CARS and SHG microscopy to follow collagen production in living human corneal fibroblasts and mesenchymal stem cells in fibrin hydrogel 3D cultures

Abstract

Coherent anti-Stokes Raman scattering (CARS) microscopy is combined with second harmonic generation (SHG) technique in order to follow the early stage of stem cell differentiation within a 3D scaffold.One of the first evidence of hMSCs differentiation is the formation of an extracellular matrix (ECM) where the collagen protein is its main component.This work demonstrated the multimodal CARS and SHG microscopy as a powerful non-invasive label free technique to follow the collagen production in living cell 3D cultures. Its ability to image the cell morphology and the produced collagen distribution on the same sample at the same time, on a long term (4 weeks) experiment allowed to obtain important information about the cell-scaffold interaction and the ECM production. The very low limit reached in detecting collagen has permitted to map even the small amount of collagen produced by the cells in few hours of culture. This demonstrates multimodal CARS and SHG microscopy as a novel method to follow cells collagen production and cells differentiation process in both short and long term experiments. In addition the experiment shows that the technique is a powerful tool for imaging of very thick sections (about 4 mm) with several advantages in its applications. As collagen production is considered a biomarker for ECM production and also a signal of initial stem cells differentiation, the study conducted on 1 mesenchymal stem cell in 3D cultures confirmed that differentiation stimulus is induced by the fibrin gel scaffold.Coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) microscopy techniques, allow a label free non-invasive chemical selection of target species, deep tissue penetration and high 3D spatial resolution at a cellular level. CARS microscopy can detect lipid membranes and droplet compartments in living cells and SHG microscopy enables a strong imaging contrast for molecules with a non-centrosymmetric ordered structure like collagen. In addition, CARS and SHG techniques can be easily combined together in the same microscopy allowing multiple chemical contrasts.This study aims to demonstrate the efficacy of the CARS-SHG combined technique to investigate, in a non-invasive and non-destructive experiment, the dynamics and the distribution of the collagen produced by living stem cells seeded in a 3D fibrin scaffold. The monitoring of stem cell differentiation within a scaffold in a non-destructive way will be an important advantage in regenerative medicine and tissue engineering field.

Micro‐ and Macrostructured PLGA/Gelatin Scaffolds Promote Early Cardiogenic Commitment of Human Mesenchymal Stem Cells In Vitro

“…Employing the multimodal CARS/TPEF microscope realized as described in a previous paper [36], the PLGA/gelatin scaffold was 3D imaged simultaneously with the hMSCs using CARS and TPEF techniques, respectively. CARS signal was measured in forward detection, in a spectral range between 716 nm and 770 nm, while TPEF signal in epidetection in a spectral range between 510 nm and 570 nm.…”

Recent advances in linear and nonlinear Raman spectroscopy. Part VII

“…Mortati, Divieto, and Sassi applied CARS and second‐harmonic generation microscopy to follow collagen production in living human corneal fibroblasts and mesenchymal stem cells in fibrin hydrogel 3‐D cultures. Their findings indicate that multimodal CARS and second‐harmonic generation imaging can be used as a new way of monitoring the differentiation of stem cells by evaluating the production of collagen in both short and long‐term experiments . Shapiro, Konorov, and Milner described interference spectroscopy associated with CARS from noisy broadband pulses.…”

Synthesis, structure and non-linear optical properties of new isostructural β-D-fructopyranose alkaline halide metal–organic frameworks: a theoretical and an experimental study

“…In our previous work (Marabello et al, 2015), we reported a theoretical and experimental study on the NLO properties of two MOFs obtained from fructose and calcium chloride, [Ca(C 6 H 12 O 6 ) 2 (H 2 O) 2 ]Cl 2 ÁH 2 O (CaFruCl) and [Ca(C 6 H 12 O 6 )-(H 2 O) 2 ]Cl 2 (CaFruCl-2). We measured (Mortati et al, 2012) their SH intensity by SHG microscopy and showed that the coordination of fructose on the calcium ion improves the SH efficiency with respect to fructose itself, probably by both the induced lowering of symmetry and the dissociation of CaCl 2 . In this work we extended our investigation on MOFs obtained from fructose and other alkali-earth halogenides to better understand the effect of the size of the cation and the different polarizability of the anion in isomorphic structures on the calculated hyperpolarizability and optical susceptibility, which in turn are related to the SHG efficiency.…”