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In this study, polarization second harmonic generation (SHG) imaging is used and data analysis is developed to gain contrast and to discriminate with pixel resolution, in the same image, SHG source architectures. We use mammalian tissue in which both skeletal muscle and fibrilar collagen can be found. The images are fitted point by point using an algorithm based on a biophysical model, where the coefficient of determination is utilized as a filtering mechanism. For the whole image we retrieve for every pixel, the effective orientation, θe, of the SHG active structures. As a result a new image is formed which its contrast depends on the values of θe. Collagen presented in the forward direction for a predefined region of interest (ROI), peak distribution of angles θe centered in the region of ∼45°, while muscle in the region of ∼65°. Consequently, collagen and muscle are represented in different colors in the same image. Thus, here we show that it is possible to gain contrast and to discriminate between collagen and muscle without the use of any exogenous labeling or any co-localization with fluorescence imaging. © 2009 SPIE-OSA.This work is supported by the Generalitat de Catalunya and by the Spanish government grant TEC2006-12654 SICO. Authors also acknowledge The Centre Innovacio i Desenvolupament Empresarial-CIDEM (RDITSCON07-1-0006), Grupo Ferrer and the European Regional Development Fund. This research has been partially supported by Fundació Cellex Barcelona.Peer Reviewe

Contrast enhancement in second harmonic imaging: discriminating between muscle and collagen