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標題: | 利用雙光子螢光及二倍頻顯微術觀察綠膿桿菌對角膜的感染作用 The Observation of Interaction Between Pseudomonas aeruginosa and Cornea by the Use of Two-Photon Fluorescence and Second Harmonic Generation Microscopy |
作者: | Yu-Lin Chang 張鈺林 |
指導教授: | 董成淵(Chen-Yuan Dong) |
關鍵字: | 雙光子螢光,二倍頻,顯微術,角膜,感染,綠膿桿菌, two-photon fluorescence,second harmonic generation,microscopy,cornea,infection,Pseudomonas aeruginosa, |
出版年 : | 2007 |
學位: | 碩士 |
摘要: | 眼睛是我們用來接收與探索浩瀚世界最直接的感官,而角膜則位於眼睛的最前線,因此也最容易受到外來異物的侵襲與破壞,造成角膜炎,嚴重會影響視力造成失明的傷害。在隱形眼鏡愈來愈普及的趨勢下,因隱形眼鏡配戴習慣不正確造成角膜感染的議題也逐漸受到重視,而綠膿桿菌正是其中最重要的一種感染病菌,其會附著在角膜上,然後伺機地破壞角膜組織,綠膿桿菌毒性相當猛烈,故其破壞的效率相當地快,角膜大概一至二天就會被摧殘潰爛,因此即時診斷出角膜感染的病原體是相當重要的。利用低破壞性的雙光子螢光及二倍頻非線性光學技術可以讓我們清楚地觀察角膜的表皮細胞及膠原蛋白纖維組織的型態及結構。我們選擇牛眼角膜做組織培養,然後再對各個樣品注射細菌溶液或生理緩衝液(PBS),對個別感染的角膜樣品注射的區域附近在不同的時間點取影像。隨著注射後的時間增加,角膜基質的膠原蛋白被破壞的程度也顯著的增加,細菌也有增殖的趨勢以及向周圍散落蔓延,並且在角膜內有大量的自發螢光物質產生。而在對照組中,角膜基質的膠原蛋白訊號沒有衰減的很多,並且沒有大量的自發螢光訊號產生。我們可以藉此初步的實驗做一些型態上的探討,至於定性定量的分析,以及定位的影像可能還要再做實驗方法的改良。我們期盼此多光子顯微術在日後可以應用在醫學臨床上,輔助醫師不需做破壞性的組織切片來直接診斷角膜感染的程度並加以即時治療。 The eyes are the most direct sensory organ in our body to receive and explore the world, and the corneas residing in the outmost frontline easily result in suffering invasion and destruction by external matter, lead to keratitis, affect vision ability and lose vision seriously. In the generalization of contact lens develops day by day, on the issue associated with corneal infection due to incorrect habit of contact lens is put much emphasis. Pseudomonas aeruginosa is one of the most important infectious pathogens, it will adhere on the cornea and destroy the corneal tissue opportunistically with its quite violent virulence. The infected corneas will be attacked to necrosis by one to two days with very fast destructive speed. It is very important to diagnosis the infectious pathogen in time. We can investigate the morphology and the structure of the corneal epithelium and stroma collagen by the use of minimally invasive two-photon fluorescence and second harmonic generation microscopy. We culture the bovine cornea as our specimens, and inject into each specimen with bacteria suspension or PBS, and image the region nearby the injection hole of each cornea at different time. As the time went by, there are more increase in the severity of the stromal collagen destruction, the more outspreading pattern and proliferation occurred in bacteria activity, and more abundant substance with auto-fluorescence within the cornea. For our controls, there are a little decay of the signals of stromal collagen, and no emergence of the huge auto-fluorescence signals. We can make the morphological discuss via these preliminary experiment, and as for the quality, quantity analysis, and the fixed point observation should need the refinement of the experimental methods. We expect that this multi-photon microscopy could apply in clinic in the future, to assist doctors in diagnosing the degree of corneal infection and treating in time without destructive histology biopsy. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/28065 |
全文授權: | 有償授權 |
顯示於系所單位: | 物理學系 |
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