Imaging of collagen matrix remodeling in three-dimensional space using second harmonic generation and two photon excitation fluorescence

Second harmonic generation (SHG) a nonlinear optical phenomenon exhibits several in-common characteristics of twophoton excited fluorescence (TPEF) microscopy. These characteristics include identical equipment requirements from experiment to experiment and the intrinsic capability of generating 3-dimensional (D) high resolution images. Structural protein arrays that are highly ordered such as collagen produce strong SHG signals without the need for any exogenous label (stain). SHG and TPEF can be used together to provide information on structural rearrangements in 3D space of the collagen matrix associated with various physiological processes. In this study we used SHG and TPEF to detect cellmediated structural reorganization of the extracellular collagen matrix in 3D space triggered by dimensional changes of embedded fibroblasts. These fibroblasts were cultured in native type I collagen gels and were stimulated to contract for a period of 24 hours. The gels were stained for cell nuclei with Hoechst and for actin with phalloidin conjugated to Alexa Fluor 488. We used non-de-scanned detectors and spectral scanning mode both in the reflection geometry for generating the 3D images and for SHG spectra respectively. We used a tunable infrared laser with 100-fs pulses at a repetition rate of 80-MHz tuned to 800-nm for Hoechst and Alexa 488 excitations. We employed a broad range of excitation wavelengths (800 to 880-nm) with a scan interval of 10 nm to detect the SHG signal. We found that spectrally clean SHG signal peaked at 414-nm with excitation wavelength of 830-nm. The SHG spectrum has a full width half maximum (FWHM) bandwidth of 6.60-nm which is consistent with its scaling relation to FWHM bandwidth 100-fs excitation pulses. When stimulated to contract we found the fibroblasts to be highly elongated as well as interconnected in 2D space and the collagen matrix in the form of a visibly clear fibril structure accumulated around the cells. In the absence of contraction on the other hand the cells were predominantly round in shape and no sign of collagen accumulation around the cell was evident despite the presence of SHG signal as well as the fibrillar collagen morphology in the collagen matrix. We here conclude that SHG in conjunction with TPEF can serve as a noninvasive method to provide spatially resolved 3D structural reorganization of collagen matrices triggered by various physiological processes.