以多光子顯微技術分析二甲基亞碸對果蠅幼蟲的影響

Abstract

研究神經科學的重要性已與日俱增，這個領域最具吸引力的生物組織研究就是大腦的研究，在此一環最重要的模式動物之一就是果蠅幼蟲。腦的研究之所以重要不僅是其具備其他組織上能表現的接收、傳遞與反應外，最重要的是具備了額外的思考機制。雖然在許多傑出的研究成果顯示大腦神經網路地圖已被基因工程學完整的標定出來，但這樣的結果只顯示了神經網路系統形式上的連結，而非真正功能上的連結。為了更進一步去證明兩者連結關係的關聯性，我們將利用鈣離子訊號染劑 (calcium dye) 與電位訊號染劑 (voltage sensitive dye) 配合共焦顯微技術 (confocal microscopy) 做為我們量測神經訊號的工具，然而這些染劑都必須透過二甲基亞碸 (DMSO) 此有機溶劑幫助溶解，因此我們希望透過非線性光學技術來檢驗DMSO是否會對果蠅幼蟲樣本的分子結構造成影響。根據二倍頻 (second harmonic generation-SHG) 信號產生必須與材料的分子結構有密切關係，因此二倍頻信號強弱變化將被分子結構的變化影響。我們分析了浸泡在等滲透壓的生理緩衝液 (PBS) 與DMSO中的樣本，發現浸泡DMSO樣本的二倍頻信號將明顯增強，這暗示樣本的分子結構已經在不同溶液下產生改變。我們也另外發現浸泡DMSO會導致基因轉殖的綠螢光蛋白 (green fluorescence protein-GFP) 在近紅外光激發下，雙光子螢光 (two-photon fluorescence) 信號有消失的跡象。相信這些結果對未來以果蠅幼蟲做為研究神經科學樣本的學者們提供了在細胞染色上值得注意的方向，也讓基因轉殖工程多了一份參考資料。

Neuron science research has been more and more popular recently. The most attractive organism in this study is brain, and one of the most famous model samples is drosophila larva. Although almost whole brain structure map studied by genetic engineering has been constructed, it still only provides structural connection, but not functional connection. In order to further prove the correlation of these two connections, we use calcium dye and voltage sensitive dye as our neuron signal indicator and construct a confocal microscopy to detect it. Both of these dyes should be dissolved into dimethyl sulfoxide (DMSO) solution, thus, analyzing the influence of DMSO on our sample drosophila larva is also important. According to the principle of second harmonic microscopy (SHG), signal intensity will depend on the molecular structure of samples. We analyze the SHG signal of our sample after soaking in phosphate buffered saline (PBS) solution and DMSO solution respectively. Samples soaked in DMSO lead to the enhancement of SHG signal, and that implies there would be some structural changes in our organism samples. We also find that the performance of green fluorescence protein (GFP) of two-photon fluorescence (TPF) will vanish after DMSO soaked. This result may provide a new perspective for those who want to load dyes on drosophila larvae.