Two-photon spectral imaging microscopy of skin tissues

The last two decades saw the emergence of spectroscopy and microscopic imaging as techniques for tissue diagnostics. The biochemical state of the tissue is revealed by spectroscopy, while the morphological information is visualized by microscopic imaging. Little research has been carried out to diagnose tissues based on the combination of spectroscopy and microscopic imaging. Here, we report on tissue spectroscopy and microscopic imaging employing two-photon excitation of tissue autofluorescence and second harmonic generation. We designed and constructed a prism-based spectral imaging system coupled to a two-photon microscope. Full emission spectra with a 1-7 nm spectral resolution covering 330nm to 600nm can be recorded at a maximum rate of 500 spectra per second equivalent to about 0.5 frames/min (224x224 pixels). We present results on spectral imaging of human skin sections and in-depth imaging of pig skin tissue. Different skin layers show clear differences in their intrinsic emission spectral signature that can be used for diagnosis.