Journal
JOURNAL OF BIOMEDICAL OPTICS
Volume 17, Issue 1, Pages -Publisher
SPIE-SOC PHOTO-OPTICAL INSTRUMENTATION ENGINEERS
DOI: 10.1117/1.JBO.17.1.016007
Keywords
chondrocytes; oxygen gradients; ruthenium; extracellular; fluorescence lifetime; multi-photon microscopy; fluorescence lifetime imaging microscopy; time-correlated single photon counting
Funding
- Biotechnology and Biological Sciences Research Council
- Engineering and Physical Sciences Research Council
- Engineering and Physical Sciences Research Council [EP/E046975/1] Funding Source: researchfish
- EPSRC [EP/E046975/1] Funding Source: UKRI
Ask authors/readers for more resources
Fluorescence lifetime imaging microscopy offers a non-invasive method for quantifying local oxygen concentrations. However, existing methods are either invasive, require custom-made systems, or show limited spatial resolution. Therefore, these methods are unsuitable for investigation of pericellular oxygen concentrations. This study describes an adaptation of commercially available equipment which has been optimized for quantitative extracellular oxygen detection with high lifetime accuracy and spatial resolution while avoiding systematic photon pile-up. The oxygen sensitive fluorescent dye, tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate [Ru(bipy)(3)](2+), was excited using a two-photon excitation laser. Lifetime was measured using a Becker & Hickl time-correlated single photon counting, which will be referred to as a TCSPC card. [Ru(bipy)(3)](2+) characterization studies quantified the influences of temperature, pH, cellular culture media and oxygen on the fluorescence lifetime measurements. This provided a precisely calibrated and accurate system for quantification of pericellular oxygen concentration based on measured lifetimes. Using this technique, quantification of oxygen concentrations around isolated viable chondrocytes, seeded in three-dimensional agarose gel, revealed a subpopulation of cells that exhibited significant spatial oxygen gradients such that oxygen concentration reduced with increasing proximity to the cell. This technique provides a powerful tool for quantifying spatial oxygen gradients within three-dimensional cellular models. (c) 2012 Society of Photo-Optical Instrumentation Engineers (SPIE). [DOI: 10.1117/1.JBO.17.1.016007]
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available