3D CARS image reconstruction and pattern recognition on SHG images

Nonlinear optical imaging techniques based e.g. on coherent anti-Stokes Raman scattering (CARS) or second-harmonic generation (SHG) show great potential for in-vivo investigations of tissue. While the microspectroscopic imaging tools are established, automized data evaluation, i.e. image pattern recognition and automized image classification, of nonlinear optical images still bares great possibilities for future developments towards an objective clinical diagnosis. This contribution details the capability of nonlinear microscopy for both 3D visualization of human tissues and automated discrimination between healthy and diseased patterns using ex-vivo human skin samples. By means of CARS image alignment we show how to obtain a quasi-3D model of a skin biopsy, which allows us to trace the tissue structure in different projections. Furthermore, the potential of automated pattern and organization recognition to distinguish between healthy and keloidal skin tissue is discussed. A first classification algorithm employs the intrinsic geometrical features of collagen, which can be efficiently visualized by SHG microscopy. The shape of the collagen pattern allows conclusions about the physiological state of the skin, as the typical wavy collagen structure of healthy skin is disturbed e.g. in keloid formation. Based on the different collagen patterns a quantitative score characterizing the collagen waviness - and hence reflecting the physiological state of the tissue - is obtained. Further, two additional scoring methods for collagen organization, respectively based on a statistical analysis of the mutual organization of fibers and on FFT, are presented.