Abstract

Fluorescence lifetime measurements of long excited-state lifetime, oxygen-quenched ruthenium dyes are emerging as methods for intracellular oxygen sensing. Fluorescence lifetime imaging microscopy (FLIM) studies in cells have been reported previously. Many current FLIM systems use high repetition rate (∼107 Hz) lasers optimized for nanosecond lifetime measurements, making measurement of long, microsecond lifetime fluorophores difficult. Here, we present an experimental approach for obtaining a large temporal dynamic range in a FLIM system by using a low repetition rate (101 Hz), high output, nitrogen pumped dye laser and a wide-field, intensified CCD camera for image detection. We explore the feasibility of the approach by imaging the oxygen-sensitive dye tris(2,2′-bipyridyl)dichloro-ruthenium(II) hexahydrate (RTDP) in solution and in living cells. We demonstrate the ability of the system to resolve 60% variations in RTDP fluorescence lifetime upon oxygen cycling in solution. Furthermore, the FLIM system was able to resolve an increase in RTDP fluorescence lifetime in cultured human epithelial cells under diminished oxygen conditions. The technique may be useful in developing methods for quantifying intracellular oxygen concentrations.

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call

Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.