可見光飛秒雷射對細菌活性降低之研究

Abstract

在近年來的研究中，可見光飛秒雷射被發現可應用於降低廣泛種類的微生物活性，其作用機制亦被發現與功率密度與雷射脈衝寬度有關，然而對於飛秒雷射與細菌間的交互作用而言，詳細的作用機制與理論基礎上仍有懸而未解的問題，在這篇論文中，將探討經雷射照射後大腸桿菌（Escherichia coli ）的細胞膜表面性質與完整性，細菌的新陳代謝率以及質體去氧核醣核酸的構形變化，我們的研究結果顯示當受到60分鐘的雷射照射後，細菌將出現細胞質洩漏、蛋白質聚集的現象，以及細胞膜的物理性質改變，同時亦能觀察到一受到雷射功率密度倚變的超螺旋質體去氧核醣核酸之弛豫現象。而在10分鐘的短時間雷射照射下，細菌的有氧葡萄糖細胞呼吸率可在細胞質洩漏並未被觀測到的情況下損失75%的活性，針對細胞呼吸電子傳遞鍊的進一步測試中，氧化還原酶的測試結果顯示飛秒雷射對於不同種類的酶與輔酶分子造成程度不一的破壞，細菌經雷射照射後，該迅速產生的呼吸抑制效應被認為在細菌活性降低的早期過程中扮演著重要的角色。Visible femtosecond laser is shown to be capable of selectively inactivating a wide spectrum of microorganisms in a power density and pulse width dependent manner. However, the mechanism of how visible femtosecond laser affects the viability of bacteria is still elusive. In this thesis, the cellular surface properties, membrane integrity, metabolic rate and plasmid DNA conformation of Escherichia coli (E. coli) irradiated by a visible femtosecond laser with different power density and exposure time were investigated. Our results showed that femtosecond laser treatment for 60 minutes (min) led to cytoplasmic leakage, protein aggregation, and alternation of the physical properties of E. coli cell membrane. A power density dependent genetic damage from laser induced relaxation of supercoiled plasmid DNA was observed as well. In comparison, a 10 min exposure of bacteria to femtosecond laser irradiation induced an immediate reduction of 75% of the glucose-dependent respiratory rate, while the cytoplasmic leakage was not detected. Results from enzymatic assays showed that oxidase and dehydrogenases involving in E. coli respiratory chain exhibited divergent susceptibility after laser irradiation. This early commencement of respiratory inhibition after a short irradiation is presumed to play a dominant effect on the early stage of bacteria inactivation.