Design of a confocal fluorescence spectrometer for transdermal observation of green fluorescent protein expression in cartilage

We have developed a noninvasive confocal fluorescence spectrometer to measure in vivo fluorescence spectra in mice. Our motivation is the study of the healing process of wounded cartilage in the murine model. The spectrometer measures the expression of green fluorescent protein (GFP) which is linked to the promoter of type II collagen as a marker for the expression of the chondrocyte phenotype. The confocal system uses an argon ion laser (488 nm) to excite fluorescence in a confocal volume about 200 um below the skin surface in the cartilage of the xyphoid process. In vitro studies showed how the refractive mismatch at the surface boundary and the light scattering of the tissue affect the depth and size of the confocal volume. A thin fluorescent layer was detected through a 130 um tissue phantom (murine skin) by moving the confocal volume axially through the phantom. The presence of skin increased the axial full width at half maximum of the confocal response from 7 um to 72 um. In conclusion, we achieve a low-spatial-resolution confocal spectrometer, which yields a fluorescence spectrum that maximizes the GFP fluorescence of cartilage and minimizes the skin autofluorescence.