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首页> 外文会议>Reporters, markers, dyes, nanoparticles, and molecular probes for biomedical applications III >Fluorescence lifetime imaging to quantify sub-cellular oxygen measurements in live macrophage during bacterial invasion
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Fluorescence lifetime imaging to quantify sub-cellular oxygen measurements in live macrophage during bacterial invasion

机译：荧光寿命成像可量化细菌入侵过程中活巨噬细胞中亚细胞氧的含量

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Fluorophore concentration, the surrounding microenvironment, and photobleaching greatly influence the fluorescence intensity of a fluorophore, increasing the difficulty to directly observe micro-environmental factors such as pH and oxygen. However, the fluorescence lifetime of a fluorophore is essentially independent of both the fluorophore concentration and photobleaching, providing a viable alternative to intensity measurements. The development of fluorescence lifetime imaging (FLI) allows for the direct measurement of the microenvironment surrounding a fluorophore. Pt-porphyrin is a fluorophore whose optical properties include a very stable triplet excited state. This energy level overlaps strongly with the ground triplet state of oxygen, making the phosphorescent lifetime directly proportional to the surrounding oxygen concentration. Initial experiments using this fluorophore involved the use of individual micro-wells coated with the porphyrin. Cells were allowed to enter the micro-wells before being sealed to create a diffusionally isolated volume. The decrease in the extracellular oxygen concentration was observed using FLI. However, this isolation technique provides only the consumption rate but cannot indicate the subcellular oxygen distribution. To improve upon this, live macrophages are loaded with the porphyrin and the fluorescence lifetime determined using a Lambert Instruments Lifa-X FLI system. Initial results indicate that an increase in subcellular oxygen is observed upon initial exposure to invasive bacteria. A substantial decrease in oxygen is observed after about 1 hour of exposure. The cells remain in this deoxygenated state until the bacteria are removed or cell death occurs.
机译：荧光团的浓度，周围的微环境和光漂白极大地影响了荧光团的荧光强度，增加了直接观察微环境因素（例如pH值和氧气）的难度。但是，荧光团的荧光寿命基本上与荧光团的浓度和光漂白无关，为强度测量提供了可行的替代方法。荧光寿命成像（FLI）的发展允许直接测量荧光团周围的微环境。铂卟啉是一种荧光团，其光学性质包括非常稳定的三线态激发态。该能级与氧气的基态三重态强烈重叠，从而使磷光寿命与周围的氧气浓度成正比。使用该荧光团的初始实验涉及使用涂有卟啉的单个微孔。在密封之前，允许细胞进入微孔，以创建扩散隔离的体积。使用FLI观察到细胞外氧浓度的降低。但是，这种隔离技术仅提供消耗速率，而不能指示亚细胞氧分布。为了改善这一点，在活的巨噬细胞中加载了卟啉，并使用Lambert Instruments Lifa-X FLI系统确定了荧光寿命。初步结果表明，初次接触侵入性细菌后，观察到亚细胞氧的增加。暴露约1小时后，观察到氧气大量减少。细胞保持脱氧状态，直到细菌被清除或细胞死亡。

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