自動對焦平台結合誘導螢光系統應用於細胞生長觀測之可行性研究

Feasibility Study of Application of Autofocus Platform Combined with Induced Fluorescene System in Cell Growth Observation

金怡傑(Jin Yi-Jie)

指導教授 ： 王耀男

國立屏東科技大學/工學院/車輛工程系所/碩士(2020年)

https://doi.org/10.6346/NPUST202000348

摘要

本研究開發了一個基於對比度檢測方法所製作的被動式自動對焦平台，使其可放置在細胞培養箱內，觀測微觀尺度下的已染色細胞之明視野/螢光強度衰減/螢光顏色變化的生長情形。其中本平台在不同時段下拍攝相同孔位位置的視野時，其水平或垂直方向上的平均誤差都在10 µm以內，而在對已染色細胞的長時間拍攝中(總拍攝時間最少20小時/最多40小時以上)，本研究經過圖像清晰度算法比較分析後，採用Sobel算法去計算明視野圖像的清晰度；螢光圖像的清晰度則是使用NVAR算法去進行計算，以提高成功對焦之機率，結果發現在所有拍攝到的圖像中，都沒有出現失焦或模糊致無法辨認細胞輪廓之情形，並且都可以很清楚地看出每個時間點下所拍攝圖像的細胞生長、螢光強度變化及螢光顏色變化等現象，可見這二算法應用於生物微觀圖像對焦作業的優異性。在分析各時間點下所拍攝的已染色細胞圖像中，發現了在染CFSE染劑的STO細胞中添加LPS(脂多醣)的話，其細胞生長狀況與無添加藥劑組(CTRL組)幾乎無異，但若添加BAC+AXI(黃芩苷+血管內皮細胞生長因子抑制劑)的話，則會造成細胞生長初期時，抑制其生長速率之作用；而在染FUCCI染劑的STO細胞中，則可知STO細胞的G1/ S過渡期，至少會持續23小時以上，其中還可以看出少數細胞的顏色變化現象。另外在基於圖像處理方法的細胞檢測法之評估中，得知了使用圖像處理方法對細胞進行計數是可行的；但若是將其方法使用在平均螢光強度(MFI)計算的話，則將會是非常具有挑戰性的一項作業，而在對染CFSE染劑的STO細胞之計數結果中，得出CTRL組LPS組BAC+AXI組的細胞倍增時間分別為16.1小時、17.4小時及20.1小時；細胞分裂指數則為84.8 %、82.5 %及77.9 %。

關鍵字

自動對焦 ； 圖像清晰度算法 ； LabVIEW ； 細胞生長 ； 平均螢光強度

並列摘要

This research has developed a passive autofocus platform based on the contrast detection method, which can be placed in a cell incubator to observe the bright field/fluorescence intensity attenuation/fluorescent color change of stained cells at the microscopic scale growth situation. Among them, when the platform was shooting the field of view of the same hole position at different time periods, the average error in the horizontal or vertical direction is within 10 µm, while in the long-term shooting of stained cells (the total shooting time was at least 20 hours/up to 40 hours or more), after comparing and analyzing image sharpness algorithms, this study uses Sobel algorithm to calculate the sharpness of bright field images; the sharpness of fluorescent images was calculated using NVAR algorithm to improve success probability of focusing. As a result, it was found that in all the captured images, there was no out-of-focus or blurring to make it impossible to recognize the cell outline, and it was possible to clearly see the cell growth, fluorescence intensity changes and fluorescence color changes in the images taken at each time point, show the superiority of these two algorithms when applied to biological microscopic image focusing tasks. In analyzing the stained cell images taken at each time point, it was found that
when LPS (Lipopolysaccharide) was added to STO cells stained with CFSE stain, the cell growth status was almost the same as that of the non-additive group (CTRL group), but if BAC+AXI (Baicalin+Vascular Endothelial Growth Factor Inhibitor) was added, it will inhibit the growth rate of cells in the early stage of cell growth; while in STO cells stained with FUCCI, it will it can be seen that the G1/S transition period of STO cells will last at least 23 hours, and the color change of a few cells can also be seen. In addition, in the evaluation of the cell detection method based on the image processing method, it was known that feasible to use the image processing method to count cells; but if the method is used in the calculation of the mean fluorescence intensity (MFI), the it will be a very challenging task, and in the counting results of STO cells stained with CFSE stain, the cell doubling time of the CTRL groupLPS groupBAC+AXI group was 16.1 hours, 17.4 hours and 20.1 hours; cell division index was 84.8 %, 82.5 % and 77.9 %.

並列關鍵字

Autofocus ； Image sharpness algorithm ； LabVIEW ； Cell growth ； Mean Fluorescence Intensity

參考文獻

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