多模態顯微鏡觀察經紫外線照射損傷果蠅之研究

Abstract

果蠅一直是發展生物學亮眼的活體模型。其體積小，容易繁殖並可大量飼養，因此果蠅成了生物學家研究的新寵兒。已有許多研究說明紫外線對於生物體造成傷害，在果蠅模型上也指出照射紫外線會造成DNA 的變異，導致其子代會有基因缺損的問題。太陽光90%的波長來自於紫外線光(UVA)，其波長介於315-400 奈米(nm)之間，波長越長穿透力越深，可以達到真皮層導致曬黑並造成生物體表的傷害。 本研究選用UVA 400nm 紫外光刺激，觀察不同時間點累積性的UVA刺激果蠅幼蟲，使幼蟲產生曬傷(sunburn)，並利用多種非侵入式顯微鏡系統，研究果蠅幼蟲的行為模式變化與細胞狀態的改變及肌肉結構與影像強度的分析。藉由非侵入式的顯微鏡觀察，從巨觀的果蠅行為到微觀的細胞層次，發現經UVA刺激後的果蠅幼蟲在溫水逃脫實驗下，幼蟲的行為會隨著UVA照射時間的累加，其扭曲身體的次數會增加，運動速率也有明顯的差異，代表其曬傷後導致幼蟲對於水溫的感受越劇烈。而雙光子顯微鏡下觀察到表皮細胞經UVA傷害後的影像，表皮細胞逐漸壞死，血球細胞因發炎反應而聚集。果蠅體內的肌肉結構也可透過二倍頻顯微鏡觀察到隨著時間UVA 的累積傷害，其肌球蛋白越來越鬆散，並觀察到有雙重帶(double band)的產生。 結合多種模式的影像分析方法，提供了非侵入式觀察果蠅幼蟲經UVA 傷害後巨觀到微觀的影像，提供對於研究果蠅或是其他生物體長期照射UVA的影像並瞭解曬傷的組織改變，除了表皮細胞損傷及發炎之外，對於淺層肌球蛋白的結構與功能也有相當程度的影響。

Drosophila larvae model is wildly used in the field of developmental biology.Since Drosophila are small in size, easily bred and can be kept in a large amount, they are now the new favorite of the biologists. There has been a many of researches on the mutant effect ultraviolet rays have on DNA, and there is no exception on the Drosophila model; ultraviolet rays cause damages on the DNA of the offspring. 90% of the sunlight consists of UVA, whose wavelength falls within the range of 315-400 nm. The longer the wavelength, the stronger the penetration through the skin, and UVA causes sun-tanned and damage to the surface of the body when it reaches dermis. In this study we used 400 nm UVA as the stimulus to cause sunburn on the Drosophila larvae, observe its effect at different duration time points with multimodal non-invasive microscopy systems, and investigate the changes in the behavioral pattern and the status of the cells of the larvae, as well as analyze the muscle structure of the larvae and its image intensity. Through non-invasive microscope observation of the Drosophila from macro behavioral aspect to micro cellular aspect, this study found that the larvae struggled more times in the warm water escape experiment after the UVA stimulation; its movement rate also increased significantly, suggesting that sunburn causes larvae to be more sensitive to the water temperature. The necrosis of the epidermal cells and the aggregation of the hemocyte due to inflammation can be observed on the image of the epidermis layer under the two-photon microscope. The muscle structure and function of the larvae as the UVA exposure accumulated can also be observed through second harmonic generation microscopy, the myosin loosened and double band appeared. The combination of various image analyzing methods yielded macro and micro images of Drosophila larvae after UVA exposure in a non-invasive imaging, and provided insight into the tissue change after long-term UVA exposure and sunburn of Drosophila or other organisms; besides cellular damage and inflammation, UVA also exert an influence on superficial myosin structure and muscle function.