胞外泌體從細胞產生，可以用來與相鄰細胞或遠處細胞溝通，胞外泌體係一含有跨膜蛋白的脂雙層結構，內含細胞質內的蛋白、核醣核酸、去氧核醣核酸及脂質；根據細胞狀態，會分泌不同種類胞外泌體。現今，胞外泌體影像面臨一些挑戰，如：微小的胞外泌體成像，以及在細胞及活體之間，觀察胞外泌體釋放及運送的動態，為了克服這些問題，觀察胞外泌體的影像需仰賴特定的報導基因，以及健全的活細胞分子影像技術做為輔助；在此，我們使用一個光學的報導基因去標定胞外泌體，優化胞外泌體追蹤影像；我們融合了增強綠色螢光蛋白(enhanced fluorescence protein; eGFP) 和Nanoluc® (Nluc) 冷光素酶，以及參考過去棕櫚酰化的基因片段PalmGFP，精確標定於細胞膜內，使胞外泌體產出時帶有更高比率之報導基因蛋白。藉由基質furimazine的利用，產生的冷光經由生物發光共振能量轉移（bioluminescence resonance energy transfer, BRET）激發螢光，使報導蛋白兼具有螢光強度的優勢及冷光訊號雜訊比低的長處，發展高檢測靈敏度與長時間穩定的光源，作為研究胞外泌體分子影像的工具。
在本篇研究指出，棕櫚酰化的生物發光共振能量轉移的報導蛋白，可運用在胞外泌體及細胞，是一個創新且優化標定胞外泌體的工具，在研究胞外泌體上，希望可以克服在體內之胞外泌體觀測深度、動態胞外泌體釋放及交換情形；藉由此嶄新之報導基因蛋白，搭配高解析度的共軛焦顯微鏡技術，可以在細胞和胞外泌體兩者作為即時觀測使用。

Cells can communicate with neighboring and distant cells via extracellular vesicles (EVs). EVs are lipid bilayered structures composed of cytosolic proteins, RNAs, DNAs and lipids. Cells secrete different types of EVs from different sub-cellular origin. Recently, EV imaging needs optimal reporter to visualize EVs subpopulations, observing specific labelled EVs in vitro and in vivo and dynamics of EV biogenesis. Due to these reasons, EVs delivery requires specific reporters and robust live-cell imaging technology. Here we create an optical reporter to label EVs for optimal tracking of EVs utilizing eGFP fused with Nluc sequence and palmitoylation sequence. Palmitoylation can anchor a reporter to the inner leaflet of a lipid bilayer. Using its substrate furimazine, bioluminescence light produces and excites fluorescent light via bioluminescence resonance energy transfer (BRET). Fluorescence emits higher intensity light and bioluminescence offers better signal-to-noise ratio. This reporter provides a long-term and brighter light for studying EVs.
In this study, GpNluc and PalmGpNluc reporter protein can be applied in both cells and EVs. We believed that these advantages combined together is an optimal tool can be applied in EV tracking and visualization. Acting as an imaging platform for both in vitro and in vivo compare with other state-of-the-art EV labeling methods.

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