The structural origin of second harmonic generation in fascia

Abstract

Fascia tissue is rich in collagen type I proteins and can be imaged by second harmonic generation (SHG) microscopy. While identifying the overall alignment of the collagen fibrils is evident from those images, the tridimensional structural origin for the observation of SHG signal is more complex than it apparently seems. Those images reveal that the noncentrosymmetric (piezoelectric) structures are distributed heterogeneously on spatial dimensions inferior to the resolution provided by the nonlinear optical microscope (sub-micron). Using piezoresponse force microscopy (PFM), we show that an individual collagen fibril has a noncentrosymmetric structural organization. Fibrils are found to be arranged in nano-domains where the anisotropic axis is preserved along the fibrillar axis, while across the collagen sheets, the phase of the second order nonlinear susceptibility is changing by 180 degrees between adjacent nano-domains. This complex architecture of noncentrosymmetric nano-domains governs the coherent addition of 2ω light within the focal volume and the observed features in the SHG images taken in fascia.

Image formation in CARS microscopy: effect of the Gouy phase shift

“…Parametric nonlinear microscopy of subwavelength structures may introduce distortions into the image that cannot always be predicted by models. For example, recent microscopy experiments using another parametric process, second harmonic generation (SHG), showed that image formation was not simply related to the concentration of the species being probed [8]. Similar distortions were previously discussed for backward direction-detected CARS signals [9].…”

Imaging the noncentrosymmetric structural organization of tendon with Interferometric Second Harmonic Generation microscopy

“…Collagen fibrils have a C 8 symmetry [21] and their c (2) tensor is characterized by three main elements in SHG microscopy, c zzz , c zxx and c xzx , where Z is the direction parallel to the fibrillar axis. All these tensor elements are achiral in nature [27,28] and can be probed using ISHG microscopy.…”

“…They are focussed by a 0.85 NA objective and collected by a 0.55 NA condenser. The use of a slightly lower NA condenser does not significantly alter SHG signal collection [21]. The reference beam interferes with the SHG generated in the sample.…”

Imaging of biological tissues with pixel-level analysis of second-order susceptibility

“…30 A number of biological materials, such as cartilage, tendons, myosin-rich striated muscle, crystallized microtubules of mitotic spindles, and plant chloroplast, have been found capable of generating the second harmonic signal. [31][32][33][34][35][36] Thus, the identification of SHG sources within biological media is of primary importance for its further application in studies of the characteristics of biological organisms. The basis of SHG can be explained from molecular hyperpolarizability β, 19 given as…”