The structural remodeling of collagens is important in several biological processes including wound healing, tendon repair and adaptation, fibrosis and morphogenesis. Multiphoton microscopy is efficient in the induction of highly specific second harmonic generation (SHG) signal from non-centrosymmetric macromolecules such as fibrillar collagens. Although the detectors in the reflection geometry have been normally employed for capturing the backward scattered SHG considering the wide range of engineered thick tissue applications, there are still questions about the generated 3D collagen structures because of the directional pattern of SHG signals. The present study dealt with an in vitro collagen-fibroblast raft or bioartificial tendon model where the stimulation of fibroblast cells induced lateral orientation of collagen Type I fibers. The SHG signals originating from 3D collagen matrix were captured simultaneously in both forward and backward scattering directions. Our structural analysis indicates that collagen fibers formed in such in vitro model systems are predominantly of uniform sizes and are aligned preferentially in the lateral direction. The criss-cross arrangements of laterally oriented fibers are evident in the initial stages of contraction but eventually those laterally oriented collagen fibers are found to be aligned in parallel to each other as well as to the fibroblasts after an extended period of contraction. Our comprehensive quantitative assessment of simultaneously captured forward and backward 3D SHG image datasets, which includes the SHG signal decay, fiber diameter, cell dimensions, colocalization profiles, the 3D voxel volumes and Fourier analysis, indicates strong correlation of structural features identified in forward and backward directions. (C) 2012 Elsevier Inc. All rights reserved.