the intensity of the light is modulated by the proposed gradient light intensity modulation pattern. After two sets of 4 complementary images are acquired, the phase contrast value of a specimen is obtained by using lab developed Matlab software. Compared with the method proposed by other research groups, which utilized contrast structured light for multiple image acquisition to achieve an isotropic phase transfer function. The color gradient light intensity modulation pattern further makes the system achieve isotropic phase transfer functions at high acquisition speed. The proposed method improves the efficiency and accuracy of phase reconstruction.
For experimental verification, micro-plastic spheres have been used standard targets to verify the quantitative measurement capability and accuracy of proposed QDPC system. With the measured phase contrast value and the refractive index of the microspheres, the geometric thickness of the microspheres can be calculated. Furthermore, the developed automatic microscopic image-acquisition is used for acquiring time-lapse QDPC photography of mouse label-free 3T3 fibroblasts cells and human lung cancer cells CYL2 . QDPC reconstructed images provide high contrast and the reconstruction result is independent of viewing angles. The detailed structures and apoptosis process of cells can be clearly observed. While the 3T3 fibroblasts cells shrinks, their phase difference value gradually increases from ~1.5 to ~2.5 rad. While the lung cancer cells CYL2, their phase difference value gradually decrease from ~4.5 to ~2.5 rad. The optical thickness of the cells can be further calculate, and quantitative thickness change data is helpful for the analysis of the cells’ long-time monitoring.
Keyword：Optical microscopy, Structured illumination, Variable pupil control, Phase retrieval, Differential phase contrast imaging

並列關鍵字

Optical microscopy ； Structured illumination ； Variable pupil control ； Phase retrieval ； Differential phase contrast imaging

參考文獻

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