全域式光學同調斷層掃描術用於角膜細胞影像與頻譜資訊分析

Abstract

全域式光學同調斷層掃描術(Full-field optical coherence tomography; FF-OCT)是一種無標定、高解析度的三維影像技術，廣泛應用於生物醫學影像領域。本研究採用摻鈰釔鋁石榴石(Ce3+:YAG)晶體光纖作為寬頻光源，中心波長560 nm、頻寬為99 nm，使用米繞式干涉儀與高數值孔徑的物鏡，搭配壓電控制器及高速攝影機，空間解析度約為1 μm，非常適合細胞影像的量測。 動態全域式光學同調斷層掃描術(Dynamic full-field optical coherence tomography; D-FF-OCT) 是一種基於現有FF-OCT技術的頻譜資訊分析方法。本研究優化實驗室現有的D-FF-OCT分析方法，利用適當的影像降噪及歸一化方式，建立一種分析速度快，時間解析度7.6 ms，高色彩鮮豔度的平均頻率暨標準差D-FF-OCT影像分析流程，可用於細胞動態信號判讀以及細胞型態觀察。 研究探討B4G12角膜內皮細胞株在不同狀態下的動態特性，結果顯示，活細胞的動態資訊在頻率分析區間0.062-129 Hz與固定後細胞未顯現出明顯差異。施用不同抑制劑於B4G12細胞，發現糖解作用抑制劑(2-DG)對角膜內皮細胞的型態有顯著影響，故推論糖解作用為B4G12角膜內皮細胞相當重要的代謝路徑。 研究中發現，利用平均頻率暨標準差D-FF-OCT影像與白光顯微鏡相比，更能觀察細胞內部構造，我們發現細胞膜在低頻表現、細胞質在中頻表現、細胞核在高頻表現，利用此特性結合信號標準差大小，可作為一細胞影像標註的方式。頻譜分析中，我們發現位於低頻0.252 Hz與高頻120 Hz均有特徵頻率，這些特徵頻率在實驗中證實為外界擾動導致，代表本系統對於外界擾動非常敏感，有潛力用於光學同調斷層掃描彈性量測(Optical coherence elastrography; OCE)，我們透過相位分析的方式，嘗試解讀其細胞在特徵頻率的相位資訊。

Full-field optical coherence tomography (FF-OCT) is a label-free, high-resolution 3D imaging technique widely used in the biomedical imaging field. In this study, we used Ce3+crystal fiber as a broadband light source with a central wavelength of 560 nm and a bandwidth of 99 nm. A mirau interferometer and high numerical aperture objective were employed, along with a piezo controller and a high-speed camera, achieving a spatial resolution of approximately 1 μm, making it highly suitable for cellular imaging. Dynamic full-field optical coherence tomography (D-FF-OCT) is a spectral information analysis method based on the existing FF-OCT technology. This study optimized the lab's existing D-FF-OCT analysis method by employing appropriate image denoising and normalization techniques. We established an analysis process for mean frequency and standard deviation D-FF-OCT imaging that is quick to perform, with a temporal resolution of 7.6 ms and high color saturation, suitable for interpreting cell dynamic signals and observing cell morphology. The study explored the dynamic characteristics of B4G12 corneal endothelial cells in different states. The results showed that the dynamic information of live cells did not differ significantly from fixed cells within the frequency analysis range of 0.062-129 Hz. Moreover, the glycolysis inhibitor 2-DG significantly affected the morphology of corneal endothelial cells, suggesting that glycolysis is a crucial metabolic process for corneal cells. We also found that using mean frequency and standard deviation D-FF-OCT imaging, compared to white light microscopy, better identifies internal cell structures. The cell membrane shows in low frequencies, the cytoplasm in mid frequencies, and the nucleus in high frequencies. Spectral analysis revealed specific frequencies at low (0.252 Hz) and high (120 Hz) frequencies, which were confirmed to be caused by external disturbances in the experiments. This sensitivity to external disturbances indicates potential for the system to be used in optical coherence elastography (OCE) . We attempt to interpret the phase information of cells at characteristic frequencies through phase analysis.