Polarization-Resolved SHG towards Collagen Imaging

Abstract

Collagen is a triple-helix protein that self-assembles into supramolecular fibrils, essential for the biomechanical integrity and physical properties of biological tissues and organs. Second Harmonic Generation(SHG) microscopy exploits a second order nonlinear coherent optical process to image macromolecules with non-centrosymmetric structure, as collagen fibers(1). Such a label-free approach provides three-dimensional(3D) imaging of the structural organization of the collagen fibers in tissues at a sub-cellular resolution with intrinsic optical sectioning, high deep penetration and reduction of out-of-focus photobleaching and phototoxicity(2). Here, we collected the endogenous SHG signal of the collagen to evaluate the hierarchical structure, organization and orientation of collagen fibers in different types of specimen, from ex-vivo biological tissues to bioengineered samples(3). To conduct this study, we coupled a femtosecond Ti:Sapphire Chameleon Ultra II laser(Coherent, Santa Clara, CA, USA) with a Leica TCS STED-CW confocal microscope(Leica-Microsystems, Mannheim, Germany). This allowed collecting the forward, the backward SHG signal and the TPE autofluorescence of the samples(2). Moreover, we also focused our attention on the SHG signal as function of polarization states of the incoming light to distinguish among different collagen fiber types and orientations(4). Analysis of the polarization of the outcoming light and the ratio FSHG/BSHG was carried out due to its potential in providing more detailed information about the sample, for instance a relationship with the diameter and the functional and structural organization of the collagen fibers variability in various tissues. For example, this allows realizing a set of features useful to evaluate the assembly of artificial tissues depending on the scaffold role in the collagen fibers assembly when mimicking a target tissue.1. Chen X. et al. Nat Protoc. 7(4),654(2012).2. Bianchini P. et al., J. Biophotonics, 1,6(2008).3. Scaglione S. et al. Nature Sci. Rep. 2,(274): 1-6(2012).4. Odin C. et al, Opt.Express, 16,20(2008).