利用拉曼顯微光譜技術與成像法觀測在分子層級下活體的分裂酵母之細胞活性

Abstract

此篇論文從許多會影響分裂酵母菌的細胞活性的因素中，針對酵母菌生長的溫度與其在細胞生長曲線中所處的成長階段作為探討的主軸，並且利用拉曼顯微光譜技術與成像法深入探討分裂酵母菌在分子層級下的活體生物活性。首先，我們探討1602 cm-1的拉曼訊號在各種溫度下訊號強度的變化，由文獻得知，1602 cm-1的拉曼訊號的強度和酵母菌的代謝活性是息息相關的。而我們的結果顯示，酵母菌的1602 cm-1的拉曼訊號在32 ℃的環境中，會達到最高值，而後此訊號隨著溫度上升逐漸地下降。在35 ℃時，降幅達到最大值約45%。但當培養環境超過35 ℃時，1602 cm-1的拉曼訊號將不再下降。此處1602 cm-1的拉曼訊號的在高溫的培養環境中逐漸地下降，代表著分裂酵母菌在高溫下具有較低的細胞活性。接著，我們利用拉曼光譜成像法探討分裂酵母菌在對數生長期與穩定生長期兩種不同生長階段時，生物分子在細胞中的分佈與組成的變化。實驗結果顯示，在穩定生長期時，酵母菌中的磷脂質和蛋白質生物分子是區域化分佈於酵母菌的兩端。然而，在對數生長期時，磷脂質和蛋白質生物分子的分佈卻呈現均勻分佈於整個酵母菌。對於此生物分子組成的變化，我們認為也許是和酵母菌的能量儲存作用相關。因為在穩定生長期時，酵母菌會將磷脂質區域化於細胞的兩端，以利於能量的儲存。然而，對數生長期中的酵母菌是處於快速分裂且不斷生長的狀態，生產出的蛋白質和脂質大部分馬上被當作複製DNA 的成分來源，所以它們不需要像在穩定生長期一樣被大量儲存。是故拉曼光譜影像顯示，對數生長期中的酵母菌並不具有明顯地脂質區域化分佈。

A number of physiological parameters and conditions profoundly influence the bioactivity of cells in various ways. Here, among these parameters and conditions, we investigated the effects of growth temperature and phase of the growth curve on the cellular activities of Schizosaccharomyces pombe by using Raman microspectroscopy and imaging, in vivo and at the molecular level. First, in the study of the temperature effects on yeast Raman spectra, we focus on the variation of the intensity of the band at 1602 cm−1, which is a Raman band that is highly relevant to metabolic activities of the yeast cells. The result shows that at ~32 °C, the intensity of the 1602 cm−1 band begins to decrease gradually by approximately 45% and seems to reach a plateau above ~ 35 °C. This decrease in the 1602 cm−1 band intensity is indicative of lowered metabolic activity of the yeast cells at such high temperatures. Second, Raman imaging was performed on S. pombe cells in different growth phases, i.e., log and stationary phases, in order to examine how these growth phases affect molecular compositions and distributions in S. pombe cells. The observed Raman images revealed that the distributions of phospholipids and proteins are usually localized at the two sides of yeast cells in the stationary phase, while in the log phase, they appear to be more uniformly distributed over the whole cell. We interpret the localization of lipids in the stationary phase in terms of substantial energy storage in some particular regions of S. pombe cells. Compared with the stationary phase, the produced proteins and phospholipids may be consumed immediately for DNA duplication and other events, there may be no need for them to be stored in a large amount in the log phase. As a result, the cells do not show lipid localization as evidently as those in the stationary phase.