Department of Chemical Engineering and Materials Science, University of California, Irvine, Irvine, CA, 92697, USA.
Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, Irvine, CA, 92697, USA.
Sci Rep. 2017 Jun 16;7(1):3743. doi: 10.1038/s41598-017-04032-w.
Bacterial populations exhibit a range of metabolic states influenced by their environment, intra- and interspecies interactions. The identification of bacterial metabolic states and transitions between them in their native environment promises to elucidate community behavior and stochastic processes, such as antibiotic resistance acquisition. In this work, we employ two-photon fluorescence lifetime imaging microscopy (FLIM) to create a metabolic fingerprint of individual bacteria and populations. FLIM of autofluorescent reduced nicotinamide adenine dinucleotide (phosphate), NAD(P)H, has been previously exploited for label-free metabolic imaging of mammalian cells. However, NAD(P)H FLIM has not been established as a metabolic proxy in bacteria. Applying the phasor approach, we create FLIM-phasor maps of Escherichia coli, Salmonella enterica serovar Typhimurium, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus epidermidis at the single cell and population levels. The bacterial phasor is sensitive to environmental conditions such as antibiotic exposure and growth phase, suggesting that observed shifts in the phasor are representative of metabolic changes within the cells. The FLIM-phasor approach represents a powerful, non-invasive imaging technique to study bacterial metabolism in situ and could provide unique insights into bacterial community behavior, pathology and antibiotic resistance with sub-cellular resolution.
细菌群体表现出一系列受环境、种内和种间相互作用影响的代谢状态。在其天然环境中识别细菌的代谢状态和状态之间的转变，有望阐明群落行为和随机过程，例如抗生素耐药性的获得。在这项工作中，我们采用双光子荧光寿命成像显微镜 (FLIM) 为单个细菌和细菌群体创建代谢指纹图谱。以前曾利用自发荧光还原型烟酰胺腺嘌呤二核苷酸 (磷酸)，即 NAD(P)H，对哺乳动物细胞进行无标记代谢成像。然而，NAD(P)H FLIM 尚未在细菌中确立为代谢替代物。通过应用相位向量方法，我们在单细胞和群体水平上创建了大肠杆菌、鼠伤寒沙门氏菌、铜绿假单胞菌、枯草芽孢杆菌和表皮葡萄球菌的 FLIM-相位向量图谱。细菌的相位向量对环境条件（如抗生素暴露和生长阶段）敏感，这表明观察到的相位向量的变化代表了细胞内的代谢变化。FLIM-相位向量方法是一种强大的、非侵入性的原位研究细菌代谢的成像技术，它可以提供对细菌群落行为、病理学和抗生素耐药性的独特见解，具有亚细胞分辨率。
