在眾多生質柴油原料中，微藻具有高含油量且生長快速的優點，因此被視為未來最適合的生質柴油來源。然而，使用測量程序複雜且耗時的氣相層析法或克重法測量油量及篩選藻種，使得篩選過程相當費時。自1940年代建立了光學方式，利用Nile red分析微藻細胞內的含油量後，Nile red就被廣泛地運用在偵測細胞內的脂質體。然而，Nile red與油脂結合後散射出的螢光強度會被許多變數影響(例如:染色時間及樣品濃度)，進而影響實驗結果的正確性。且傳統Nile red染色法並無法測得單細胞油量，提供篩選所需的資訊。微流體系統具有快速檢測與可準確控制變數的優點，因此本研究致力於發展快速且準確分析微藻細胞內油含量及篩選的技術。
本研究使用Chlorella vulgaris(小球藻)來進行油量測量，首先利用二維聚焦微流體流式細胞儀測量其被Nile red染色後的螢光強度，其檢測的速率可達1000 細胞/分鐘。然而，藉由染色後的平均細胞螢光強度與油含量比較，發現訊號強度並不隨著油量增加而有明顯的線性關係。為了避免微藻細胞通過偵測區時，因z軸位置上的不同造成的誤差，所以使用三維聚焦微流道做進一步檢測。並利用螢光染劑與螢光粒子測試三維微流體流式細胞儀聚焦效果。進行油量測量實驗後發現，染劑吸附所造成的背景過高，以致無法正確判別訊號。本研究的最後，利用三維微流體流式細胞儀中產生的離心力分離不同大小與形狀的小球藻與柵藻。

Among various sources of biodiesels, microalgae have high lipid contents and fast growth rate; therefore, they have been considered promising candidates. However, conventional methods for screening and measuring lipid amounts such as gas chromatograph and gram measurement are often complex or time consuming. An optical method that uses Nile red to analyze the lipid abundance inside cells was established in 1940. Since then, Nile red has been extensively applied to detect lipid bodies inside cells. However, the induced fluorescence intensity varies with the labeling durations or concentrations of reagents. It is challenging to find a universal correlation for lipid amounts and the fluorescence intensity. Additionally, conventional ensemble detection could not measure signal from single microalgae cell. Single cell information provides fundamentals for the screening of biological samples. Microfluidic devices provide advantages such as precisely controlled reaction parameters, high throughput and single cell analysis. Therefore, this study focused on developing a rapid and straightforward analysis for measuring the neutral lipid abundance inside microalgae and for cell screening using microfluidic systems.
Microalgae (Chlorella vulgaris) containing different lipid abundances were first analyzed using two-dimensional focusing cytometry and the throughput can be as high as 1000 cells/min. However, the signal intensity from microalgae did not increase proportionally with the lipid abundance. We subsequently used a three-dimensional focusing cytometry to prevent the signal shift due to the variation of sample positions in the z-direction. The three-dimensional focusing was demonstrated using fluorescein solution and fluorescent microbeads. Although the three-dimensional focusing was achieved, Nile red adsorbed on PDMS surface and caused a high floor of noise. The poor signal-to-noise ratio prevented us from further investigation of the relationship between signal intensities and lipid amounts. In the last part of this study, we used the three-dimensional focusing flow cytometry to separate microalgae that have different sizes and shapes.

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