Fluorescence lifetime imaging of articular cartilage

Introduction  Fluorescence lifetime imaging (FLIM) provides a contrast parameter for tissue components independent of wavelength, spectrum or polarization. It has been shown that contrast between autofluorescent matrix components such as collagen and elastin is available through FLIM (Siegel et al. 2003; Dowling et al. 1998), which is not clear using other fluorescent techniques. The lifetime of collagen has been studied whilst in solution; however, research is ongoing to quantify its fluorescence whilst in tissue. The aim of this study was to investigate the contrast available by applying fluorescence lifetime imaging (FLIM) to articular cartilage, which has extracellular matrix containing collagen type-II and proteoglycan.

Materials and methods  A time-gated FLIM system was used to analyse the articular cartilage from unstained sections of human femurs as well as from the knee joints young and old sheep. The sections were also observed under a conventional fluorescence microscope, and for the human femurs, adjacent H&E-stained sections were also obtained. Details of the FLIM system can be found elsewhere (Dowling et al. 1998), with the laser source being tuneable, allowing excitation over a range 350–600 nm. Fluorescence half-lives were obtained using single exponentials applied to the intensity decay data.

Results  It was found that younger cartilage was generally less fluorescent than older. For example, when exciting at 401 nm and observing above 450 nm, it was found that the young sheep samples provided a signal to noise ratio too low to obtain lifetime data, whereas other samples fluoresced brightly. It was also found that within the matrix, FLIM provided contrast unavailable with conventional fluorescence microscopy and, in the case of human femurs, with H&E stains.

Discussion  In the study of collagen, it is believed that the cross-linkages are responsible for fluorescence (Richards-Kortum & Sevick-Muraca 1996). Because the cross-linkages increase with age, the fluorescence intensity is also expected to increase. The lesser amount of fluorescence from the younger samples therefore suggests that the dominant fluorophore in cartilage is collagen. The contrast obtained with FLIM in the cartilage indicates nonuniformity in the matrix structure.

Acknowledgements  This work was supported by the EPSRC, BBSRC, Kentech Instruments, a DTI Beacon award and a Wellcome Trust Showcase award.