Fluorescence lifetime imaging to quantify sub-cellular oxygen measurements in live macrophage during bacterial invasion

Abstract

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 microwells 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.

Intracellular probes for imaging oxygen concentration: how good are they?

“…Not many icO 2 probes have been evaluated with representative panels of different cell types. Some probes were only demonstrated with easy-to-load phagocytic cells which effectively ingest particles and solutes from surrounding media [42][43][44]. Also staining of heterogeneous tissue samples containing different cell types or multi-cellular aggregates can be complex, or limited mainly to the surface layer (spheroids) or one particular cell type.…”

Millisecond lifetime imaging with a europium complex using a commercial confocal microscope under one or two-photon excitation

“…However, lifetime measurements and imaging on micro-to millisecond timescales under the microscope remain much less common. Recently, long-lifetime imaging methods were reported by using multiple frequency phase modulation in wide eld microscopy 48,49 or time-correlated single photon counting in laser scanning 2P microscopy. 50 These advances are possible due to instrumental improvements of the intensied CCDs and pulsed laser control, but they remain technically challenging, rather costly and require substantial modications to the microscope conguration.…”