Confocal fluorescence spectroscopy of subcutaneous cartilage expressing green fluorescent protein versus cutaneous collagen autofluorescence

Optically monitoring the expression of green fluorescent

protein (GFP) in the cartilage underlying the skin of a mouse allows

tracking the expression of the chondrocyte phenotype. This paper

considers how confocal microscopy with spectral detection can sense

GFP fluorescence in the cartilage despite light scattering and collagen

autofluorescence from the overlying skin. An in vivo experiment

tested the abilities of a topical optical fiber measurement and a confocal

microscope measurement to detect GFP in cartilage under the

skin versus the collagen autofluorescence. An ex vivo experiment

tested the ability of a confocal microscope without and with its pinhole

to detect a fluorescent microsphere underneath an ex vivo skin

layer versus the collagen autofluorescence. In both systems, spectroscopic

detection followed by linear analysis allowed spectral discrimination

of collagen autofluorescence (MC) and the subdermal green

fluorescence (MG) due to either GFP or the microsphere. Contrast

was defined as MG /(MG1MC). The in vivo contrast for GFP using

optical fiber and confocal measurements was 0.16 and 0.92, respectively.

The ex vivo contrast for a fluorescent microsphere using a confocal

system without and with a pinhole was 0.13 and 0.48, respectively.

The study demonstrates that a topical optical fiber

measurement is affected by collagen autofluorescence, while a confocal

microscope can detect subdermal fluorescence while rejecting

collagen autofluorescence.