Custom Multiphoton/Raman Microscopy Setup for Imaging and Characterization of Biological Samples

Year: 2019

Authors: Marchetti M., Baria E., Cicchi R., Pavone F.S.

Autors Affiliation: Department of Physics, University of Florence, 50019 Sesto Fiorentino, Italy;
National Institute of Optics, National Research Council (CNR-INO), 50019 Sesto Fiorentino, Italy;
European Laboratory for Non-linear Spectroscopy (LENS), 50019 Sesto Fiorentino, Italy

Abstract: Modern optics o ers several label-free microscopic and spectroscopic solutions which are useful for both imaging and pathological assessments of biological tissues. The possibility to obtain similar morphological and biochemical information with fast and label-free techniques is highly desirable, but no single optical modality is capable of obtaining all of the information provided by histological and immunohistochemical analyses. Integrated multimodal imaging o ffers the possibility of integrating morphological with functional-chemical information in a label-free modality, complementing the simple observation with multiple specific contrast mechanisms. Here, we developed a custom laser-scanning microscopic platform that combines confocal Raman spectroscopy with multimodal non-linear imaging, including Coherent Anti-Stokes Raman Scattering, Second-Harmonic Generation, Two-Photon Excited Fluorescence, and Fluorescence Lifetime Imaging Microscopy. The experimental apparatus is capable of high-resolution morphological imaging of the specimen, while also providing specific information about molecular organization, functional behavior, and molecular fingerprint. The system was successfully tested in the analysis of ex vivo tissues a ected by urothelial carcinoma and by atherosclerosis, allowing us to multimodally characterize of the investigated specimen. Our results show a proof-of-principle demonstrating the potential of the presented multimodal approach, which could serve in a wide range of biological and biomedical applications.


Volume: 2 (2)      Pages from: 51-1  to: 51-14

More Information: The research leading to these results has received funding from Tuscany Region (program PAR FAS 2007-2013 -Bando FAS Salute 2014), from the Italian Ministry for Education, University and Research (Flagship Project NANOMAX), from the Italian Ministry of Health (GR-2011-02349626), from EU Horizon 2020 research and innovation programme (grant H2020-ICT-2016-1 732111 PICCOLO, and grant 654148 Laserlab-Europe) and from Ente Cassa di Risparmio di Firenze (private foundation).
KeyWords: microscopy; multi-photon processes; Raman; Imaging systems; tissue characterization
DOI: 10.3390/mps2020051