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

Joe Dragavon; Megdouda Amiri; Benoit Marteyn; Philipe Sansonetti; Spencer Shorte

doi:10.1117/12.875430

11 February 2011 Fluorescence lifetime imaging to quantify sub-cellular oxygen measurements in live macrophage during bacterial invasion

Joe Dragavon, Megdouda Amiri, Benoit Marteyn, Philipe Sansonetti, Spencer Shorte

Author Affiliations +

Proceedings Volume 7910, Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications III; 791019 (2011) https://doi.org/10.1117/12.875430
Event: SPIE BiOS, 2011, San Francisco, California, United States

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.

Citation Download Citation

Joe Dragavon, Megdouda Amiri, Benoit Marteyn, Philipe Sansonetti, and Spencer Shorte "Fluorescence lifetime imaging to quantify sub-cellular oxygen measurements in live macrophage during bacterial invasion", Proc. SPIE 7910, Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications III, 791019 (11 February 2011); https://doi.org/10.1117/12.875430

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