Coherent Anti-Stokes Raman Scattering, Second Harmonic Generation and Two-Photon Excitation Fluorescence Multimodal Microscope: Realization, Metrological Characterization and Applications in Regenerative Medicine

In the frame of the research in biology and in particular in regenerative medicine, it is widely requested the ability to perform measurements that have a low impact on the observed biological systems. Many measurements imply sample modifications and also sample fixation avoiding living samples measurements. In this doctoral thesis it is presented the realization of an advanced optical multimodal microscope that integrates coherent anti-Stokes Raman scattering, second harmonic generation and two-photon excitation fluorescence techniques in a single powerful tool. The combination of all these microscopy techniques in a single microscope allows gathering more information during samples imaging, implementing fluorescence technique with label free techniques. A description of the experimental setup of the realized multimodal microscope is presented together with the metrological characterization of the instrument, evaluating the main uncertainty sources that influence the measurement processes. Label free microscopy techniques allow performing measurements on biological samples with low invasiveness, since to image the specimens it is not demanded any specific sample preparation. This characteristic leads to measurements on living samples with a true low impact, opening new avenues on the research in biology and in particular way in regenerative medicine. Novel applications of these microscopy techniques are presented to study the extracellular matrix production in both fixed and living samples, as well as to characterize the scaffolds topology and the scaffold-cells interactions in a time-lapse experiment using living samples. This doctorate thesis is composed by a state of the art chapter in which are discussed the advanced nonlinear optical microscopy techniques from a theoretical point of view, the main experimental implementations of CARS microscopy and the main parameters and properties to be measured relevant in regenerative medicine applications and products. A chapter is dedicated to the experimental setup for the realization of the multimodal CARS-SHG-TPEF microscope at the I.N.Ri.M. laboratory. A specific chapter is dedicated to the study of the main sources of uncertainty of the measurements using CARS, TPEF and SHG techniques. A chapter in which are discussed the biological experiments realized using the multimodal CARS-SHG-TPEF microscope and at the end a final chapter with the conclusion of this doctorate thesis. This study has been conducted at the Italian institute of metrology (Istituto Nazionale di Ricerca Metrologica, I.N.Ri.M.) as part of the projects REGENMED, METREGEN and ACTIVE with funding respectively from the UE (ERA-NET plus Grant Agreement No 217257) and from Piedmont Region on UE under the programs CIPE 2007- converging technologies, grant 0126000010-METREGEN and POR-FESR I-I.1.3-I1.1 - ACTIVE. The main accomplished results are: • The development of the whole experimental multimodal CARS-SHG-TPEF microscopy system • The theoretical study of the main sources of uncertainty in the measurements with CARS, TPEF and SHG techniques. • The realization of biological experiments using these microscopy techniques: o To study the collagen production from fixed histological sections of human dermal fibroblasts cultured in fibrin gel scaffold using CARS and SHG techniques o To study the collagen production by live human fibroblasts and mesenchymal stem cells cultured in fibrin gel scaffold using CARS and SHG techniques o To characterize polymeric scaffolds in culture media with a label-free method using CARS and SHG techniques o To study the colonization in a two days time-lapse experiment of a polymeric scaffold by human mesenchymal stem cells stained with calcein using CARS and TPEF techniques