Extracellular oxygen concentration mapping with a confocal multiphoton laser scanning microscope and TCSPC card

Neveen A. Hosny; David A. Lee; Martin M. Knight

doi:10.1117/12.842281

26 February 2010 Extracellular oxygen concentration mapping with a confocal multiphoton laser scanning microscope and TCSPC card

Neveen A. Hosny, David A. Lee, Martin M. Knight

Author Affiliations +

Proceedings Volume 7569, Multiphoton Microscopy in the Biomedical Sciences X; 756932 (2010) https://doi.org/10.1117/12.842281
Event: SPIE BiOS, 2010, San Francisco, California, United States

Abstract

Extracellular oxygen concentrations influence cell metabolism and tissue function. Fluorescence Lifetime Imaging Microscopy (FLIM) offers a non-invasive method for quantifying local oxygen concentrations. However, existing methods show limited spatial resolution and/or require custom made systems. This study describes a new optimised approach for quantitative extracellular oxygen detection, providing an off-the-shelf system with high spatial resolution and an improved lifetime determination over previous techniques, while avoiding systematic photon pile-up. Fluorescence lifetime detection of an oxygen sensitive fluorescent dye, tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate [Ru(bipy)₃]²⁺, was measured using a Becker&Hickl time-correlated single photon counting (TCSPC) card with excitation provided by a multi-photon laser. This technique was able to identify a subpopulation of isolated chondrocyte cells, seeded in three-dimensional agarose gel, displaying a significant spatial oxygen gradient. Thus this technique provides a powerful tool for quantifying spatial oxygen gradients within three-dimensional cellular models.

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Neveen A. Hosny, David A. Lee, and Martin M. Knight "Extracellular oxygen concentration mapping with a confocal multiphoton laser scanning microscope and TCSPC card", Proc. SPIE 7569, Multiphoton Microscopy in the Biomedical Sciences X, 756932 (26 February 2010); https://doi.org/10.1117/12.842281

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