Second harmonic generation imaging of collagen matrix remodeling in a stimulated 3D cellular environment: forward versus backward detection

The structural remodeling of collagen is important in several biological processes such as wound healing, tendon repair, fibrosis and developmental morphogenesis. Multiphoton microscopy, which uses ultra-short femto-second laser pulses as an excitation source, is efficient in the multiphoton excitation fluorescence (MPEF) of exogenous fluorescent labels tagged to various cellular macromolecular objects, as well as in the induction of a highly specific second harmonic generation (SHG) signal from non-centrosymmetric macromolecules such as fibrillar collagens. Although the non-descanned detectors in the reflection geometry have normally been employed for capturing the backward scattered SHG as well as the MPEF signals, considering the wide range of engineered thick tissue imaging applications, there are still un-answered 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 model in which the stimulation of fibroblast cells induced the lateral orientation of collagen molecules. The SHG signals originating from the 3D collagen matrix were captured simultaneously in both forward and backward scattering directions to understand the collagen structural differences and to generate a comprehensive understanding of collagen matrix remodeling.